Twenty-one dried samples of rhubarb root and rhizome were collected from different herbal stores in Taiwan and labeled as RH-1 to RH-9 and SP-1 to SP-12. All samples were identified using TLC according to the method described in CP 2020. Samples containing rhaponticin were divided into the unofficial group including RH-5 to RH-8, SP-1, SP-2, SP-3, and SP-5 to SP-12, whereas those that did not contain rhaponticin were classified into the official rhubarb group, including RH-1 to RH-4, RH-9, and SP-4 (Supplementary Figure S1). The photos of samples SP-1 and SP-4, which were used for method validation, are shown in Figure 1. The voucher specimens of all samples were deposited at the Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Chinese Medine, China Medical University, Taichung, Taiwan.

Eight reference markers including five anthraquinones and three stilbenes were purchased from different companies as follows: aloe-emodin (purity >95%) and emodin (purity >90%) from Sigma-Aldrich Chemie Gmbh., Taufkirchen, Germany; rhein (purity >98%), chrysophanol (purity >98%), physcion (purity >98%), rhapontigenin (purity >95%), and resveratrol (purity >98%) from Chengdu Purify Phytochemicals Ltd., Chengdu, China; and rhaponticin (purity >98%) from Cayman Chemical Company, Ann Arbor, Michigan, United States of America. Structures of the markers are shown in Supplementary Figure S2. Acetonitrile, methanol, phosphoric acid, hydrochloric acid, acetone, and formic acid were purchased from Merck KGaA, St. Louis, Missouri, United States of America; pentane was procured from ITW Reagents, Monza, Italy; and ethyl acetate was acquired from Honeywell International Inc., Charlotte, North Carolina, United States of America.

Six reference standards (rhapontigenin, rhein, aloe-emodin, emodin, chrysophanol, and physcion) were dissolved in methanol to obtain 50 mg/L solution. For the rhubarb extraction method, 0.5 g powder of the sample was extracted in 20 mL of 2% HCl in 70% MeOH, refluxed for 45 min, and filtered to obtain the sample solution (self-development). The development solvent was pentane-ethyl acetate–acetone–formic acid in 15–5–1–0.7 (v/v) (self-development). In the TLC analysis, we used TLC silica gel F254 plate, with 3 µL application as 6 mm bands using a TLC sample semi-automatic applicator Linomat 5 (CAMAG, Muttenz, Switzerland) with Wincat program and Linomat syringes (3 mL; CAMAG, Muttenz, Switzerland). The plate was developed at 80 mm from the lower edge and visualized under 366 nm UV light.

For network pharmacology analysis and KEGG analysis, we collected data from different databases as follows: TCMSP for bioactive components, screened the components with OB >20%, DL > 0.1; PubChem and BindingDB for bioactive components—target; UniProt for target—UniProt genes; DisGeNET for UniProt genes—diseases; and KEGG: Kyoto Encyclopedia of Genes and Genomes for genes—pathways.

In this section, we used Cytoscape 3.9.1 and RStudio 4.2.2 software to analyze our results.

To compare the content of eight markers between two groups of samples, we used the t-test method using R 4.3.2 and RStudio 4.2.2 to generate boxplots of the data.

Network pharmacology and KEGG analyses revealed that anthraquinones, including aloe-emodin, emodin, rhein, chrysophanol, physcion, and their glycosides, targeted 13 proteins similarly, and stilbenes, including rhapontigenin and resveratrol, targeted 29 proteins. Protein target nomenclature followed that of BindingDB. Both groups of active components had four targets in common. In Figure 8, the target nodes with a darker green color are nodes with a greater degree, indicating that the number of compounds acting on them was higher. The anthraquinone group targets that had the largest degrees were ESR1 (estrogen receptor 1), LCK (human lymphocyte-specific protein tyrosine kinase), and ACHE (acetylcholinesterase). Similarly, for compound nodes, the thicker the border, the more targets that the compound node has. In the anthraquinones group, chrysophanol, physcion, and emodin had the majority of targets. Stilbenes also had a huge target number. Stilbenes had a wide effect, whereas anthraquinones displayed a relatively concentrated effect on a few targets. These results showed that the effects of the two groups were different. The component–target network highlighted the pharmacological differences between stilbenes and anthraquinones (Figure 8).

From the above network, the KEGG pathway of anthraquinones and stilbenes was conducted to clarify the difference between the two groups of active components. The greater the rich factor, the greater the intensiveness, and the −log10 (p-values) represents the level of significance of the pathway. Anthraquinones mainly had anti-inflammatory and anti-cancer effects, whereas stilbenes had ovarian regulatory and anti-cancer effects (Figure 9).

Many Rheum species have already been identified, including R. rhabarbarum, R. emodi, R. hotaoense, and R. wittrochii (Wei et al., 2013). Comparing the pharmacological effects of R. palmatum and R. rhabarbarum L., it was found that R. rhabarbarum L. has a stronger small intestinal propulsion-promoting effect but exhibits higher cytotoxicity and acute toxicity in mice (Tianshi et al., 2012). The EP cites R. rhaponticum as a species easily confused with the official rhubarb; however, this species is primarily found in Europe and America, whereas R. rhabarbarum and others are widely distributed throughout mainland China, Tibet, Malaysia, and other countries. Given the geographical proximity and close cultural history between Taiwan and mainland China, many species, such as R. rhabarbarum, R. emodi, and R. australe, are more likely to surface in the Taiwanese market. In this study, we collected 21 rhubarb samples from the market. Using testing methods based on the pharmacopeia, we discovered that 15 rhubarb samples contained rhaponticin—an active ingredient with anti-bacterial properties, which does not exist in the three official rhubarb varieties listed in the pharmacopeias. Through many previous minor investigations, we found that over 70% of rhubarbs in the market are not official. However, determining the species of these unofficial rhubarbs is difficult because of the diversity of species in genus Rheum. Moreover, rhubarb is imported in dried form, making them appear similar, and many genes in the material are damaged during transportation and preservation, rendering gene identification challenging. To date, quantifying eight markers remains the viable method for differentiating unofficial rhubarb in the Taiwanese market. The unofficial rhubarb samples have great similarities, which may be attributed to their homogeneity in the Taiwanese market or that these samples are of the same species. Owing to the economic value of rhubarb in the market, further research is imperative to determine and identify official and unofficial rhubarb.

Our research was based on the understanding of market rhubarb, resulting in a quantitative determination method using eight essential markers, including stilbenes and anthraquinones. Although the quantitative HPLC method has yet to be established, changing market conditions necessitates a faster and simpler method with broad applications. According to many pharmacopeias, traditional rhubarb extraction procedure includes reflux, concentration, hydrolysis with HCl, heating, and separation. This procedure provides a cleaner sample with less contamination but eliminates several important active components, especially stilbenes, owing to their high polarity. Eight markers we want to detect are aglycones, and it is necessary to extract them in an acidic solvent at high temperatures to hydrolyze the glycosides. Based on the principles of glycoside hydrolysis, we devised a more straightforward method and validated through HPLC, which demonstrated minimal reduction in anthraquinones content while enabling the detection of stilbenes. This could help in differentiating official rhubarb from unofficial rhubarb.

Additionally, the HPLC conditions used acetonitrile (solvent A) and 0.1% phosphoric acid (solvent B) instead of methanol: 0.1% phosphoric acid. Acetonitrile is considered to have less absorbance, lower pressure requirement, and higher elution capacity than methanol. Therefore, it is generally safe and suitable for HPLC with a UV detector. In addition, the calibration parameters demonstrated the accuracy of our proposed HPLC method.

Based on the results of the HPLC quantitative determination, 13 samples of rhubarb, including six official rhubarb samples and seven unofficial rhubarb samples, showed no significant difference in the total anthraquinones content. Compared with the standard for total anthraquinones in pharmacopeia (NLT 1.5%), only RH-3 was found to be within the limit. However, the anthraquinones content obtained using our extraction method was slightly lower than that obtained using the method described in the THP 4th, so adhering to the extraction method from THP 4th may enable compliance with standards. Notably, all samples exhibited higher concentrations of chrysophanol than other active ingredients, especially in the unofficial rhubarb. Official rhubarb samples contained significant amounts of rhein, whereas the unofficial rhubarb samples contained exceptionally high levels of stilbenes. This result is consistent with the conclusion of a previous study by Xu Jing from China (Jing, 2001), which showed the importance of stilbenes and rhein in distinguishing the two groups of rhubarb. Stilbenes are commonly found in various species, including R. rhaponticum, R. rhabarbarum, and R. emodi (Mohtashami et al., 2021). EP 10.0 indicates rhaponticin as one of the markers for R. rhaponticum (European Pharmacopoeia, 2020). In our study, in addition to quantitative HPLC, to compare the two groups of rhubarb, we developed a TLC method using rhapontigenin, the aglycone of rhaponticin with lower polarity, together with five anthraquinones as markers for official and unofficial rhubarb. Based on the TLC results, we successfully differentiated 21 batches into two groups in the rhubarb market. This result demonstrates the applicability of this method to rhubarb in the market.

Rhubarb contains many active components, in both official and unofficial rhubarb, encompassing anthraquinones, stilbenes, and flavonoids such as myricetin, epicatechin, catechin, epicatechin-3-O-gallate, procyanidin B-2-3,3′-di-O-gallate, procyanidin B1, gallic acid, 1-o-galloyl-b-D-glucose, resveratrol, rhapontigenin, piceatannol, and methoxy resveratrol (Table 1). Anthraquinones is a class of phenolic compounds comprising the 9,10-anthraquinone skeleton and can be found in various species in the Rheum genus (Komatsu et al., 2006; Smolarz et al., 2013; Ahmad et al., 2014; Mohtashami et al., 2021). They also exist in many natural herbs, such as Aloe vera with main ingredients such as aloin, aloe-emodin, and rhein (Sadiq et al., 2022); Fallopia multiflora containing emodin, emodin-8-O-beta-D-glucoside, physcion, and physcion-8-O-beta-D-glucoside (Yan et al., 2010); Semen cassiae consisting of eight major anthraquinones (obtusifolin-2-glucoside, aurantio-obtusin, aloe-emodin, rhein, obtusifolin, emodin, chrysophanol, and physcion) (Xu et al., 2012); and Cassia obtusifolia L. and Cassia tora L. with sennoside A and sennoside B (Franz, 1993; Chen et al., 2023). Anthraquinones include monomers, bianthraquinones, and anthraquinone glycosides, which exhibit several pharmacological effects such as anti-inflammatory (De Oliveira et al., 2021) and anti-obesity properties (Zhang et al., 2018; Lee et al., 2019; Liudvytska and Kolodziejczyk-Czepas, 2022). Most anthraquinones in TCM exist in the form of glycosides (Zhao et al., 2014; Jintao et al., 2018) and the anti-constipation effect is mainly due to the action of anthraquinone glycosides (de Witte, 1993; Le et al., 2021). Sennosides induce the effect of a laxative by boosting intestinal motility, which may be connected with promoting the release of motilin in the intestines, lowering the level of enteric somatostatin, and inhibiting Na+–K + -ATPase activity in the intestinal mucosa (Wu et al., 2020). Free anthraquinones can be absorbed by the small intestine (Feng et al., 2013). As previously mentioned, the efficacy of rhubarb’s active components stems from free anthraquinone derivatives rather than anthraquinone glycosides (Fairbairn, 2011; Lin et al., 2017). Hydroxyl substitution can enhance most of the bioactivities of anthraquinones, such as anti-tumor, anti-pathogenic microorganism activities, antioxidant, and anti-osteoporosis effects (Li and Li, 2018).

KEGG pathway is a database system used to clarify the pharmacological pathways of pharmaceutical substances. To expand our understanding and compare the two groups of rhubarb in the scope of pharmacology in humans, we analyzed the KEGG pathway, in which the rich factor is the % of genes enriched in the total number of genes in the category. According to our result in the KEGG pathway, anthraquinones in rhubarb, including aloe-emodin, emodin, rhein, chrysophanol, and physcion, significantly affected the NF-kappa B signaling pathway—a prototypical pro-inflammatory signaling pathway implicated in chemical carcinogenesis. Emodin, an important anthraquinone with strong anti-inflammatory properties, protects human nucleus pulposus cells against IL-1 beta-induced apoptosis and inflammation via inhibiting ROS-mediated activation of NF-kappa B (Zhu et al., 2023). Emodin also suppresses tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), iNOS, and COX-2 expression, and inhibits LPS-induced NF-kappa B activation, IKB alpha degradation, phosphorylation of ERK, JNK, and P38 (Yang et al., 2014). In addition, aloe-emodin and its derivatives might be involved in inhibiting Akt, NF-kappa B, and JNK signaling pathways (Shang et al., 2022). Chrysophanol has an anti-inflammatory effect on osteoarthritis in mice in vitro by regulating the SIRT6/NF-kappa B and Nrf2/NF-kappa B signaling pathways (Lu et al., 2023). Rhein exerts anti-inflammatory effects by regulating the PPAR-gamma-NF-kappa B-HDAC3 axis (Wen et al., 2020). Physcion glucoside may decrease the expression of pro-inflammatory cytokines and mediators by inhibiting the TGF-beta/NF-kappa B/mitogen-activated protein kinase pathways (Geng et al., 2018).

In addition to anthraquinones, stilbenes can be found in other species of the Rheum genus (unofficial rhubarb). Stilbenes, including rhaponticin, resveratrol, and piceatannol, are a class of phenolic compounds with a basic skeleton containing 14 carbons (C6–C2–C6), in which a double-bonded ethylene bridge links the two phenyl moieties (Teka et al., 2022). Stilbenes exhibit several effects like antioxidant (Dou et al., 2017), anti-inflammatory (Tan et al., 2022), and anti-viral properties (Mattio et al., 2020). The three stilbenes discussed in this study were rhaponticin, rhapontigenin, and resveratrol. Resveratrol reduces oxidative stress and improves ovarian function in a rat model (Li and Li, 2018), plays a crucial role in preventing and treating ovarian cancer (Xu et al., 2021), exhibits antioxidant activity with the potential to induce apoptosis, and plays a chemopreventive role in UVR-induced skin carcinogenesis (Aziz and Aziz, 2018). Rhaponticin and desoxyrhaponticin can inhibit fatty acid synthase and are considered potential therapeutic agents to treat cancer (Li et al., 2014). Rhaponticin treatment improves lung cancer in vitro (Wang et al., 2021), and it has a protective effect on ovariectomy-induced osteoporosis in rats (Yang et al., 2021).

Generally, anthraquinones exhibit a strong anti-inflammatory effect, followed by anti-cancer effects. However, stilbenes have broader spectrum of effects, such as anti-cancer activities related to ovaries and ovarian hormones. The high stilbenes content in unofficial rhubarb indicates its potential value in the Taiwanese market. With more than 50% of rhubarb on the market being unofficial rhubarb, leveraging this resource can maximize the potential of unofficial source of rhubarb. Figure 10 illustrates the phytochemical differences between the three Rheum species mentioned in the pharmacopeias (R. palmatum, R. tanguticum, and R. officinale) and other Rheum species. This provides a more comprehensive overview of the application of the two groups of rhubarbs and the potential value of unofficial rhubarbs.

The relationship between KEGG pathway analysis and qualitative and quantitative analysis can be discussed in several aspects: the presence of active ingredients, the number of active ingredients, the content of active ingredients, and their specific effects.

First, regarding the presence and the number of active ingredients, it can be understood that the herb can act on specific KEGG pathways when there is an active ingredient. When there are many active ingredients, the number of KEGG pathways might differ, and the effect may be more potent. One example is the unofficial rhubarb group, which includes stilbenes and anthraquinones; under examining the KEGG pathway, the unofficial rhubarb group can act on more KEGG pathways than the official rhubarb group, which only contains anthraquinones (Figure 9). However, there is a limitation in determining how strong the effects are. Second, quantitative analysis would provide more information, as many studies have found the drug’s effects are through a dose-dependent pharmacological pathway, with the higher the dose, the stronger the activity. For example, anti-inflammatory research shows that the effect depends on the dose of free anthraquinones (Xiong et al., 2018) or the higher rhaponticin also showed a dose-dependent effect on anti-inflammatory (Zhou et al., 2021). These conclusions suggest that the content of active ingredients and their extraction are also two factors affecting the pharmacological activity of TCM herbs through many pharmacological pathways. Third, through the p-value and count index in the KEGG pathway, stilbenes and anthraquinones have strong associations with several pathways, such as anthraquinones with NF-kappa B signaling pathway and stilbenes with ovarian steroidogenesis pathway; thus, with a high concentration of active ingredients, the herb may have some special outstanding medicinal effects rather than the pathway with insignificant p-value, such as NF-kappa B signaling pathway and ovarian steroidogenesis pathway.

Qualitative and quantitative analysis methods are used to determine the presence of active ingredients and simultaneously determine the content of that active ingredient in the plant. Meanwhile, the KEGG pathway is a database through which we can know the function and pathway of that active ingredient. Hence, both have a complementary relationship to improve our understanding of active ingredients and drugs.