Qualitative and quantitative analysis methods for quality control of rhubarb in Taiwan's markets

Abstract

Introduction: Rhubarb is a traditional Chinese medicine (TCM) used to release heat and has cathartic effects. Official rhubarb in Taiwan Herbal Pharmacopeias 4th edition (THP 4th) and China Pharmacopeia 2020 (CP 2020) are the roots and rhizomes of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf., and Rheum officinale Baill. However, the Rheum genus is a large genus with many different species, and owing to the similarity in appearance and taste with official rhubarb, there needs to be more clarity in the distinction between the species of rhubarb and their applications. Given the time-consuming and complicated extraction and chromatography methods outlined in pharmacopeias, we improved the qualitative analysis and quantitative analysis methods for rhubarb in the market. Hence, we applied our method to identify the species and quality of official and unofficial rhubarb. Method: We analyzed 21 rhubarb samples from the Taiwanese market using a proposed HPLC-based extraction and qualitative analysis employing eight markers: aloe-emodin, rhein, emodin, chrysophanol, physcion, rhapontigenin, rhaponticin, and resveratrol. Additionally, we developed a TLC method for the analysis of rhubarb. KEGG pathway analysis was used to clarify the phytochemical and pharmacological knowledge of official and unofficial rhubarb. Results: Rhein and rhapontigenin emerged as key markers to differentiate official and unofficial rhubarb. Rhapontigenin is abundant in unofficial rhubarb; however, rhein content was low. In contrast, their contents in official rhubarb were opposite to their contents in unofficial rhubarb. The TLC analysis used rhein and rhapontigenin to identify rhubarb in Taiwan's markets, whereas the KEGG pathway analysis revealed that anthraquinones and stilbenes affected different pathways. Discussion: Eight reference standards were used in this study to propose a quality control method for rhubarb in Taiwanese markets. We propose a rapid extraction method and quantitative analysis of rhubarb to differentiate between official and unofficial rhubarb.

Keywords: anthraquinones; quality control; rhubarb; stilbenes; unofficial rhubarb.

FIGURE 1

Rhubarb samples from Taiwan market. (A) Official rhubarb SP-4 and (B) unofficial rhubarb SP-1.

FIGURE 2

Typical chromatogram of eight reference standards at 225 nm. 1: rhaponticin, 2: resveratrol, 3: rhapontigenin, 4: aloe-emodin, 5: rhein, 6: emodin, 7: chrysophanol, 8: physcion.

FIGURE 3

HPLC chromatograms of 13 market rhubarb samples.

FIGURE 4

Eight marker content in six official and seven unofficial rhubarb samples. (A) Comparison of five anthraquinones content (%) between the two groups of rhubarb. (B) Comparison of three stilbenes content (%) between the two groups of rhubarb. (C) Comparison of total stilbenes and total anthraquinones content (%) between the two groups. Statistic method: pair t-test, ns p-value >0.05; *p-value ≦ 0.05; **p-value ≦ 0.01; ***p-value ≦ 0.001; ****p-value ≦ 0.0001.

FIGURE 5

TLC visualization condition for rhubarb: TLC analysis of SP-1 and SP-4 with six reference markers. (A) 366 nm UV light, (B) 254 nm UV light, and (C) white light. 1–2: SP-4, 3: aloe-emodin, 4: emodin, 5: physcion, 6: chrysophanol, 7: rhein, 8: rhapontigenin, 9–10: SP-1, and 11: spike (RH-6 + rhapontigenin).

FIGURE 6

TLC analysis of SP-1 and SP-4 under two development solvents: (A) pentane-ethyl acetate–acetone–formic acid 15–5–1–0.7 (v/v) and (B) pentane-ethyl acetate–formic acid 15–5–0.5 (v/v). 1: rhein, 2: rhapontigenin, 3–4: SP-4, 5–6: SP-1, 7: spike (SP-1 + rhapontigenin).

FIGURE 7

TLC analysis of 21 market rhubarb samples under 366 nm UV light, development solvent A pentane-ethyl acetate–acetone–formic acid 15–5–1–0.7 (v/v). 1: blank (methanol), 2: aloe-emodin, 3: emodin, 4: chrysophanol, 5: rhein, and 6: rhapontigenin. (A) Sample RH-1 to RH-6 (B) sample RH-7 to RH-9, SP-1, SP-2, and SP-6 (C) sample SP-1, SP-5 to SP-9 (D) sample SP-10 to SP-12.

FIGURE 8

Compound–protein target network of stilbenes and anthraquinones.

FIGURE 9

KEGG pathway of (A) anthraquinones and (B) stilbenes.

FIGURE 10

Phytochemical differences between the three Rheum species mentioned in Pharmacopeias (R. palmatum, R. tanguticum, and R. officinale) and unofficial rhubarb in Taiwan’s market.