Study on the Spectral Effect Relationship of Total Polyphenols and Total Anthraquinone Content in Rhubarb and Its In Vitro Antioxidant Activity
In 1956, Harman proposed the theory of free radical aging, in which oxidative reactions are the main cause of inducing biological aging and various diseases. Free radicals are mainly generated by electron leakage from mitochondrial respiratory chains or catalyzed by enzymatic reactions, including oxygen free radicals and other free radicals. Some active substances can directly or indirectly act on free radicals to reverse their oxidative attacks on body cells. Currently, synthetic antioxidants such as butyl hydroxyanisole, dibutyl hydroxymethyl ether, and propyl gallate all have certain cytotoxicity. Therefore, the search for efficient and low toxicity antioxidants from food, spices, and natural products has also attracted much attention. The DPPH method and ABTS method can be used for large sample determination and antioxidant capacity determination of hydrophilic and lipophilic substances. Therefore, it is necessary to search for efficient and low toxicity antioxidants from natural products. Rhubarb is a traditional Chinese medicinal herb, originating from the Polygonaceae plant Rheum palmatum L. and Rheum tanguticum Maxim The dried roots and rhizomes of ex Balf. or medicinal Rheum officinale Baill. mainly contain free and bound anthraquinone derivatives, tannins, stilbene glycosides, polyphenols, and other components, which have pharmacological effects such as diarrhea, antibacterial, hemostatic, and antioxidant. Previous studies have shown that emodin -8-O – β – D-glucoside and total anthraquinone glycosides are positively correlated with in vitro antioxidant activity. Eight components, including gallic acid, catechins or epicatechins, and 2-O-cinnamoyl-galloyl glucose, have strong scavenging abilities against ABTS free radicals.
The traditional method of screening active compounds often involves separation, purification, etc., which undoubtedly increases labor intensity and is costly and time-consuming. Currently, the spectral effect relationship has been widely used in the screening of active ingredients in traditional Chinese medicine. It refers to the establishment of fingerprint spectra and pharmacological experiments, using mathematical software and computer technology to link “spectra” and “effects”. This method is in line with the theoretical system of traditional Chinese medicine and makes up for the shortcomings of chemical fingerprint spectra in controlling the quality of traditional Chinese medicine. The correlation between the quality and quantity characteristics of traditional Chinese medicine fingerprint spectra and the biological activity research results of Chinese medicinal materials can be used to establish a mathematical expression for evaluating the quality of Chinese medicinal materials. Therefore, this study established a “spectrum” using HPLC method, and established an in vitro antioxidant “effect” using DPPH method and ABTS method. The total polyphenol content and total anthraquinone content of 20 batches of rhubarb were determined to investigate the relationship between the chemical composition of rhubarb and its in vitro antioxidant activity, in order to obtain the potential antioxidant active ingredient group of rhubarb.

The total anthraquinone content of 20 batches of rhubarb decoction pieces is 1.06 ± 0.08~4.82 ± 0.12 mg/g, and the total polyphenol content is 4.92 ± 0.21~10.12 ± 0.11 mg/g. The 20 batches of rhubarb slices can be divided into four categories, namely Longxi County and Lintao County, Dangchang County and Li County, Min County and Zhang County, and Weiyuan County. Peaks 7 (aloe emodin 8-O-glucoside), 1, 10 (rhein), 2, and 9 (aloe emodin) have the activity of scavenging DPPH free radicals, while peaks 7, 2, 9, 10, 3, 8, 12 (emodin), and 14 (emodin methyl ether) have the activity of scavenging ABTS free radicals. This is consistent with the changes in peak area of in vitro antioxidant chromatography, indicating that the antioxidant effect of rhubarb is the result of the combined action of multiple components. Peak 10 (rhein) showed a positive correlation with ABTS free radical scavenging, while peak 7 (aloe emodin 8-O-glucoside) showed a negative correlation. This may be due to the fact that the shared peak 10 (rhein) can form a complete large π – conjugated system. In ethanol solution, rhein complexes may be unstable and prone to form free radical intermediates, resulting in a high inhibition rate and a positive correlation. Peak 7 (aloe emodin 8-O-glucoside) is relatively stable in ethanol solution and is less likely to form free radical intermediates, resulting in a lower inhibition rate and a negative correlation. There is a negative correlation between peaks 2 and 11 in terms of ABTS free radical scavenging activity. These two compounds are highly likely to contain no or fewer hydroxyl groups that provide active hydrogen. The ABTS free radical scavenging activity depends on the structure of the compound molecule, and the number and position of phenolic hydroxyl groups are the main factors affecting its antioxidant activity. The more active hydrogen that can be provided, the stronger its antioxidant ability. Therefore, this project will further improve the identification and characterization of the unknown common peaks mentioned above, in order to further clarify the pharmacological basis of the antioxidant effect outside the corpus luteum.
This experiment considers the instability of polyphenolic components and chooses ultrasonic extraction to avoid the loss of components under high temperature conditions. Comparing the components with significant changes in chromatographic peak area, subsequent antioxidant verification was carried out by preparing liquid phase separation of monomers. LC-MS and nuclear magnetic resonance techniques were used to identify the separated products and clarify the single components that play a role, which is also a direction for future research. The correlation between the quality and quantity characteristics of traditional Chinese medicine fingerprint spectra and the biological activity research results of medicinal materials can be used to establish a mathematical expression for evaluating the quality of traditional Chinese medicine. On the basis of establishing a fingerprint map of rhubarb and determining the quality and quantity of total anthraquinone and total polyphenol content in rhubarb decoction pieces, this article further establishes the spectrum effect relationship of the antioxidant activity outside the corpus luteum, clarifies the effective groups of rhubarb for scavenging DPPH free radicals and ABTS free radicals, and identifies the components of rhubarb that exert antioxidant effects, such as gallic acid, emodin, and emodin methyl ether. This provides a theoretical basis for screening and distinguishing the active ingredients of rhubarb antioxidant substances from different regions of rhubarb.