Antioxidant substance extracted from rabdosia lophanthide as well as preparation method and application of antioxidant substance

Abstract

The preparation method comprises the following steps: (1) extraction: extracting a serrate rabdosia herb raw material by adopting an ethanol hot dipping method to obtain a concentrated solution, extracting the concentrated solution by using an organic solvent to obtain a water part and an organic part, adsorbing the water part by using a resin column, carrying out gradient elution by using an ethanol solution, and collecting the organic part; performing vacuum freeze drying to obtain total polyphenol; (2) separation: carrying out gradient elution on the total polyphenol by using acetonitrile-0. 1% formic acid water as an elution system, and collecting to obtain 6 fractions; performing isocratic elution and purification on each fraction to obtain an antioxidant substance, and the antioxidant substance comprises Rabdosiin; an ethanol hot dipping method is combined with extraction, polyphenol active ingredients in rabdosia lophanthide can be effectively enriched, and the purity of total polyphenols is remarkably improved through resin column elution and vacuum freeze drying; and isocratic purification is further utilized, so that the antioxidant substances can be directionally separated from the total polyphenol, and the yield is high.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and separation technology, specifically relating to a method for extracting antioxidant substances from *Hedyotis diffusa*, its preparation method, and its application. Background Technology

[0002] Oxidative stress refers to an imbalance between the production of reactive oxygen species (ROS) and the body's antioxidant defense system. Among these imbalances are superoxide anions (SOS). 2- Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radicals (·OH), are common byproducts of biological metabolism. These molecules are highly reactive and can directly cause oxidative damage to intracellular lipids, proteins, and DNA, thereby triggering cellular dysfunction and accelerating disease progression. Existing research has confirmed that oxidative stress is closely related to the occurrence and development of many major diseases, including neurodegenerative diseases, cardiovascular diseases, and inflammatory diseases. Therefore, finding efficient and safe exogenous antioxidants to balance oxidative stress in the body has become a research hotspot in the field of disease prevention and treatment.

[0003] *Hedyotis diffusa*, a perennial herb belonging to the genus *Hedyotis diffusa* in the Lamiaceae family, is commonly used in traditional Chinese medicine for clearing heat and dampness, relieving jaundice, cooling the blood and dispersing blood stasis. It can treat acute jaundice hepatitis, acute cholecystitis, dysentery, enteritis, and traumatic injuries with bruises and swelling. Modern pharmacological studies have shown that *Hedyotis diffusa* contains various chemical components such as phenols, flavonoids, terpenes, and amino acids. Polyphenols are one of its core active ingredients, possessing strong antioxidant properties. Further research has confirmed that the antioxidant effects of *Hedyotis diffusa* polyphenols can slow the progression of cancer, coronary artery sclerosis, and age-related degenerative brain diseases, while also exhibiting anti-inflammatory, antibacterial, and vasodilatory effects.

[0004] However, current research on the antioxidants in *Hedyotis diffusa* remains insufficient. On the one hand, existing extraction processes mostly focus on the crude extraction of total polyphenols, lacking targeted separation and purification techniques for single highly active compounds, resulting in low purity of active ingredients and unclear mechanisms of action. On the other hand, most studies only reach the stage of in vitro antioxidant activity evaluation, lacking activity verification at the cellular level and in vivo in animals, thus failing to fully demonstrate its application potential in the treatment of actual diseases. Therefore, developing a method for efficiently extracting and accurately separating highly active antioxidants from *Hedyotis diffusa* is of great significance for promoting drug development for oxidative stress-related diseases. Summary of the Invention

[0005] To address the problems of low purity in the extraction of antioxidant substances from *Rhizoma Cynanchi*, difficulty in isolating single highly active compounds, and insufficient in vitro and in vivo activity verification in existing technologies, the present invention aims to provide a method for preparing antioxidant substances from *Rhizoma Cynanchi*. This method can efficiently extract the active ingredients from *Rhizoma Cynanchi* and accurately separate compounds including highly active Rabdosiin. Simultaneously, through in vitro antioxidant experiments, the antioxidant activity and protective effect against oxidative stress-related diseases of the obtained substances are comprehensively verified, providing high-quality raw materials for drug development for liver diseases and other oxidative stress-related diseases.

[0006] In a first aspect, the present invention provides a method for preparing antioxidant substances extracted from *Hymenochloa crus-galli*, the steps of which include: (1) Extraction: The raw material of *Hedyotis diffusa* was extracted by hot ethanol extraction to obtain a concentrated solution. The concentrated solution was then extracted with an organic solvent to obtain an aqueous fraction and an organic fraction. The aqueous fraction was adsorbed onto a resin column, eluted with a gradient of ethanol solution, and then freeze-dried under vacuum to obtain total polyphenols. (2) Separation: The total polyphenols were subjected to gradient elution using a medium-pressure preparation liquid. The elution system was acetonitrile-0.1% formic acid water. Six fractions were collected, including TP.A~TP.F. Each fraction was purified by isocratic elution using reversed-phase high-performance liquid chromatography to obtain the antioxidant substances, including Rabdosiin.

[0007] Further, in the separation step (2), the elution ratio was gradually adjusted from 13:87 to 37:63. Specifically, TP.A was eluted with acetonitrile-0.1% formic acid water (15:85) to obtain Vicenin 2, sorghum oleanum, and Camellignanoside; TP.B was eluted with acetonitrile-0.1% formic acid water (17:83) to obtain Icariside E3; TP.C was eluted with acetonitrile-0.1% formic acid water (23:77) to obtain Rabdosiin; and TP.D was eluted with methanol-0.1% formic acid water (45:55) to obtain Iabiserin A, rosmarinic acid, and 1,3-carboxyblumenol. C; TP.E was eluted isocratically with acetonitrile-0.1% formic acid water (30:70) to obtain Homoplantaginin; TP.F was eluted isocratically with methanol-0.1% formic acid water (50:50) to obtain Labiserin B. This invention uses different elution ratios for isocratically eluted reactions to achieve precise separation of 10 single compounds.

[0008] Further, in the extraction step (1): the raw material of *Hedyotis diffusa* is extracted by hot soaking in an ethanol aqueous solution with a volume fraction of 50%~90%, the extraction temperature is 40~80℃, the volume of the extraction solvent is 2~4 times the volume of the medicinal material, the number of extractions is 2~4 times, the extraction time is 2~4 hours each time, the extraction filtrate is combined, the ethanol is recovered under reduced pressure until there is no alcohol taste, and a concentrated solution is obtained; the concentrated solution is extracted with an organic solvent in equal volume, the number of extractions is 2~6 times, and the water part and the organic part are collected respectively.

[0009] Further, in the extraction step (1): the water fraction is eluted sequentially with 10%~30%, 30%~50%, and 50%~70% ethanol through a macroporous adsorption resin column D101, the fractions are combined and recovered under reduced pressure, and then freeze-dried under vacuum to obtain total polyphenols.

[0010] Furthermore, the raw material of the herb *Hymenochloa chinensis* is selected from the aerial parts of the herb *Hymenochloa chinensis*, belonging to the genus *Hymenochloa* of the Lamiaceae family, and is cut into 0.5cm segments after being washed, dried and processed before extraction.

[0011] Furthermore, the organic solvent includes at least one of petroleum ether and ethyl acetate, and the organic part includes at least one of the petroleum ether part and the ethyl acetate part.

[0012] Secondly, the present invention also provides an antioxidant substance prepared according to the above method, wherein the antioxidant substance includes Vicenin 2, sage, Camellignanoside, Icariside E3, Iabiserin A, rosmarinic acid, Rabdosiin, 1,3-carboxyblumenol C, Homoplantaginin, and Labiserin B. In the preparation method of the present invention, compounds such as Vicenin 2, sage, Camellignanoside, Icariside E3, Iabiserin A, rosmarinic acid, Rabdosiin, 1,3-carboxyblumenol C, Homoplantaginin, and Labiserin B all have antioxidant properties, but relatively speaking, Rabdosiin has the highest antioxidant activity.

[0013] Thirdly, the present invention also provides the application of an antioxidant substance in the preparation of antioxidant drugs.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a combination of ethanol hot maceration and extraction to effectively enrich the polyphenolic active ingredients in *Hedyotis diffusa*. Resin column elution and vacuum freeze-drying further significantly improve the purity of total polyphenols (TP). Furthermore, using medium-pressure preparative liquid chromatography gradient elution and reversed-phase high-performance liquid chromatography (RP-HPLC) for isocratic purification, Rabdosiin can be directionally separated from the total polyphenols at a yield of 3.00%. Therefore, the preparation method of this invention has high extraction efficiency and high purity.

[0015] The antioxidant substance prepared by this invention includes Rabdosiin. Rabdosiin's DPPH free radical scavenging ability is 10 times that of the positive drug vitamin C, and its total antioxidant capacity is twice that of the positive drug Trolox. Its antioxidant activity is significantly superior to that of commonly used antioxidants. Furthermore, Rabdosiin is non-toxic to HepG2 cells and can significantly improve the survival rate of cells damaged by t-BHP-induced oxidative stress, increase SOD activity, reduce ROS and MDA levels, effectively alleviate cell damage caused by oxidative stress, and improve liver tissue pathological damage. It has significant potential application prospects in the field of antioxidant liver-protective drugs. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the process for extracting total polyphenols from *Hymenochloa crus-galli*. Figure 2 A process flow diagram for separating antioxidants from total polyphenols; Figure 3 The chemical formulas of the antioxidants numbered 1 to 10 are listed below. Figure 4 A comparison chart of total phenolic content (TPC) in different extracts of *Hedyotis diffusa*. Figure 5 The figure shows the experimental results of testing the antioxidant activity of different extracts of *Hedyotis diffusa*. Figure 5 -a: dose-effect of t-BHP-induced cellular oxidative damage; Figure 5 -b: Cell vitality of different extracts of *Hedyotis diffusa*; Figure 5 -c: Total antioxidant capacity of different parts; Figure 5 -d: Free radical scavenging rate at different sites; Figure 5 -e: Protective effect of different sites on oxidatively damaged cells; Figure 6 The figure shows the experimental results of evaluating the antioxidant activity of different compounds. Figure 6 -a: DPPH free radical scavenging rate; Figure 6 -b: Total antioxidant capacity; Figure 6 -c: Free radical scavenging rate as a function of concentration; Figure 7Figure showing the results of cytotoxicity assays of different doses of Rabdosiin on HepG2 cells; Figure 8 Figure 1 shows the experimental results of the protective effect of Rabdosiin against t-BHP-induced oxidative damage in HepG2 cells. Figure 9 The figure shows the experimental results of the regulatory effects of different doses of Rabdosiin (Camp.7) on t-BHP-induced cellular oxidative stress-related indicators. Figure 9 -a: Flow cytometry distribution of ROS fluorescence intensity; Figure 9 -b: Effect of Rabdosiin on ROS levels; Figure 9 -c: Plot showing the effect of Rabdosiin on MDA levels; Figure 9 -d: Effect of Rabdosiin on SOD levels; Figure 10 Figure showing the effect of different doses of Rabdosiin on liver index in mice with t-BHP-induced acute liver injury; Figure 11 Figure showing the effect of different doses of rabdosiin on AST and ALT levels; Figure 12 Figure showing the effect of different doses of rabdosiin on MDA levels; Figure 13 Image of mouse liver stained with hematoxylin and eosin (HE). Detailed Implementation

[0017] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0018] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0019] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the invention.

[0021] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0022] Example 1 (1) Extraction: See Figure 1 80 kg of the above-ground parts of *Hedyotis diffusa* were extracted using a hot extraction method with 80% ethanol (60℃, 3 times the volume of the herb, 3 extractions, 3 hours each time). The filtrates were combined and purified under reduced pressure until no alcohol odor remained, yielding a concentrated solution. The concentrated solution was extracted three times sequentially with equal volumes of petroleum ether (PE) and ethyl acetate (EtOAc) to obtain the petroleum ether fraction (PE), ethyl acetate fraction (EA), and aqueous fraction (AS). The aqueous fraction was eluted sequentially with 20%, 40%, and 60% ethanol using a macroporous resin column (D101). The elutions were combined and purified under reduced pressure at 40℃ until no alcohol odor remained. The solution was then freeze-dried under vacuum to obtain 570 g of total polyphenols (TP).

[0023] (2) Separation: See Figure 2 Chromatographic separation flowchart and Figure 3 The molecular formulas of 10 antioxidants were obtained. 20g of TP was processed using a medium-pressure preparative liquid chromatography (MPLC) column with ODS, eluted with a gradient of acetonitrile-0.1% formic acid and water (13:87→37:63), yielding 6 fractions (TP.A–TP.F). TP.A was purified by RP-HPLC (acetonitrile-0.1% formic acid isocratic elution = 15:85) to obtain Vicenin 2 (…). Figure 3-3 ), rough perilla ( Figure 3-4 ), Camellignanoside Figure 3-5 ); TP.B was purified by RP-HPLC (acetonitrile-0.1% formic acid isocratic elution = 17:83) to obtain Icariside E3 ( Figure 3-6 ); TP.C was purified by RP-HPLC (acetonitrile-0.1% formic acid isocratic elution = 23:77) to obtain Rabdosiin. Figure 3-7 ); TP.D was purified by RP-HPLC (methanol-0.1% formic acid isocratic elution = 45:55) to obtain Iabiserin A ( Figure 3-1 ), rosmarinic acid ( Figure 3-9 ), 1,3-carboxyblumenol C ( Figure 3-10); TP.E was purified by RP-HPLC (acetonitrile-0.1% formic acid isocratic elution = 30:70) to obtain Homoplantaginin ( Figure 3-8 ); TP.F was purified by RP-HPLC (methanol-0.1% formic acid isocratic elution = 50:50) to obtain Labiserin B. Figure 3-2 ).

[0024] In this embodiment, 20 g of the TP fraction was processed by MPLC to obtain 6 fractions (TP.A-TP.F), including 4.0 g of TP.C. Each fraction was then separated and purified by RP-HPLC (acetonitrile-0.1% formic acid eluted at different ratios) to obtain Vicenin 2 (30.1 mg, 0.15%), sage (20.3 mg, 0.10%), Camellignanoside (13.5 mg, 0.07%), Icariside E3 (4.6 mg, 0.02%), Iabiserin A (2.32 mg, 0.01%), rosmarinic acid (14.5 mg, 0.07%), 1,3-carboxyblumenol C (4.5 mg, 0.02%), Homoplantaginin (16.5 mg, 0.08%), Labiserin B (10.3 mg, 0.05%), and Rabdosiin, which exhibited the best antioxidant activity. 600mg, with a yield of 3.00%.

[0025] (a) Antioxidant activity test (1) DPPH free radical scavenging ability determination: The fractions and compounds obtained in Example 1 were measured using a Solarbio reagent kit. 25 μL of samples or standards of different concentrations and 975 μL of DPPH working solution were added to 1.5 mL EP tubes, vortexed, and incubated at room temperature in the dark for 30 min. The absorbance of the reactants was detected at 515 nm using a multi-functional microplate reader. A blank group (25 μL ethanol + 975 μL DPPH working solution, A blank) and a control group (25 μL sample / standard + 975 μL ethanol, A control) were also set up. Each sample was measured three times. The test results for each fraction are shown below. Figures 4-5 The test results for each compound are shown in Table 1 below. Figure 6 The formula for calculating the DPPH free radical scavenging rate is as follows:

[0026] Table 1:

[0027] (2) Total antioxidant capacity determination: The total antioxidant capacity assay was performed using the Beyotime Total Antioxidant Capacity Kit (S0116). 190 μL of FRAP working solution was mixed with 5 μL of sample or standard and placed in a 96-well plate, with Trolox as a positive control. After incubation at 37℃ for 5 min, the absorbance was read at 593 nm using a microplate reader. The total antioxidant capacity of the sample was calculated based on the standard curve and expressed as mmol Fe. 2+ / mg is the unit. Test results are referenced. Figure 6 .

[0028] Experimental conclusions: From Table 1 and Figures 4-6 It is evident that the TP fraction has the highest total polyphenol content and the best antioxidant activity; Rabdosiin's IC50... 50 Rabdossin exhibited the lowest antioxidant activity and the strongest antioxidant capacity among the 10 isolated compounds. DPPH free radical scavenging assays demonstrated that Rabdossin possessed the highest overall antioxidant capacity (IC50). 50 0.43±0.05 mM) is a positive result for vitamin C (IC50). 50 It was 10 times that of (4.22±0.04 mM). In the total antioxidant capacity assay, Rabdosiin had a total antioxidant capacity of 11.78±1.3 mmol Fe. 2+ / g, is the positive control drug Trolox (6.94±1.0 mmol Fe). 2+ Twice the amount of ( / g).

[0029] II. Cytotoxicity Protection Experiment: (1) Cytotoxicity assay: HepG2 cells in logarithmic growth phase were seeded into 96-well plates (5 × 10⁻⁶ cells / well). 3 Cells / well were treated with different concentrations of Rabdosiin, and cell viability was determined using the MTT assay to confirm the cytotoxicity of Rabdosiin to HepG2 cells. The test results are as follows: Figure 7 As can be seen, compared with the control group, 20-80 μM Rabdosiin had no significant effect on the viability of HepG2 cells (P>0.05) and no cytotoxicity.

[0030] (2) Oxidative damage protection experiment: A cell oxidative damage model was induced using 300 μM t-BHP, and 20, 40, and 80 μM Rabdosiin were added. Cell viability was measured using the MTT assay, and the levels of ROS, SOD, and MDA were also measured. The test results are shown below. Figures 8-9The cell viability in the model group was significantly reduced (33.63% ± 6.86%, P < 0.01). Different concentrations of t-BHP treatment significantly increased cell viability, with the 80 μM Rabdosiin group showing a viability of 97.69% ± 11.0% (P < 0.01). Compared to the control group, t-BHP treatment significantly increased intracellular MDA and ROS levels (P < 0.01) and significantly decreased SOD activity (P < 0.01). Compared to the model group, 80 μM significantly reduced t-BHP-induced increases in MDA and ROS (P < 0.01) and increased SOD activity (P < 0.01). These data indicate that Rabdosiin can improve t-BHP-induced oxidative damage in HepG2 cells.

[0031] III. Animal Experiments: Forty 6-8 week old SPF-grade C57BL / 6 mice were randomly divided into four groups: a control group, a model group, a low-dose rabdosiin group (50 mg / kg), and a high-dose rabdosiin group (100 mg / kg), with 10 mice in each group. The low- and high-dose groups were administered the drug via gavage daily, while the control and model groups were administered an equal volume of physiological saline via gavage for 7 consecutive days. One hour after the last administration, the model and drug-treated groups were intraperitoneally injected with 300 mg / kg t-BHP to establish an acute liver injury model. Liver index, AST, ALT, and MDA levels were measured continuously after administration, and the liver was stained with hematoxylin and eosin (HE) for histopathological examination. The experimental results are as follows: Figure 10-13 As shown.

[0032] As can be seen, compared with the control group, the liver index of mice in the model group was significantly increased (P<0.01); compared with the model group, the liver index of the high-dose group was significantly decreased (P<0.01), while there was no significant difference in the liver index of the low-dose group. The results indicate that high-dose rabdosiin can significantly reduce the liver index in mice with t-BHP-induced acute liver injury.

[0033] Compared with the control group, the serum AST and ALT levels in the model group mice were significantly increased (P<0.01); compared with the model group, the serum AST and ALT levels in the high-dose group mice were significantly decreased (P<0.05). These results indicate that rabdosiin has a protective effect against t-BHP-induced acute liver injury.

[0034] Compared with the control group, the MDA level in the liver tissue of mice in the model group was increased (P<0.05); compared with the model group, the MDA level in the high-dose treatment group was significantly decreased (P<0.01). The results indicate that rabdosiin can reduce t-BHP-induced oxidative damage.

[0035] Furthermore, HE staining was used to study the effects of rabdosiin on the histopathology of liver tissue in mice with t-BHP-induced acute liver injury. The results showed that the liver tissue structure of mice in the blank group was normal and the hepatic cords were neatly arranged; the liver lobules in the model group were unclear, the hepatic cords were disordered, and hepatocytes were degenerated; the degree of hepatocyte necrosis was reduced in the low-dose group, and the high-dose group had normal hepatic cord structure and no hepatocyte necrosis.

Claims

1. A method for preparing antioxidant substances extracted from *Hymenochloa crus-galli*, characterized in that, The preparation method includes the following steps: (1) Extraction: The raw material of *Hedyotis diffusa* was extracted by hot ethanol extraction to obtain a concentrated solution. The concentrated solution was then extracted with an organic solvent to obtain an aqueous fraction and an organic fraction. The aqueous fraction was adsorbed onto a resin column, eluted with a gradient of ethanol solution, and then freeze-dried under vacuum to obtain total polyphenols. (2) Separation: The total polyphenols were subjected to gradient elution using a medium-pressure preparation liquid. The elution system was acetonitrile-0.1% formic acid water. Six fractions were collected, including TP.A~TP.F. Each fraction was purified by isocratic elution using reversed-phase high-performance liquid chromatography to obtain the antioxidant substances, including Rabdosiin.

2. The preparation method according to claim 1, characterized in that, In the separation step (2), the elution ratio was gradually adjusted from 13:87 to 37:

63. Specifically, TP.A was eluted with acetonitrile-0.1% formic acid water (15:85) to obtain Vicenin 2, sorghum oleanum, and Camellignanoside; TP.B was eluted with acetonitrile-0.1% formic acid water (17:83) to obtain Icariside E3; TP.C was eluted with acetonitrile-0.1% formic acid water (23:77) to obtain Rabdosiin; and TP.D was eluted with methanol-0.1% formic acid water (45:55) to obtain Iabiserin A, rosmarinic acid, and 1,3-carboxyblumenol. C; TP.E was eluted isocratically with acetonitrile-0.1% formic acid water (30:70) to obtain Homoplantaginin; TP.F was eluted isocratically with methanol-0.1% formic acid water (50:50) to obtain Labiserin B.

3. The preparation method according to claim 1, characterized in that, In step (1), the raw material of *Hedyotis diffusa* is extracted by hot soaking in an ethanol aqueous solution with a volume fraction of 50% to 90%, the extraction temperature is 40 to 80°C, the volume of the extraction solvent is 2 to 4 times the volume of the medicinal material, the number of extractions is 2 to 4 times, the extraction time is 2 to 4 hours each time, the extraction filtrates are combined, the ethanol is recovered under reduced pressure until there is no alcohol odor, and a concentrated solution is obtained; the concentrated solution is extracted with an organic solvent in equal volume, the number of extractions is 2 to 6 times, and the water part and the organic part are collected respectively.

4. The preparation method according to claim 1, characterized in that, In the extraction step (1), the aqueous fraction is eluted sequentially with 10%~30%, 30%~50%, and 50%~70% ethanol through a macroporous adsorption resin column D101. The fractions are then combined, recovered under reduced pressure, and freeze-dried under vacuum to obtain total polyphenols.

5. According to the preparation method of claim 1, the raw material of *Hymenochloa crus-galli* is the aerial part of *Hymenochloa crus-galli*, belonging to the genus *Hymenochloa* of the Lamiaceae family, and is cut into 0.5cm segments after being washed, dried and cut before extraction.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The organic solvent includes at least one of petroleum ether and ethyl acetate, and the organic part includes at least one of petroleum ether and ethyl acetate.

7. An antioxidant substance prepared by the method according to any one of claims 1-6, characterized in that, The antioxidants include Rabdosiin, Vicenin 2, chamomile, Camellignanoside, Icariside E3, Iabiserin A, rosmarinic acid, 1,3-carboxyblumenol C, Homoplantaginin, and Labiserin B.

8. The use of the antioxidant substance according to claim 7 in the preparation of antioxidant drugs.

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