A method for screening antioxidant active ingredients of fructus alpiniae oxyphyllae based on a caenorhabditis elegans model

Abstract

The application belongs to the technical field of medicine, and discloses a method for screening antioxidant active ingredients of Fructus Schisandrae based on a Caenorhabditis elegans model. The method comprises the following operation steps: transferring wild-type N2 Caenorhabditis eleganss that are synchronously grown to the L4 stage to NGM culture plates to which active ingredient samples of Fructus Schisandrae are added and blank control NGM culture plates, as a sample group and a control group; observing and recording the survival rate, heat stress life span, antioxidant stress life span, swallowing frequency, head shaking frequency, movement ability, egg laying rate, lipofuscin and cellular reactive oxygen species level, antioxidant enzyme defense capacity of the nematodes, and completing antioxidant gene correlation analysis through transcriptome sequencing, and when the sample group treated by the active ingredients of Fructus Schisandrae has a significant difference compared with the control group, the active ingredients of Fructus Schisandrae have antioxidant activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for screening antioxidant active ingredients in Alpinia oxyphylla based on a Caenorhabditis elegans model. Background Technology

[0002] Increasing work and life pressures, unhealthy lifestyles, and other factors contribute to the rising incidence of many chronic diseases, such as metabolic disorders, cardiovascular and cerebrovascular diseases, and cancer. These chronic diseases are also showing a trend of affecting younger people, seriously impacting the quality of life for the population. These diseases are closely related to the instability of the body's antioxidant system. Under normal conditions, the level of free radicals in the body is in dynamic equilibrium and plays an important role in cell signaling, homeostasis, autophagy, and cell division. However, when the body is subjected to external stimuli or is in a disease state, free radicals and reactive oxygen species (ROS) are released and accumulate rapidly, disrupting the body's normally stable oxidation and antioxidant system. This can ultimately lead to chronic diseases such as neurodegenerative diseases, inflammation, cancer, and cardiovascular diseases. Therefore, regulating the body's antioxidant defense system to reduce oxidative stress is of great significance for maintaining health.

[0003] Given the mixed quality of antioxidant products on the market, people are increasingly turning their attention to natural active ingredients in hopes of finding effective, low-toxicity, and safe new methods of anti-oxidation. Natural plants, especially those used in both food and medicine, contain a variety of bioactive components, such as flavonoids, polysaccharides, polyphenols, and heptanes, thus possessing excellent antioxidant, anti-inflammatory, and anti-cancer biological activities. Ingesting these medicinal and edible plants through diet, or developing functional foods using their specific active ingredients, can help improve the body's antioxidant capacity and reduce inflammatory responses, becoming a popular research direction in the food and pharmaceutical fields.

[0004] Alpinia oxyphylla is one of the "Four Great Southern Chinese Herbs" and one of the first batch of Chinese medicinal herbs listed as both food and medicine by the National Health Commission of my country. As a traditional Chinese medicine, it is mainly used clinically to treat symptoms such as kidney deficiency causing enuresis, frequent urination, and abdominal pain due to cold. As a food, Alpinia oxyphylla has the effects of stimulating appetite, strengthening the spleen, increasing appetite, and aiding digestion, and it has a high safety profile. Modern pharmacological research also shows that, in addition to its traditional effects, Alpinia oxyphylla also possesses various pharmacological activities such as neuroprotection, antioxidation, antitumor activity, and immunomodulation. Therefore, it has high development and utilization value and broad application prospects in the fields of food and health products. Currently, scholars at home and abroad have conducted in-depth research on the chemical composition of Alpinia oxyphylla, discovering that it contains a variety of active ingredients, such as terpenes, sterols, flavonoids, and diphenylheptanes. Current research on Alpinia oxyphylla mainly focuses on its chemical composition and pharmacological activities, but research on the active ingredients corresponding to different biological effects, such as its antioxidant and anti-inflammatory effects, is still lacking. This greatly restricts a comprehensive understanding of the biological functions of Alpinia oxyphylla and further limits its better development and utilization.

[0005] Diphenylheptane compounds are a class of compounds unique to plants in the ginger family (Zingiberaceae). They possess a parent structure of 1,7-disubstituted phenyl groups with a heptane backbone and can be divided into linear and cyclic diphenylheptane compounds. All diphenylheptane compounds have phenolic, hydroxyl, or olefinic chain structures and possess potential anti-inflammatory, antioxidant, antibacterial, antiviral, and antitumor effects.

[0006] Among the diphenylheptane compounds of Alpinia oxyphylla, yakuchinone A, yakuchinone B, oxyphyllacinol, and neonootkatol are the main representatives. However, current research on the antioxidant properties of diphenylheptane compounds of Alpinia oxyphylla is lacking, limited to simple in vitro chemical model activity verification or antioxidant activity studies of Alpinia oxyphylla extracts rather than pure compounds. For example, Lin et al. demonstrated that yakuchinone A can effectively scavenge DPPH and ABTS+ free radicals and inhibit lipid oxidation; Yang Fan et al. found that Alpinia oxyphylla extract extracted with 95% ethanol can increase the lifespan of nematodes, increase their high-temperature resistance, and increase the expression of related antioxidant genes. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a method for screening antioxidant active ingredients of Alpinia oxyphylla based on the Caenorhabditis elegans model.

[0008] Current research on Alpinia oxyphylla primarily focuses on its chemical composition and pharmacological activities, but studies on its antioxidant active components are still lacking, particularly on the antioxidant properties of its diphenylheptane compounds. Research is limited to simple in vitro chemical model activity verification or studies on the antioxidant activity of Alpinia oxyphylla extracts rather than pure compounds. This significantly restricts a comprehensive understanding of the antioxidant activity of Alpinia oxyphylla and further limits its development and utilization. Caenorhabditis elegans shares 60%–80% gene homology with human genes, making the use of nematode models for antioxidant research a hot topic. This invention is the first in the field to utilize the Caenorhabditis elegans model to evaluate the antioxidant active components and mechanisms of Alpinia oxyphylla, providing a preliminary research foundation for future in vivo animal experiments and even human clinical trials. This has significant research implications for the body's antioxidant processes through dietary nutrition strategies, and also lays the material foundation and provides a theoretical basis for further development of antioxidant functional foods and health products based on Alpinia oxyphylla.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A method for screening antioxidant active ingredients of Alpinia oxyphylla based on the Caenorhabditis elegans model includes the following steps: wild-type N2 Caenorhabditis elegans, synchronized to the L4 stage, are transferred to NGM culture plates containing Alpinia oxyphylla active ingredient samples and blank control NGM culture plates as sample groups and control groups, respectively; the normal lifespan, heat stress lifespan, antioxidant stress lifespan, swallowing frequency, head shaking frequency, mobility, oviposition rate, lipofuscin and cellular reactive oxygen species levels, and antioxidant enzyme defense capacity of the nematodes are observed and recorded; antioxidant gene association analysis is performed by transcriptome sequencing; when the sample group treated with Alpinia oxyphylla active ingredients shows a significant difference compared with the control group, the Alpinia oxyphylla active ingredients have antioxidant activity.

[0011] The active ingredients of Alpinia oxyphylla include Alpinia oxyphylla A, Alpinia oxyphylla alcohol, and Alpinia oxyphylla B.

[0012] The wild-type N2 Caenorhabditis elegans that has been synchronized to the L4 stage was obtained according to the following steps: pregnant nematodes were collected with M9 buffer, and after lysis, centrifugation, precipitation, and washing, they were cultured at 20°C for 12 h to obtain L1 stage nematodes; the L1 stage nematodes were transferred to NGM plates containing Escherichia coli OP50 and cultured for 52-60 h to obtain L4 stage nematodes.

[0013] The concentration of the active ingredient sample of Alpinia oxyphylla was 50–100 μM.

[0014] When the active ingredient sample of Alpinia oxyphylla is Alpinia oxyphylla A, its concentration is 100 μM; when the active ingredient sample of Alpinia oxyphylla is Alpinia oxyphylla alcohol, its concentration is 50 μM.

[0015] The principle of this invention:

[0016] (1) This invention verifies whether the two substances extend the lifespan of nematodes when they exert their antioxidant effects by detecting the effects of active ingredients of Alpinia oxyphylla (including Alpinia oxyphylla A and Alpinia oxyphylla alcohol) on the lifespan of nematodes.

[0017] (2) This invention verifies whether the two substances enhance the motility of nematodes when exerting antioxidant effects by detecting the effects of active ingredients of Alpinia oxyphylla (including Alpinia oxyphylla A and Alpinia oxyphylla alcohol) on the motility of nematodes (including head shaking frequency, swallowing frequency and movement ability).

[0018] (3) This invention verifies whether the two substances have an adverse effect on the reproductive capacity of nematodes when they exert their antioxidant effects by detecting the effects of the active ingredients of Alpinia oxyphylla (including Alpinia oxyphylla A and Alpinia oxyphylla alcohol) on the reproductive capacity of nematodes.

[0019] (4) This invention verifies whether these two substances exert their antioxidant effect by reducing the ROS content in nematodes by detecting the effect of active ingredients of Alpinia oxyphylla (including Alpinia oxyphylla A and Alpinia oxyphylla alcohol) on the ROS content in nematodes.

[0020] (5) This invention verifies whether the two substances exert their antioxidant effects by increasing the activity of SOD and GSH enzymes or decreasing the activity of CAT enzymes by detecting the effects of active ingredients of Alpinia oxyphylla (including Alpinia oxyphylla A and Alpinia oxyphylla alcohol) on oxidases (SOD, GSH, CAT) in nematodes.

[0021] (6) This invention uses q-PCR molecular biology technology to detect the effects of active ingredients of Alpinia oxyphylla (including Alpinia oxyphylla A and Alpinia oxyphylla alcohol) on the antioxidant genes (daf-2, daf-16, age-1, sod-3, hsp16.2 and skn-1) of Nematodes, and reveals the antioxidant mechanism of these two substances;

[0022] The present invention has the following advantages and effects compared with the prior art:

[0023] (1) Literature indicates that Alpinia oxyphylla has antioxidant and anti-inflammatory effects, but research on its corresponding bioactive substances is insufficient. One innovation of this study is that it will be the first in the field to use the Caenorhabditis elegans antioxidant model to evaluate the antioxidant effects of two major representative diphenylheptane compounds in Alpinia oxyphylla—alpinoxone A and Alpinoxone L. By detecting the effects of these two substances on the lifespan, motility, reproductive capacity, ROS content, and antioxidant enzyme activity of nematodes, the antioxidant activity of specific components of Alpinia oxyphylla will be clarified, as well as the structure-activity relationship between antioxidant activity and components, thereby improving the understanding of the antioxidant material basis of Alpinia oxyphylla.

[0024] (2) Currently, research on the antioxidant activity of diphenylheptane in Alpinia oxyphylla is limited to in vitro chemical antioxidant models or Alpinia oxyphylla extracts rather than pure compounds, and there is a lack of in-depth exploration of its mechanism of action. Another feature and innovation of this invention is that it uses q-PCR molecular biology methods to study the potential antioxidant mechanism of Alpinia oxyphylla A and Alpinia oxyphylla alcohol in the Caenorhabditis elegans model, thereby providing a theoretical basis for the development of natural functional foods and health products with antioxidant effects from Alpinia oxyphylla. Attached Figure Description

[0025] Figure 1 The effects of Aldehyde A and Aldehyde B on the lifespan of Nematodes.

[0026] Figure 2 The effects of oxytocin A and oxytocin on the swallowing frequency of nematodes.

[0027] Figure 3 The effects of Aldehyde A and Aldehyde B on the head-shaking frequency of nematodes.

[0028] Figure 4 The effects of oxadoxime and oxadoxol on the mobility of nematodes.

[0029] Figure 5 The effects of Aldehyde A and Aldehyde B on nematode reproduction.

[0030] Figure 6 The effects of oxadoxime and oxadoxime on heat stress in nematodes.

[0031] Figure 7 The effects of oxytocin A and oxytocin on oxidative stress in nematodes.

[0032] Figure 8 The effects of Aldehyde A and Aldehyde B on ROS and antioxidant enzyme activity in Nematodes.

[0033] Figure 9 The effects of Aldehyde A and Aldehyde B on lipofuscin in nematodes.

[0034] Figure 10 Effects of Aldehyde A and Aldehyde B on Antioxidant Gene Expression in Nematodes Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment of the present invention are conventional reagents, methods, and equipment in this technical field.

[0036] This invention does not impose any special restrictions on the use of Aldehyde A and Aldehyde Alcohol. In the embodiments of this invention, Aldehyde A and Aldehyde Alcohol were purchased from Jinan Mingke Biotechnology Co., Ltd., with a purity of over 99%. Wild-type C. elegans (the Bristolstrain N2) and uracil-deficient Escherichia coli (E. coli) OP50 were provided by the Functional Food Research Group of South China Agricultural University.

[0037] Example 1: Nematode lifespan experiment

[0038] Specific operations:

[0039] This experiment aimed to analyze the effects of oxadoxime and oxadoxine on the lifespan of nematodes. Nematodes that had synchronized to the L4 stage were transferred to NGM culture plates containing either oxadoxime (100 μM) or oxadoxine (50 μM), with 60 nematodes per plate, marked as day 0 of the nematode lifespan. The plates were incubated at 20°C, and the nematodes were transferred to a new NGM plate daily to eliminate the influence of offspring. After the reproductive period, the NGM plates were changed every 2 days to maintain drug concentration. The number of nematodes remaining, dying, and lost were observed and recorded daily. The experiment was repeated three times, with a total of 180 individuals in each group.

[0040] Experimental results:

[0041] like Figure 1 As shown, the survival curves of nematodes treated with 100 μM oxadione or 50 μM oxadione were significantly shifted to the right compared with the control group. Compared with the control group, the median lifespan and maximum lifespan of nematodes in the oxadione group increased by 18.75% and 16%, respectively, while the median lifespan and maximum lifespan of nematodes in the oxadione group increased by 12.5% ​​and 12%, respectively. This indicates that both substances can prolong the lifespan of nematodes.

[0042] Example 2: Detection of Nematode Motility

[0043] Over time, nematodes exhibit a series of changes in physiological aging-related indicators. Among these, decreased locomotor ability and reduced response to external mechanical stimuli are manifestations of nematode aging, and locomotor ability is a commonly measured indicator for assessing the level of aging in nematodes. This invention comprehensively evaluates the effects of oxytocin A and oxytocin on the locomotor ability of nematodes by detecting swallowing frequency, head twitching frequency, and mobility.

[0044] (1) Detection of nematode swallowing ability

[0045] Specific operations:

[0046] N2 nematodes that had been synchronized to the L4 stage were transferred to NGM medium containing 50 μM oxadoxime and NGM medium containing 100 μM oxadoxime A, with a blank control group included. At days 3, 7, and 11, the swallowing frequency of 10 nematodes was randomly observed using a stereomicroscope, and the number of pharyngeal pump contractions within 30 seconds was recorded as the nematode swallowing rate.

[0047] Experimental results: such as Figure 2 As shown, the swallowing frequency of nematodes treated with oxadoxone A (100 μM) or oxadoxol (50 μM) decreased with age. Compared with the control group, oxadoxone A and oxadoxol had no adverse effects on swallowing in all three stages of nematode life, and the effect of increasing swallowing frequency was more obvious in the later stage of nematode life (11 days).

[0048] (2) Detection of nematode head-shaking ability

[0049] Specific operations:

[0050] N2 nematodes that had been synchronized to the L4 stage were transferred to NGM medium containing 50 μM oxadoxime and NGM medium containing 100 μM oxadoxime A, with a blank control group included. On days 4, 8, and 12, the number of times the nematode's head swung from one side to the other and back within 30 seconds was observed and recorded under a stereomicroscope, with a total of 45 nematodes in each group.

[0051] Experimental results:

[0052] like Figure 3 As shown, the head frequency of nematodes treated with oxadoxime (100 μM) or oxadoxine (50 μM) decreased with age, and compared with the control group, oxadoxime and oxadoxine significantly increased the head-shaking frequency of nematodes in the later stage of life (day 12) (p < 0.01).

[0053] (3) Detection of nematode mobility

[0054] Specific operations:

[0055] The mobility assay was performed to determine whether oxadoxime and oxadoxine affected the mobility of nematodes. N2 nematodes that had been synchronized to the L4 stage were transferred to NGM medium containing 50 μM oxadoxine and NGM medium containing 100 μM oxadoxime, with a blank control group included. Mobility was observed and recorded under a stereomicroscope on days 3, 7, and 11. Mobility was classified into three levels: (1) nematodes moved spontaneously without physical stimulation (Class A); (2) nematodes moved only upon physical stimulation (Class B); and (3) nematodes only wiggled their head or tail upon physical stimulation (Class C). Data were expressed as percentages.

[0056] Experimental results:

[0057] like Figure 4 As shown, treatment with oxadoxime A and oxadoxol effectively delayed the degradation of nematode mobility. On day 3, nematodes in both the control and sample groups were able to move spontaneously; on day 7, 93.3% of nematodes in the control group were able to move spontaneously (A), while 93.33% and 95.67% of nematodes in the oxadoxime A and oxadoxol treatment groups, respectively, were able to move spontaneously (A); on day 11, the proportions of nematodes in the control group rated A, B, and C for mobility were 55.67%, 17.78%, and 26.67%, respectively; the proportions in the oxadoxime A group rated A, B, and C for mobility were [not specified in the original text]. The proportions of A, B, and C were 75.56%, 13.33%, and 11.11%, respectively; in the Alpinia oxyphylla group, the proportions of nematodes rated A, B, and C for mobility were 77.78%, 13.33%, and 8.89%, respectively. The results clearly show that Alpinia oxyphylla and Alpinia oxyphylla can effectively enhance the mobility of nematodes while prolonging their lifespan, especially in the later stages of life (day 11), and Alpinia oxyphylla shows a stronger effect than Alpinia oxyphylla.

[0058] Example 3: Detection of Nematode Reproductive Capacity

[0059] Specific operations:

[0060] The reproductive experiment aimed to determine whether oxadoxime and oxadoxine would inhibit the offspring of nematodes. Synchronized hermaphroditic nematodes at the L4 stage were selected and placed in sample plates. Every 24 hours, the nematodes were transferred to new NGM plates until oviposition was lost. The oviposition plates were then incubated at 20°C. After 48 hours, the number of offspring was counted, which was the effective oviposition rate.

[0061] Experimental results:

[0062] like Figure 5 As shown, compared with the control group, the effective oviposition of the sample groups with 100 μM oxadione or 50 μM oxadione was higher on the 1st and 2nd days. The total effective oviposition of the sample groups was also slightly higher than that of the control group, but there was no significant difference. This indicates that oxadione and oxadione did not have an adverse effect on nematode reproduction.

[0063] Example 4: Detection of Acute Heat Stress in Nematodes

[0064] Specific operations:

[0065] Synchronized nematode eggs were cultured on NGM culture plates containing samples for 3 days, with a blank control plate set up. The nematodes were then transferred from a 20°C incubator to a 35°C stress environment. The number of surviving and dead nematodes was recorded every 1 hour, and dehydrated and dead nematodes were removed in a timely manner.

[0066] Experimental results:

[0067] like Figure 6 As shown, under acute heat stress at 35℃, compared with the control group, the samples of oxadixyl-A and oxadixylol significantly shifted the survival curves of nematodes to the right (p<0.01). Specifically, the median and maximum lifespan of nematodes in the oxadixyl-A group increased by 15.38% and 15.79%, respectively, while the median and maximum lifespan of nematodes in the oxadixylol group increased by 7.69% and 10.53%, respectively. These results indicate that both oxadixyl-A and oxadixylol have a protective effect on nematodes under acute heat stress and can increase the heat stress lifespan of N2 nematodes.

[0068] Example 5: Detection of Acute Oxidative Stress in Nematodes

[0069] Specific operations:

[0070] Fifty nematodes from each group were treated with 100 μM or 50 μM oxadiol for 3 days and exposed to oxidative stress induced by 0.1% H2O2 (30% by volume). The number of surviving and dying nematodes was recorded every 30 minutes until all nematodes died.

[0071] Experimental results:

[0072] like Figure 7 As shown, under acute oxidative stress from H2O2, the survival curves of nematodes in the groups of alpinoxazone A and alpinoxazone alcohol shifted to the right compared with the control group, indicating that alpinoxazone A and alpinoxazone alcohol have the potential to enhance the antioxidant stress resistance of nematodes.

[0073] Example 6: Detection of ROS and antioxidant enzyme activities in nematodes

[0074] (1) Detection of ROS in nematodes

[0075] Specific operations:

[0076] Nematodes were treated with 100 μM oxadione or 50 μM oxadione for 3 days. The nematodes were then collected in 2 mL centrifuge tubes, homogenized using a bead mill, and centrifuged at low temperature (4℃, 14000 r / min, 15 min). The supernatant was collected and stored at 4℃ for later analysis. A 10 μM working solution of the fluorescent probe H2DCF-DA in PBS buffer was prepared. 50 μL of the supernatant and 50 μL of the 10 μM H2DCF-DA solution were added to a 96-well plate, mixed thoroughly, and allowed to stand for 1 h. The OD value was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Protein concentration was standardized and quantified using a BCA assay kit.

[0077] Experimental results:

[0078] like Figure 8 As shown in (A), compared with the blank control group, the ROS content in the Aldehyde A and Aldehyde B groups was significantly reduced (p<0.05), and Aldehyde B had a better effect. This indicates that Aldehyde A and Aldehyde B can reduce the oxidative damage caused by ROS accumulation in nematodes and delay nematode aging.

[0079] (2) Detection of SOD, CAT and GSH activity in nematodes

[0080] Specific operations:

[0081] Nematodes were treated with 100 μM or 50 μM oxadiol for 3 days. The nematodes were then collected in 2 mL centrifuge tubes, homogenized using a bead mill, and centrifuged at low temperature (4℃, 12000 rpm / min, 10 min). The supernatant was collected and stored at 4℃ for analysis. The activities of SOD, CAT, and GSH were measured according to the kit instructions. The results were standardized and quantified using a BCA kit to determine protein concentration.

[0082] Experimental results: such as Figure 8 As shown in (B), (C), and (D), compared with the blank control group, the SOD enzyme activity of the alpinoxonol and alpinoxonol sample groups increased by 15.08% and 72.32%, respectively; the GSH enzyme activity increased by 28% and 48.04%, respectively; and the CAT enzyme activity increased by 23.25% and 123.53%, respectively. Alpinoxonol showed a better effect than alpinoxonol. These data suggest that alpinoxonol and alpinoxonol may have the potential to activate the antioxidant defense system of nematodes to some extent, increasing the activity of antioxidant enzymes in nematodes, reducing oxidative damage, and delaying senescence.

[0083] Example 7: Detection of lipofuscin in nematodes

[0084] Lipofuscin, a marker of cellular damage during aging, accumulates in the intestinal cells of nematodes as oxidative damage and autophagy intensify. Aldehyde methyl and aldehyde alcohol reduce lipofuscin accumulation in nematodes, possibly by reducing oxidative damage and increasing the activity of antioxidant enzymes. Lipofuscin in nematodes gradually accumulates with aging and exhibits autofluorescence within the nematode body.

[0085] Specific operations:

[0086] N2 *C. elegans* that had been synchronized to the L4 stage were transferred to NGM culture plates containing the sample group (50 μM of oxadiol and 100 μM of oxadiol A) and the blank control group. On day 8, 30 worms were randomly selected from each group and anesthetized with levamisole hydrochloride solution. The fluorescence of lipofuscin was photographed using an inverted fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ.

[0087] Experimental results: such as Figure 9 As shown, compared with the blank control group, the lipofuscin in N2 nematodes treated with oxytocin A and oxytocin showed no significant difference, but both showed a decreasing trend.

[0088] Example 8: qRT-PCR assay of nematode genes

[0089] To further determine how oxytocin A and oxytocinol exert their antioxidant effects to prolong the lifespan of nematodes, this study analyzed the effects of oxytocin A and oxytocinol on the mRNA expression levels of related genes (daf-2, daf-16, age-1, sod-3, hsp16.2, and skn-1) using RT-PCR to explore their molecular mechanisms of action.

[0090] Specific operations:

[0091] (1) RNA extraction

[0092] Synchronized N2 elegans worms were transferred to NGM culture plates containing the sample group (50 μM oxadoxetine, 100 μM oxadoxone) and the blank control group, and cultured for 7 days. The worms were then collected into EP tubes using M9 buffer, and washed three times to remove residual OP50 and larvae as much as possible. At least 2000 worms were collected from each group. Finally, the M9 buffer was removed, and the tubes were flash-frozen in liquid nitrogen and stored at -80°C. RNA extraction steps are as follows:

[0093] Step 1: Add 1 mL of Trizol to each sample EP tube, then add 3 clean magnetic beads and grind the worms into a homogenate using a bead mill. After grinding, let it stand at room temperature for 5 minutes. Then add chloroform to the homogenate, the volume of which should be 1 / 5 of the total volume of the homogenate. After mixing, let it stand at room temperature for 5 minutes, then centrifuge at 12000×g, 4℃ for 15 minutes.

[0094] Step 2: After centrifugation, aspirate the supernatant and transfer it to a new EP tube. Add an equal volume of isopropanol, mix thoroughly, and let stand at room temperature for 10 minutes. Centrifuge at 12000×g and 4℃ for 10 minutes. RNA precipitate will appear at the bottom of the tube.

[0095] Step 3: Discard the supernatant, retain the precipitate, add 1 mL of 75% ethanol, shake to wash the precipitate, centrifuge at 7500×g, 4℃ for 5 min, discard the supernatant, and retain the precipitate. After drying the precipitate at room temperature, add an appropriate amount of RNase-free water to dissolve the precipitate. Finally, determine the quality and concentration of RNA; qualified RNA will be used for subsequent reverse transcription.

[0096] (2) RNA reverse transcription

[0097] The qualified and quantified total RNA was reverse transcribed into cDNA according to the following system:

[0098] Table 1. RNA reverse transcription system 1

[0099]

[0100] After mixing all ingredients thoroughly, incubate the EP tube in a 65°C water bath for 5 minutes, then immediately place it in an ice bath for 2 minutes. Add the following ingredients:

[0101] Table 2 RNA reverse transcription system 2

[0102]

[0103] The above reaction solution was incubated in a water bath at 25°C for 5 min, then incubated at 42°C for 60 min, and finally heat-shocked at 85°C for 5 min. After the reaction was completed, it was placed on ice for subsequent experiments or frozen at -20°C for storage.

[0104] (3) Real-time PCR reaction

[0105] The internal reference gene is Actin-1, and the primer sequences for the gene were measured are shown in Table 3.

[0106] Table 3 Gene Primer Sequences

[0107]

[0108]

[0109] For each target gene and internal reference gene, a cDNA template from that sample was selected for PCR reaction.

[0110] Table 4 PCR reaction system

[0111]

[0112] PCR reaction conditions are shown in Table 5:

[0113] Table 5 PCR reaction conditions

[0114]

[0115] Experimental results:

[0116] This study detected the mRNA expression levels of the daf-2, daf-16, and age-1 genes in the IIS signaling pathway. The results are as follows: Figure 10As shown in (A), (B) and (C), this study found that compared with the blank control group, there were no significant differences in the expression of daf-2, daf-16 and age-1 genes in the nematodes in the oxytocin A sample group, and there were no significant differences in the expression of daf-2 and age-1 genes in the nematodes in the oxytocin sample group, while the expression level of daf-16 gene was significantly upregulated.

[0117] Superoxide dismutase (SOD) is a key antioxidant enzyme in the body, possessing physiological activity in scavenging free radicals. This study mainly measured the mRNA expression levels of SOD-3 by alginone A and alginol. The results are as follows: Figure 10 As shown in (D), compared with the blank control group, Aldehyde A had no significant effect on the expression of the sod-3 gene, while Aldehyde ethanol upregulated the expression of the sod-3 gene.

[0118] The hsp16.2 (heat shock protein) gene can improve the resistance of nematodes to heat stress and other stresses, and also reduce the accumulation of proteins in age-related diseases during aging. This invention mainly measured the mRNA expression levels of the hsp16.2 gene by alpinoxanone and alpinoxanol. The results are as follows: Figure 10 As shown in (E), compared with the blank control group, Aldehyde A had no significant effect on the expression of the hsp16.2 gene, while Aldehyde ethanol upregulated the expression of the hsp16.2 gene.

[0119] In nematodes, the transcription factor skn-1 is an important downstream target of the MAPK signaling pathway, playing a crucial role in regulating the expression of downstream stress-response genes and enhancing oxidative stress resistance to mitigate oxidative damage. MAPK signaling can delay stress-induced senescence by upregulating skn-1 gene expression. Figure 10 As shown in Figure (F), this study found that, compared with the blank control group, there was no significant difference in the expression of the skn-1 gene in the nematodes in the Alpinia oxyphylla A sample group, while the expression level of the skn-1 gene in the Alpinia oxyphylla sample group was significantly increased.

[0120] In summary, this invention found that, compared with the blank control group, oxadiol significantly increased the mRNA expression levels of daf-16, sod-3, hsp16.2, and skn-1, while having no significant effect on the expression levels of daf-2 and age-1 genes. Oxadiol A, however, had no significant effect on the expression levels of any of these six genes. Therefore, it is speculated that oxadiol may extend the lifespan of nematodes by upregulating daf-16, sod-3, hsp16.2, and skn-1 genes to alleviate oxidative and heat stress damage.

[0121] The above embodiments provide a method for evaluating the antioxidant activity of oxadiol or oxadiol-methyl in a *C. elegans* model. In the *C. elegans* antioxidant model, both oxadiol and oxadiol significantly prolonged the normal lifespan and stress-induced lifespan (heat stress and oxidative stress) of *N2* nematodes, improved their motility (swallowing frequency, head-shaking frequency, and movement ability), reduced the accumulation of ROS and lipofuscin in the nematodes, and enhanced their antioxidant enzyme defense capabilities, without significant toxic side effects on growth and reproduction. It is evident that 50 μM oxadiol exhibits better antioxidant activity than 100 μM oxadiol-methyl. Further analysis revealed that oxadiol upregulated the expression levels of daf-16, sod-3, hsp16.2, and skn-1 genes, while oxadiol-methyl showed an increasing trend in the expression levels of these four genes but no significant effect. Therefore, oxorubicin A and oxorubicin may extend the lifespan of nematodes by reducing ROS levels in the body, increasing the activity of antioxidant enzymes, and enhancing the expression of antioxidant-related genes, thereby mitigating oxidative damage and heat stress damage. Both of these substances are potential antioxidant dietary supplements.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for screening antioxidant active ingredients in Alpinia oxyphylla based on the Caenorhabditis elegans model, characterized in that... The procedure included the following steps: Wild-type N2 Caenorhabditis elegans, synchronized to the L4 stage, were transferred to NGM culture plates containing Alpinia oxyphylla active ingredient samples and blank control NGM culture plates as sample and control groups, respectively; the normal lifespan, heat stress lifespan, antioxidant stress lifespan, swallowing frequency, head shaking frequency, mobility, oviposition rate, lipofuscin and cellular reactive oxygen species levels, and antioxidant enzyme defense capacity of the nematodes were observed and recorded; antioxidant gene association analysis was performed by transcriptome sequencing; when the sample group treated with Alpinia oxyphylla active ingredient showed a significant difference compared with the control group, the Alpinia oxyphylla active ingredient exhibited antioxidant activity. The active ingredients of Alpinia oxyphylla include Alpinia oxyphylla A or Alpinia oxyphylla alcohol; When the active ingredient sample of Alpinia oxyphylla is Alpinia oxyphylla A, its concentration is 100 μM; when the active ingredient sample of Alpinia oxyphylla is Alpinia oxyphylla alcohol, its concentration is 50 μM.

2. The method according to claim 1, characterized in that: The wild-type N2 Caenorhabditis elegans that has been synchronized to the L4 stage was obtained according to the following steps: pregnant nematodes were collected with M9 buffer, and after lysis, centrifugation, precipitation, and washing, they were cultured at 20°C for 12 h to obtain L1 stage nematodes; the L1 stage nematodes were transferred to NGM plates containing Escherichia coli OP50 and cultured for 52-60 h to obtain L4 stage nematodes.

Images