Sodium nitroprusside improved the quality of Radix Saposhnikoviae through constructed physiological response under ecological stress

The ecological significance of secondary metabolites is to improve the adaptive ability of plants. Secondary metabolites, usually medicinal ingredients, are triggered by unsuitable environment, thus the quality of medicinal materials under adversity being better. The quality of the cultivated was heavily declined due to its good conditions. Radix Saposhnikoviae, the dried root of Saposhnikovia divaricata (Turcz.) Schischk., is one of the most common botanicals in Asian countries, now basically comes from cultivation, resulting in the market price being only 1/10 to 1/3 of its wild counterpart, so improving the quality of cultivated Radix Saposhnikoviae is of urgency. Nitric oxide (NO) plays a crucial role in generating reactive oxygen species and modifying the secondary metabolism of plants. This study aims to enhance the quality of cultivated Radix Saposhnikoviae by supplementing exogenous NO. To achieve this, sodium nitroprusside (SNP) was utilized as an NO provider and applied to fresh roots of S. divaricata at concentrations of 0.03, 0.1, 0.5, and 1.0 mmol/L. This study measured parameters including the activities of antioxidant enzymes, secondary metabolite synthesis enzymes such as phenylalanine ammonia-lyase (PAL), 1-aminocyclopropane-1-carboxylic acid (ACC), and chalcone synthase (CHS), as well as the contents of NO, superoxide radicals (O2·−), hydrogen peroxide (H2O2), malondialdehyde (MDA), and four secondary metabolites. The quality of Radix Saposhnikoviae was evaluated with antipyretic, analgesic, anti-inflammatory effects, and inflammatory factors. As a result, the NO contents in the fresh roots were significantly increased under SNP, which led to a significant increase of O2·−, H2O2, and MDA. The activities of important antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were found to increase as well, with their peak levels observed on the 2nd and 3rd days. PAL, ACC, and CHS activities were also significantly enhanced, resulting in the increased secondary metabolite contents of Radix saposhnikoviae in all groups, especially the 0.5 mmol/L SNP. The four active ingredients, prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol, increased by 88.3%,325.0%, 55.4%, and 283.8%, respectively, on the 3rd day. The pharmaceutical effects of Radix Saposhnikoviae under 0.5 mmol/L SNP were significantly enhanced. Exogenous SNP can induce the physiological response of S. divaricata under adverse conditions and significantly improve the quality of Radix Saposhnikoviae.


Samples
All the plant experiments complied with relevant institutional, national, and international guidelines and legislation.Cultivated Radix Saposhnikoviae (FF20221001) collection was done with permission.Fresh 3-year-old roots of cultivated S. divaricata, identified by Prof. Xiang-Cai Meng of Heilongjiang University of Chinese Medicine, were collected in Daqing City, Heilongjiang Province, China, collected in October 2022 and immediately wrapped in plastic to keep fresh.The cultivated Radix Saposhnikoviae (FF20221001) has been deposited in a publicly available herbarium of Heilongjiang University of Chinese Medicine.And we complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Determination of NO contents
The NO contents in fresh roots were determined with a plant NO ELISA assay kit.

Determination of ROS contents
The homogenized protein contents.The fresh roots were determined with the protein quantification (TP) assay kit Bradford method and the O 2 •− and H 2 O 2 using the O 2 •− assay kit and the plant H 2 O 2 assay kit, respectively.

Determination of MAD contents
The MDA contents were determined with TBA using a malondialdehyde kit.

Determination of antioxidant enzyme activities
The activities of antioxidant enzymes were determined with SOD, CAT, and POD assay kits, respectively.

Determination of metabolites and key enzyme activities
The contents of primary metabolite 1,3-DPG were determined with a plant 1,3-DPG ELISA detection kit, the activities of PAL ACC and CHS using a PAL, Acetyl coenzyme A carboxylase (ACC), and chalcone synthase (CHS) ELISA kits, respectively.

Solution preparation
Preparation of standard solution: Accurately weigh 1.88 mg of prim-O-glucosylcimifugin and 2.25 mg of 4′-O-βd-glucosyl-5-O-methylvisamminol into a 5 mL volumetric flask, and methanol was added to the scale to prepare 0.38 mg/mL and 0.45 mg/mL of the control solution.In addition, accurately weigh 1.03 mg of cimifugin and 1.05 mg of sec-O-glucosylhamaudol into a 25 mL volumetric flask, and methanol was added to the scale to make up 0.042 mg/mL, and 0.021 mg/mL of the control solution.Preparation of sample solution: Accurately weigh 1.5 g of the three groups of Radix Saposhnikoviae into a 50 mL conical flask, 50 mL of methanol was added, and the mass was weighed and extracted by heating reflux at 70 °C for 2 h in a water bath.After cooling, weigh it again, balance the weight loss, shake well, and filter.The filtrate was then passed through a microporous membrane (0.

Methodological investigation
The intra-day precision was calculated by taking the test solution under 2.7.1 and measuring it 6 times according to the method under 2.7.2; the inter-day precision was calculated by analyzing it for three consecutive days.The RSDs of the inter-day precision of prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-Omethylvisamminol, and sec-O-glucosylhamaudol were 1.35%, 1.63%, 1.25%, and 1.67%, respectively, indicating the precision was high.The test solution under 2.7.1 was taken and determined with the method under item 2.7.2 to obtain the RSDs of the test solutions were 0.28%, 2.75%, 1.07%, and 2.79% for prim-O-glucosyl cimifugin, cimifugin, 4′-O-β-dglucosyl-5-O-methylvisamminol, and sec-O-glucosyl hamaudol, respectively, indicating good repeatability of the analytical method.

Drug preparation
The medicinal efficacy of Radix Saposhnikoviae is closely related to the content of secondary metabolites, so the optimal group of Radix Saposhnikoviae with the combined increase of the four chromones, was selected as the high-quality Radix Saposhnikoviae herb group, i.e., 0.5 mmol/L SNP treatment.
The ordinary Radix Saposhnikoviae group (0-day group) and the high-quality Radix Saposhnikoviae group (0.5 mmol/L SNP group) were dried to constant weight, taken 9.0 g, added 10 times the amount of water, soaked for 1 h, decocted for 1 h, filtered, and the extraction was repeated three times and the filtrates were combined.Concentrated to 0.045 g botanicals/mL, and 2.0 mL solution was given by gavage to each rat and concentrated 0.065 g botanicals/mL for mice at 0.2 mL each.

Animals
Animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH guidelines) and ARRIVE guidelines and approved by the Ethical Committee of Heilongjiang University of Chinese Medicine (approval number: HUCM2014-00348).

Antipyretic effect
In male SD rats, 180 ± 20 g, the basal body temperature was first measured 3 times a day for 3 days.For rats with an average anal temperature of 36.85 ± 0.37 °C, 2.0 mL/100 g of 15% yeast suspension were injected subcutaneously, and those whose body temperature increased by > 0.8 °C were selected as the test rats.The rats were randomly divided into four groups: saline group, model group, ordinary Radix Saposhnikoviae group, and high-quality Radix Saposhnikoviae group, with 10 rats in each group.The saline and model groups were administrated at 2.0 mL of saline daily, and the Radix Saposhnikoviae group was administrated at 0.045 g botanicals/ mL for 2.0 mL daily for seven days.At the 3rd hour after the last administration, the rats were subcutaneously injected with 2.0 mL/100 g 15% yeast suspension, and their rectal temperature was measured at 0.5, 1, 2, 3, 4, and 5 h after injection.

Analgesic effect
Male Kunming mice, weighing 18 ± 2 g, were randomly divided into four groups: saline group, model group, ordinary Radix Saposhnikoviae group, and high-quality Radix Saposhnikoviae group, 10 mice in each group.2.0 mL of saline was administrated daily for the saline group and the model group, and 0.2 mL 0.065 g botanicals/ mL was administrated for the Radix Saposhnikoviae group.This operating process was done once a day for 7 consecutive days.At 3rd hour after the last administration, 0.2 mL 0.6% of acetic acid was injected intraperitoneally, and the number of twists in mice within 15 min after the injection was observed, and the twist inhibition rate was calculated.
Inhibition rate = [(numbers of twists in the control group − numbers of twists in assay group)/numbers of twists in control group] × 100%.10 mice for each group.2.0 mL of saline was administrated daily for the saline group and the model group, and 0.2 mL was administrated for each Radix Saposhnikoviae group according to 0.65 g of botanicals/kg, once a day for 7 days.At the 3rd hour after the final administration, 0.03 mL of xylene was evenly applied to the anterior and posterior sides of the right ear of each mouse for 1 h to induce inflammation.Both ears were cut along the baseline of the auricle, and the tissue of the same part of the left and right ear was weighed by punching with an 8 mm punch, and the swelling rate and swelling inhibition rate of the auricle of mice were calculated.

Determination of anti-inflammatory effect and inflammatory factors
Swelling inhibition rate (%) = (mean swelling rate in the control group − mean swelling rate in the medicated group)/mean swelling rate in the control group × 100% The contents of TNF-α and IL-6 were determined using the double antibody sandwich method with TNF-α and IL-6 kits.

Data processing
Microsoft Office Excel 2007 and SPSS 26.0 software were used to process the data, and GraphPad Prism software was used to make graphs.The measurement data were expressed as ( x ± s) and analyzed by one-way variance analysis and t-test.P < 0.05 indicated statistically significant differences, while P < 0.01 indicated extremely statistically significant differences.

Ethics declarations
Ethics approval to conduct animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH guidelines), ARRIVE guidelines and approved by the Ethical Committee of Heilongjiang University of Chinese Medicine (approval number: HUCM2014-00348).

NO contents
Compared with the 0-day, there was no significant trend in the distilled water group and no significant trend in the 0.03, 0.1, and 1.0 mmol/L treatment groups except for a slight decrease on the 1st day.The 0.5 mmol/L SNP group caused a slight decrease in NO content in the fresh roots of S. divaricata on the first day and an increase on the 2nd and the 3rd day, reaching a peak on the 3rd day, with a rise of 55.0% compared with the 0-day (P < 0.01).As shown in Fig. 1.

O 2 •− •contents
Compared with the 0-day control, different concentrations of SNP all increased the O 2 •− contents in the fresh roots of S. divaricata, showing a trend of increasing and then decreasing.The distilled water group showed no significant trend.The 0.03 and 1.0 mmol/L SNP groups peaked on the 2nd day.The 0.1 and 0.5 mmol/L treatment groups peaked on the 3rd day.The 0.5 group showed the most significant increase, with an increase of 135.3% compared with the 0-day (P < 0.01).As shown in Fig. 2.

H 2 O 2 contents
Compared with the 0-day control, except for the distilled water group, the other groups showed no significant trend, the other H 2 O 2 contents in the fresh roots of S. divaricata increased significantly, increasing and then decreasing from the 0 to the 4th day.The 1.0 mmol/L treatment group peaked on the 2nd day, and all other www.nature.com/scientificreports/treatment groups peaked on the 3rd day.The 0.5 mmol/L treatment group showed a 985.1% increase (P < 0.01).
As shown in Fig. 3.

MDA contents
Compared with the 0-day control, the MDA contents in the fresh roots of S. divaricata rose with concentrations of SNP, except for the distilled water group.The MDA contents increased as the concentration of SNP increased, with the 1.0 mmol/L treatment group reaching a peak on the 1st day and all other treatment groups reaching a peak on the 2nd day, with the most significant increase of 133.6% in the 0.5 mmol/L treatment group compared with the 0-day (P < 0.05).As shown in Fig. 4.

Antioxidant enzyme activities
Compared with the 0-day control, the antioxidant enzyme activities of all treatment groups showed a trend of increasing and then decreasing, except for the distilled water group and the 1.0 mmol/L treatment group, with a minor variation in antioxidant enzyme activities.SOD peaked on the 2nd day, with a 27.5% increase in the 0.5 mmol/L treatment group.CAT and POD activity peaked on the 3rd day, with 281.6% and 297.1% increases in the 0.5 mmol/L group, respectively (P < 0.05).As shown in Fig. 5.

Metabolic pathway 1,3-DPG contents
Compared with 0-day, there was no significant trend in the distilled water group and 1.0 mmol/L group, while the other SNP group showed an upward trend.Among them, the 0.03 and 0.5 mmol/L groups peaked on the 3rd day, increasing by 83.5% and 121.7%, respectively, compared with the 0-day.As shown in Fig. 6. www.nature.com/scientificreports/

Key enzyme activities related to secondary metabolites
The PAL increased slightly on the 3rd day in the distilled water group compared with the 0-day.Except for the 1.0 mmol/L treatment group, all treatment groups peaked successively later with increasing concentrations of SNP, 0.03, 0.1, and 0.5 mmol/L SNP treatment groups peaking on the 1st, 2nd, and 3rd day, respectively.The 0.5 mmol/L treatment group showed a 119.7% increase on the 3rd day compared with the 0-day.CHS increased in all groups compared with the 0-day.All groups reached a peak on the 2nd day except the distilled water, and the 1.0 mmol/L SNP group peaked on the 2nd day.The 0.5 mmol/L SNP-treated group showed a 114.0%increase compared with the 0-day.Compared with the 0-day, ACC has slightly elevated but a minor variation change in the distilled water group, 0.03, and 1.0 mmol/L SNP-treated groups.The 0.5 mmol/L SNP-treated groups showed a 74.7% increase compared with the 0-day.As shown in Fig. 7.

Secondary metabolites contents
The contents of prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were increased by SNP in the Radix Saposhnikoviae, with a trend of increasing and then decreasing in each treatment group, among which the total content of four chromones had the highest increase for the 0.5 mmol/L on the 3rd day, prim-O-glucosylcimifugin with a rise of 88.3%, 4′-O-β-d-glucosyl-5-Omethylvisamminol with 55.4%, cimifugin with 325.0% and sec-O-glucosyl hamaudol with 283.8%, compared with the control group, respectively.As shown in Fig. 8.

Antipyretic effect
Compared with the model group, the body temperature of rats in Radix Saposhnikoviae groups was reduced after the 2nd day.Still, the high-quality Radix Saposhnikoviae group was lower significantly than the ordinary Radix Saposhnikoviae group.As shown in Table 1.

Analgesic effects
Compared with the model group, the number of twisting in mice in the Radix Saposhnikoviae group was significantly reduced.Still, the reduction in the high-quality Radix Saposhnikoviae group was lower than that in the ordinary Radix Saposhnikoviae group; the number of twisting was decreased by 4.17 times on average, and the inhibition rate was increased by 14.45%, which was 1.5 times of the ordinary botanicals.As shown in Table 2.

Anti-inflammatory effect and inflammatory factors
Compared with the model group, the ear swelling of mice in the Radix Saposhnikoviae group was significantly reduced; among them, the reduction was more significant in the high-quality Radix Saposhnikoviae group than in the ordinary Radix Saposhnikoviae group, with a reduction of 1.18 mg in weight and an increase of 38.78% in the inhibition rate which was 3.1 times higher than that of the ordinary botanicals.The two inflammatory factors, IF-6 and TNF-α were significantly reduced in the Radix Saposhnikoviae group compared with the model group.Still, the high-quality Radix Saposhnikoviae group had a significantly higher degree of reduction than the ordinary Radix Saposhnikoviae group, with a decrease of 17.52% and 18.00%, respectively, and the contents of inflammatory factors in the high-quality Radix Saposhnikoviae group were consistent with that in the blank group, indicating excellent therapeutic efficacy of the high-quality group.As shown in Table 3.

Discussion
Effects of SNP on NO, ROS, and MDA SNP was used as an exogenous donor of NO, which resulted in a significant increase of NO contents; the 0.5 mmol/L group with a marked difference reached a peak on the 3rd day (Fig. 1).Under the effect of NO, O 2 •− contents also continued to increase from the 1st to the 3rd day (Fig. 2), and the excess O 2 •− was converted into H 2 O 2 by SOD, resulting in a rise in H 2 O 2 contents during the same period from the 1st to the 3rd day (Fig. 3).MDA as product of the destroyed bio-membrane can directly reflect ROS damage to plant cells 28 .In this study, the MDA contents remained consistently high from the 2nd day (Fig. 4).On the 4th day, the levels of NO, O 2 •− , and H 2 O 2 were all decreased, probably due to NO's generation of more O 2 •− , which might have caused damage to corresponding enzymes.O 2

•−
, H 2 O 2, and MDA as indexes of ecological stress were significantly increased (Fig. 4), indicating that SNP can induce a physiological response of S. divaricata under ecological stress.

Effects of SNP on antioxidant enzyme activities
When the ROS content is too high, it can damage nearby molecular structures, lipid bilayer, DNA single strands, proteins, etc.The strategy of living organisms avoiding ROS damage is to evolve antioxidant substances to eliminate excess ROS.Antioxidant enzymes are a large group of substances that eliminate ROS in organisms.The biosynthesis and activities of these enzymes are induced and enhanced by the presence of ROS 29 .As the most significant nonspecific antioxidant substances, these enzymes play a crucial role in maintaining cellular redox balance and protecting cells from oxidative damage caused by ROS.SOD, as the primary defense enzyme, can convert O 2 contents were significantly increased in the 0.1 and 0.5 mmol/L SNP-treated groups on the 3rd day.Meanwhile, CAT and POD activities also elevated, especially in the 0.5 mmol/L group (P ˂ 0.05).With the interaction of CAT and POD, H 2 O 2 contents decreased on the 4th day (Fig. 5), O 2 •− and H 2 O 2 contents and antioxidant enzyme activities decreased, probably resulting from increased ROS level (Figs. 2, 3, 5), which caused damage to cellular proteins 31 .

Effect of SNP on secondary metabolite and relating key enzymes
Antioxidant enzymes are proteins with -S-S-bonds and other chemical bonds that maintain the corresponding structure, allowing them to function as catalysts in neutralizing ROS.However, these chemical bonds are unstable, excessive ROS can easily alter the configuration of antioxidant enzymes and affect activities 32 .Plants face more ecological stresses and generate more ROS due to their immobility.Studies have demonstrated that under severe ecological stress, even in highly adaptive plants like Glycyrrhiza uralensis Fisch, the activities of antioxidant enzymes such as SOD, CAT, and POD are also drastically reduced 33 , not to mention other plants.Thus, relying solely on antioxidant enzymes is impossible for plants to adapt to severe ecological stress.Adaptive strategies for plants are that they evolved the secondary metabolism as a supplement.
The main secondary metabolites of S. divaricata are chromones, which have two biosynthetic pathways.One is the acetic acid-malonate pathway, in which the acetyl-CoA carboxylation enzyme (ACC) is a key and limiting enzyme that catalyzes the carboxylation of acetyl-CoA to form propionyl-CoA.The other is the shikimic acid pathway, in which phenylalanine ammonia-lyase (PAL), a key enzyme for flavonoids, catalyzes phenylalanine to form cinnamic acid.Coumaric acid under cinnamate 4-hydroxylase (C4H), followed by coumaric acyl-CoA under 4-coumaroyl-CoA ligase (4CL).Based on this, the propionyl-CoA and the coumaric acyl-CoA undergo a series of reactions under chalcone synthase and form chromone compounds [34][35][36] .As shown in Fig. 9.
The activities of the key enzymes relating to secondary metabolism could be significantly increased by increasing ROS and NO itself 37 .By applying SNP to the fresh roots, the activities of ACC, PAL, and CHS, the key enzymes of secondary metabolite biosynthesis, were increased in each treatment group, among them, the activities of CHS and ACC in the 0.5 mmol/L SNP group increased by 117.8% and 44.1% on the 2nd day and 3rd day, respectively (Fig. 7), resulting in enhancement of chromone compounds biosynthesis.For 0.5 mmol/L SNP-treated on the 3rd day, the contents of prim-O-glucosylcimifugin rose from 2.05 to 4.13 mg/g, cimifugin from 0.08 to 0.34 mg/g, 4′-O-β-d-glucosyl-5-O-methylvisamminol from 3.86 to 6.00 mg/g, and sec-O-glucosylhamaudol from 0.18 to 4.13 mg/g, respectively, with a remarkable increase of 88.3%, 325.0%, 55.4%, and 283.8%, respectively.1,3-DPG is a product of glucose catabolism in glycolysis and a material of glucose anabolism in gluconeogenesis.This study must derive from glucose since the separated fresh roots are being used.The exogenous SNP promoted glucose catabolism, provided raw materials for the biosynthesis of secondary metabolites, and ensured that the various chromone contents increased from the 1st to the 3rd day.Under mild ecological stress, the ROS contents were relatively low, and the antioxidant enzymes can exert a more significant antioxidant effect; however, under severe ecological stress, solely antioxidant enzymes have difficulty coping with too much ROS due to reduced activities by ROS, the secondary metabolites would play an important role 38 .The lower concentrations of 0.03 and 0.1 mmol/L SNP groups induced less ROS (Figs. 2, 3) and lower levels of antioxidant enzymes, key enzyme activities of secondary metabolism, which resulted in secondary metabolites being lower (Figs.5, 7, 8).In the 1.0 mmol/L SNP-treated group, the highest level of MDA had been hit on the 2nd day.It failed to produce high levels of ROS, possibly due to the damaging effect of high levels of NO 39 or/and reduced the enzymes related to ROS production or/and produced a high amount of ROS 36 , which resulted in the secondary metabolites failing to be elevated.1,3-DPG is a product of glucose catabolism.ACC and PAL serve as intermediaries between primary and secondary metabolism, while CHS is a critical enzyme in plant flavonoid biosynthesis.The presence of a series of substances indicated that the increased chromones come from biosynthesis, not biotransformation.This ensures that the various active ingredients are significantly increased.

Validation of pharmacodynamics
Botanicals contain a wide variety of active ingredients, and there are significant variations in the contents, activities, and bio-availabilities of various active ingredients 40 .Due to such diversity, it is difficult to objectively evaluate the quality of botanicals only by the contents of a few ingredients or certain ingredients 41 .Pharmacodynamics is the best method to obtain a comprehensive and accurate assessment of botanical quality.The main effects of Radix Saposhnikoviae are antipyretic, analgesic, and anti-inflammatory 2 , and the main active ingredients are chromones 42 .For 0.5 mmol/L SNP on the 3rd day, the contents of prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O -glucosylhamaudol increased by 88.3%, 325.0%, 55.4%, and 283.8%, respectively, especially, the improved high-active components such as cimifugin and sec-O-glucosylhamaudol were even more remarkable.Cimifugin is easier to enter the cell membrane to play a drug effect due to more -OH and is stronger lipophilic than glycosides 41,43 ; the pharmacological effects of sec-O-glucosylhamaudol are also stronger due to more -OH than prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-Omethylvisamminol 44 .With this, compared with the model group, the body temperature of the high-quality Radix Saposhnikoviae group (0.5 mmol/L SNP group) was reduced by 0.11-0.13°C.The inhibition rate of twisting  body and ear swelling in mice was increased by 14.45% and 38.78%, the inhibition rate being 1.5 and 3.1 times of the ordinary botanicals, respectively.The contents of IF-6 and TNF-α were reduced by 17.52% and 18.00%, and the inflammatory factor content fell into the original level of the blank group, which indicated that the efficacy of the high-quality Radix Saposhnikoviae group was significantly improved.The nano-materials as delivery carriers have been used for biological research for decades, and some significant breakthroughs have been made 45 .For example, Cerium oxide nanoparticles increase NO production in rice leaves under salt stress and enhance nitrate reductase activity and nitrate reductase activity 46 .Similarly, multilayer nanotubes have shown the ability to modulate NO production and improve salt tolerance in oilseed rape 47 .SNP is an injectable agent for the treatment of hypertension and acute heart failure, the application to herb quality improvement is also in very low concentrations and amounts.Besides, the SNP only increases the level of intrinsic ingredients of S. divaricata under drought stress without producing other toxic components.Therefore, it is safe to use SNP.SNP as an inducer of secondary metabolism has promising applications.4) High-quality Radix saposhnikoviae.Each group was administered the drug by gavage for 7 days, and 3 h after the last administration, the swelling degree of mice's ears was observed after xylene was applied for 1 h, and mice's blood was collected and analyzed for the content of IL-6 and TNF-α.

Conclusion
The exogenous NO donor SNP can increase the levels of ROS, resulting in the enhancement of antioxidant enzyme activities and secondary metabolite contents in fresh roots.The antioxidant enzymes and secondary metabolites work together and reduce the damage to the plant body caused by ROS.In the 0.5 mmol/L SNPtreated group, prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methyl visamminol, and sec-O-glucosylhamaudol increased by 88.3%, 325.0%, 55.4%, and 283.8%, respectively.Based on this, the efficacy of Radix saposhnikoviae was enhanced.This study is based on a common strategy for plants to adapt to ecological stresses, applying SNP to improve the quality of Radix Saposhnikoviae can provide a new pathway for producing other high-quality medicinal herbs.

Figure 1 .
Figure1.Effect of SNP on NO contents.The changes in NO contents were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots of S. divaricata for two days and distilled water for the last three days.

Figure 2 .Figure 3 .
Figure 2. Effect of SNP on O 2 •−•contents.The changes in O 2•− contents were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots of S. divaricata for 2 days and distilled water for the last 3 days.

Figure 4 .
Figure 4. Effect of SNP on MDA contents.The changes in MDA contents were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots ofS.divaricata for 2 days and distilled water for the last 3 days.

Figure 5 .
Figure5.Effect of SNP on antioxidant enzyme activities.The changes in SOD, CAT and POD activities were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots of S. divaricata for 2 days and distilled water for the last 3 days.

Figure 6 .
Figure6.Effect of SNP on the contents of primary metabolites.The changes in 1,3DPG contents were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots of S. divaricata for 2 days and distilled water for the last 3 days.

Figure 7 .
Figure 7. Effects of SNP on key enzyme activities relating to secondary metabolites.The changes in ACC, CHS and PAL activities were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots of S. divaricata for 2 days and distilled water for the last 3 days.

Figure 8 .
Figure 8.Effect of SNP on the contents of secondary metabolite.The changes in secondary metabolite contents were analyzed by spraying 0, 0.03, 0.1, 0.5, and 1.0 mmol/L SNP on the fresh roots of S. divaricata for 2 days and distilled water for the last 3 days.

Figure 9 .
Figure 9. Synthesis path diagram of Radix Saposhnikoviae chromones.Analysis of the synthesis of secondary metabolites of Radix Saposhnikoviae via shikimic acid pathway and acetic acid-malonate acid pathway and the key enzymes involved in the synthesis.
Animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH guidelines) and approved by the Ethical Committee of Heilongjiang University of Chinese Medicine (approval number: HUCM2014-00348).Male Kunming mice weighing 18 ± 2 g were randomly divided into four groups: saline group, model group, ordinary Radix Saposhnikoviae group, and high-quality Radix Saposhnikoviae group, Vol.:(0123456789) Scientific Reports | (2023) 13:15823 | https://doi.org/10.1038/s41598-023-43153-3 •− into H 2 O 2, then, other antioxidant enzymes, such as catalase (CAT) and peroxidase (POD), work together to convert H 2 O 2 into H 2 O and O 2 30 .SOD peaked on the 2nd day, converting O 2 •− into H 2 O 2 , and H 2 O 2

Table 1 .
Comparison of body temperature of dry yeast-induced fever in each group ( x ± s).

Table 2 .
Comparison of twisting times in mice of each group ( x ± s).Compared with the blank group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01.(1) Blank group; (2) Model group; (3.) Ordinary Radix saposhnikoviae; (4) High-quality Radix saposhnikoviae.Each group was administered by gavage for 7 days, and 3 h after the last administration, the number of writhing times in mice injected with acetic acid within 15 min was observed and analyzed.