Evaluation of Marker Compounds and Biological Activity of In Vitro Regenerated and Commercial Rehmannia glutinosa (Gaertn.) DC. Roots Subjected to Steam Processing

Rehmannia glutinosa (Gaertn.) DC., belonging to the family Scrophulariaceae, has been known since immemorial times as a prominent oriental drug in East Asia that can treat various ailments, such as kidney disorders, anemia, and diabetes. In order to be applied for medical purposes, R. glutinosa is commonly processed using steam to increase its efficacy and biological activity. The increasing demand for R. glutinosa in the traditional medicine industry encouraged many researchers to develop a fast, efficient, and high-quality production system using biotechnological approaches. This study aimed to compare the chemical and biological activities of in vitro regenerated R. glutinosa (PKR) and commercial R. glutinosa (PCR) samples subjected to steam processing. We assessed the effects of steam processing and the differences in R. glutinosa material on 5-Hydroxymethyl-2-furaldehyde (5-HMF) content, total flavonoid and phenolic content, antioxidant activity, nitric oxide (NO) levels, and anti-inflammatory activity. PKR samples showed a significantly higher content of 5-HMF (0.15%) as compared to PCR samples (0.05%). Compared to unprocessed R. glutinosa (UPR) and PCR samples, PKR again showed the highest total phenolic and flavonoid content of 41.578 mg GAE/g and 17.208 mg RUE/g, respectively. Meanwhile, both processed R. glutinosa samples (PKR and PCR) showed a significantly higher DPPH antioxidant activity ((67.095 + 1.005)% and (61.579 + 0.907)%, respectively) than unprocessed R. glutinosa ((31.452 + 1.371)%). In addition, both PKR and PCR samples showed good anti-inflammatory activity by showing similar effects such as the inhibition of NO production and the suppression of inducible nitric oxide synthase (iNOS). Based on these results, PKR fulfilled the Chinese pharmacopeia standards, in terms of the amount of the marker compounds and showed a high level of bioactivity. Therefore, these findings are expected to be useful in verifying the efficacy of herbal medicines and the availability of suitable materials for medicinal use.


Introduction
Rehmannia glutinosa (Gaertn.) DC. (Orobanchaceae) is a highly important medicinal plant belonging to the family Scrophulariaceae. It is considered a "top grade" herb in traditional medicine in eastern countries such as Korea and China. R. glutinosa is traditionally believed to nourish Yin and tonify the kidneys, including the regulation of water metabolism, reproduction and development, dominating growth, storing essence, and regulation of the neuroendocrine system using the traditional Chinese medicine (TCM) theory, showing that R. glutinosa contains various types of active compounds and exhibits several pharmacological benefts [1]. Tere are three types of R. glutinosa samples used based on their processing methods as follows: fresh R. glutinosa roots (Xian Dihuang), dried R. glutinosa roots (Sheng Dihuang), and processed R. glutinosa roots (Shu Dihuang). Several essential phytochemicals have been isolated and identifed pharmacologically from unprocessed (raw) and processed R. glutinosa including catalpol, verbascoside, and 5-Hydroxymethyl-2-furaldehyde (5-HMF).
Pharmacologically, unprocessed and processed R. glutinosa have shown to possess several biological activities, such as inhibition of tumoral processes (antitumor activity) through their action on the enzyme topoisomerase I [2] and are toxic to the lung cancer cell lines (cytotoxicity) [3]. Specifcally, catalpol shows extensive protection for neuronal ischemia [4], elevates brain angiogenesis [5], and possesses hypoglycemic and diuretic abilities [1]. On the other hand, harpagide shows leishmanicidal activity [6] and is capable of downregulating the production of enzymes, such as lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 (COX-2) through suppression of nuclear factor κB [7]. R. glutinosa roots also accumulate phenylethanoid glycosides, including verbascoside and isoverbascoside, which are proven to show various medicinal values, including cytotoxic and apoptotic activity against breast cancer cell lines [8].
Te constituents extracted from the root and aboveground biomass of medicinal herbs contain secondary metabolites, also referred to as phytochemicals, encompass a large variety of natural products including alkaloids, glycosides, phenols, favonoids, terpenoids, saponins, steroids, tannins, quinones, and coumarins [9]. Herbs can be successfully engineered as biofactories for synthesizing biomolecules with pharmaceutical and industrial interest, while these achievements require a thorough understanding of biochemical knowledge to amend large-scale production [10]. Te increasing demand for R. glutinosa herb in traditional medicine has encouraged many researchers to develop a fast, efcient, and high-quality production system. One of them is the application of biotechnological propagation to obtain an optimum growth system to produce large-scaleR. glutinosa. Previously, our research group developed an optimum in vitro propagation protocol for the high-quality production of R. glutinosa seedlings and rootstocks [11] which has been patented under patent number 10-1881305. Our protocol has also been evaluated in a large feld farming system and was able to produce greater rhizome biomass than the commercially available R. glutinosa seedlings [12]. To provide high-quality processed R. glutinosa for commercialization purposes in the traditional medication system, we further evaluated our processed R. glutinosa products by comparing the roots of R. glutinosa obtained from in vitro and commercial plants.
Here, we examined some pivotal parameters for herbal drug development, such as marker compound, antioxidant, and anti-infammatory properties.

Collection of R. glutinosa Root Samples.
We compared two diferent R. glutinosa root samples ( Figure 1). Te frst one was the in vitro regenerated R. glutinosa roots, a patented sample based on in vitro propagation protocol developed by the Korea Institute of Oriental Medicine (KIOM) [11]. Te optimized in vitro propagation protocol consists of WPM + IAA 1.0 mg/L + IBA 0.5 mg/L combination. Te sample was harvested from the indoor KIOM production and propagation laboratory in Naju, Jeollanamdo, South Korea. Secondly, commercial R. glutinosa roots, commercially available as R. glutinosa sample, was harvested from an indoor KIOM production and propagation laboratory in Naju, Jeollanam-do, South Korea. All samples were collected 6 months after planting in the commercially available soil.

2.2.
Processing of R. glutinosa Roots. Raw R. glutinosa root samples (in vitro regenerated and commercial) were washed using distilled water and dried in an oven at 60°C for 48 h. All the dried samples were then cut into a length of 2 cm and soaked in water to separate the bad samples. All samples were steamed (KSP-240 L, Kyungseo E&P, South Korea) for 1 h at 120°C four times ( Figure 2). Tis optimized steam processing method was developed and patented by Korea Institute of Oriental Medicine (KIOM). After processing, three diferent types of samples were obtained for further analysis, namely, unprocessed R. glutinosa (UPR), processed commercial R. glutinosa (PCR), and processed in vitro R. glutinosa (PKR).

Samples Extraction.
All samples (UPR, PCR, and PKR) were ground into powder using a steel pulverizing machine (250G New Type Pulverizing Machine, Model RT-N04-2V, Taiwan) at 25,000 rpm. Te maceration method was applied to extract 2 g of fne powder from each sample in 35 mL of 70% ethanol (JT Baker Inc., Phillipsburg, NJ, USA), followed by sonication at 40°C for 1 h. All extracts were flteredusing a 0.45 μm syringe flter (PALL Corporation, Ann Arbor, MI, USA) and concentrated in a reduced pressure rotary evaporato(EYELA N-1200B, Tokyo Rikakikai Co. Ltd., Japan) at 40°C. Each concentrated extract was then vacuumdried.

DPPH Antioxidant Measurement.
Approximately, 50 mg of each concentrated extract sample was dissolved in 5 mL 70% ethanol to obtain a stock solution (10000 μg/mL). Diferent concentrations (125, 250, 500, and 1000 μg/mL) of each sample were prepared by diluting the stock solution for antioxidant testing.
Te antioxidant activity of all samples (UPR, PCR, PKR) was established using 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich (St. Louis, MO, USA)), adapted from Okello et al. [13]. About 0.1 mL of each sample (at diferent concentrations) was added to 0.1 mL of 200 μM DPPH solution (Sigma-Aldrich, St. Louis, MO, USA), wherein gallic acid was used as the positive control. Te mixture was then incubated for 30 min at 37°C. Absorbance analysis was performed at a wavelength of 517 nm using a spectrophotometer (Spectramax i3x (Molecular Devices, Wokingham, UK)). Te radical scavenging ability of these samples was calculated as follows:   Moreover, a simple regression analysis was performed to calculate the value of IC 50 .

Total
Flavonoid. Te total favonoid content was obtained by modifying the methodology of Okello et al. [13]. 90% diethyl glycol (0.8 mL) and 1 N sodium hydroxide (10 μL) were mixed with 0.1 mL of each sample extract (1000 ppm concentration) in a 1.5 mL microcentrifuge tube. A vortex was then used to homogenize the solution, and a water bath system was used for incubation (60 min, 37°C). Sample absorbance was measured using a spectrophotometer (Spectramax i3x (Molecular Devices, Wokingham, UK)) in triplicates at 420 nm wavelength. A calibration curve between the standard and rutin was constructed to obtain the total favonoid content as mg rutin equivalent (Rue)/g.

Total Phenolic.
Total phenolic content was measured using the modifed methodology of Derakhshan et al. [14]. A Folin−Ciocalteu's reagent was added to a 0.5 mL sample solution (0.3 mg/mL) in a 1.5 mL tube. A 10% Na 2 CO 3 (0.5 mL) solution was added to the mixture, followed by incubation for 60 min at 25°C in the dark. Absorbance was measured at 725 nm using a spectrophotometer (Spectramax i3x (Molecular Devices, Wokingham, UK)). Te TPC was determined by interpolation of the standard curve using gallic acid with other samples. Te total phenolic content was presented as mg gallic acid equivalent (GAE) g − 1 .
2.7. HPLC Analysis. All concentrated sample extracts (100 mg each) were dissolved in 80% HPLC-grade methanol (1 mL) and sonicated for approximately 1 h. Samples were fltered using a 0.22 μM syringe flter (PALL Corporation, Ann Arbor, MI, USA) and placed in an HPLC tube. HPLC analysis was used to determine the 5-HMF and verbascoside content. Approximately, 1 mg of each 5-HMF and verbascoside was dissolved in 80% methanol to form a 10,000 ppm standard solution. HPLC analysis was performed using an HPLC machine (Waters Corporation, Milford, MA, USA) with a 2695 separation module and 2996 photodiode array detector. A Waters Capcell Pak UG120 C18 analytical column (250 × 4.6 mm, 5 μm; Shiseido, Japan) was used for separation at 30°C. Te mobile phase consisted of HPLC grade water with 0.1% formic acid (solvent A) and HPLC grade acetonitrile with 0.1% formic acid (solvent B) was used. Te gradient program was set as follows: 0-15 min, 95% A; 15-25 min, 85% A; 25-45 min, 70% A; 45-60 min, 95% A. For bride sample analysis, the column was equilibrated using 99% B for 10 min. Te fow rate was set to 0.8 ml/min with an injection amount of samples and standards set to 10 μL, and the analytical wavelength set to 320 nm (verbascoside) and 280 nm (5-HMF). Te detection of 5-HMF and verbascoside was performed by comparing their UV spectra, peak retention time, and atom masses for reference. Te concentration of each compound in the samples was expressed as metabolites per gram of processed dried roots (mg/g DW).

Preparation of RAW 264.7 Cells and Cell Viability Assay.
Culture of RAW 264.7 macrophage cell lines were done in Dulbecco's Modifed Eagle Medium (DMEM) (Gibco, Invitrogen) supplemented with 1% (v/v) streptomycin/ penicillin and 10% fetal bovine serum (FBS) and then incubated at 37°C in an atmosphere containing 5% CO 2 . Te cell lines were then treated with the sample extracts at various concentrations (0, 100, and 200 μg/mL) and further cultured with and without LPS (1 μg/mL) for 24 h at 37°C. A cell counting kit (CCK-8, Dojindo, Kumamoto, Japan) assay was used to determine cell line proliferation on the substrates. All samples were seeded at a density of 2 × 10 5 cells/ mL per well in the 96-well plate. Te absorbance was measured at 450 nm using ELISA.

Nitric Oxide (NO) Level
Measurement. NO levels were determined following the modifed methodology given by . RAW 264.7 cells (2 × 10 5 cells/mL) were pretreated with R. glutinosa extracts (0 and 200 μg/mL) for 1 h, followed by lipopolysaccharide (1 μg/mL) treatment for 24 h. After 24 h, the supernatant was collected to determine the NO levels. Te Griess reaction method was used to measure nitrite accumulation as an indicator of NO value in the LPS-induced RAW 264.7 macrophage cell lines. Absorbance was measured using a spectrophotometer (Spectramax i3x (Molecular Devices, Wokingham, UK)) in triplicates at 540 nm.

Western Blot Analysis.
Pretreatment of RAW 264.7 cell lines were pretreated with 200 μg/mL sample extracts for 1 h followed by LPS stimulation (1 μg/mL) for 24 h. All samples were seeded in a 6-well plate (1 × 106 cells/well). Treated cells were lysed using cold phosphate-bufered saline (PBS) and a lysis bufer containing protease inhibitors. Te cell lysed were collected and centrifuged, and the protein concentration of the lysate was determined using a BCA ™ protein assay kit. Equal amounts of protein were loaded onto an SDS-PAGE gel. Proteins were then transferred to a polyvinylidene fuoride membrane and incubated overnight at 4°C with specifc primary antibodies at a dilution of 1/2000 in 5% w/v skimmed milk in tris bufered saline with tween 20 (TBST) followed by 1 h incubation with the HRP-conjugated secondary antibodies (1/2500 dilution in 1X TBST) at room temperature.
2.11. Statistical Analysis. One-way analysis of variance (ANOVA) was applied to all experimental data using Tukey's post hoc test using Prism (Graph Pad software, v5.03). Diferences between compared means were considered statistically signifcant at p ≤ 0.05.

Marker Compound Composition.
According to Chinese pharmacopoeia, the quality of processed R. glutinosa is determined by its 5-HMF content. Te 5-HMF content of all R. glutinosa samples in this study was determined by comparing the HPLC retention time, UV absorption, and mass spectra with the standard (Figure 3(a)). As shown in Figure 3(a), 5-HMF was detected in PCR and PKR samples and was present in a very small amount (negligible) in the UPR sample. Further quantitative analysis of 5-HMF was performed at a wavelength of 280 nm. Te highest 5-HMF content was observed in PKR (1.518 ± 0.004 mg/g). Te results were signifcantly higher (p < 0.05) than the rest of the R. glutinosa samples (Figure 3(a)). Furthermore, we assessed the content of verbascoside compound in unprocessed R. glutinosa roots (both in vitro and commercial samples) to evaluate the raw material quality. Te result showed that in vitro UPR sample had slightly higher verbascoside content than commercial UPR sample (Figure 3(b)). Tis indicating higher quality of our in vitro R. glutinosa roots sample compared with commercially available R. glutinosa roots. Hence, this result becomes the basis foundation for development of processed R. glutinosa roots using in vitroderived samples.

Antioxidant Activity.
Te DPPH antioxidant activity of R. glutinosa samples was evaluated at diferent concentrations. Te results showed that the antioxidant activity of all R. glutinosa samples increased as the concentration of the sample increased ( Figure 4). Overall, the highest antioxidant activity was obtained in PKR (67.095%) and PCR (61.579%) samples at a concentration of 1000 mg/mL with the values of these two samples which were not signifcantly diferent (Figure 4(a)). Moreover, compared to the positive control (gallic acid, IC 50 � 16.65 μg/mL), the IC 50 values of all these samples showed a lower DPPH radical scavenging efects (Figure 4(b)). Among all these samples, the PKR sample showed the lowest IC 50 value (654.516 μg/mL), although it was not signifcantly diferent from the PCR sample (749.646 μg/mL), validating its high DPPH antioxidant activity.

Total Phenolic and Total Flavonoid Content.
Te total phenolic and favonoid concentration varies dramatically in all R. glutinosa samples (Figure 5(a)). Te total phenolic compound ranged from 15.880 to 41.578 mg GAE/g, whereas the total favonoid content ranged from 4.233 to 17.208 mg RUE/g ( Figure 5(b)). PKR samples showed the highest total phenolic and favonoid content of 41.578 mg GAE/g and 17.208 mg RUE/g, respectively. Tese values were signifcantly higher than the values obtained by PCR and UPR samples.

Efect of Samples Extracts on Cell Viability.
Te data showed that treatment with concentrations of 100 and 200 μg/mL of all UPR, PCR, and PKR extracts resulted in the viability of cell lines compared with the control. Tere were no signifcant diferences in cell viability among these samples ( Figure 6).

Efects of Samples Extracts on NO Production Level.
UPR, PCR, and PKR samples inhibited NO production in LPS-activated RAW 264.7 macrophage cell lines. Cells treated with processed R. glutinosa extracts (PCR and PKR) showed a stronger reduction in NO levels than the samples treated with unprocessed R. glutinosa extract (UPR) (Figure 7). However, there were no signifcant diferences in NO levels after treatment with PCR and PKR ( Figure 5).

Efects of Sample Extracts on the Expression of iNOS.
iNOS expression was strongly inhibited by processed R. glutinosa samples (PKR and PCR) ( Figure 8). However, there were no signifcant diferences in iNOS inhibition levels between PKR and PCR assay. Tese results are in line with the inhibition of NO levels, which strongly suggest that the reduction in NO production is caused due to the suppression of iNOS expression.

Discussion
R. glutinosa roots have been used for centuries in China, Korea, and Japan as one of the top herbal medicines. It has several therapeutic benefts for a wide range of medical conditions. Commonly, there are three types of R. glutinosa roots in oriental medicine, namely, fresh R. glutinosa roots, dried R. glutinosa roots, and processed R. glutinosa roots by steam processing [15][16][17] and the addition of supplements. All these types of roots are used in diferent therapeutic applications based on traditional medicine theory and practices. Previously, our team conducted some optimization studies to produce good quality R. glutinosa roots using biotechnological approaches (Figure 9). Te processing of R. glutinosa roots is aimed to reduce the toxicity and side efects to maximize the biological activities, change chemical properties or functions, preserve the active compounds, or maintain the purity from contaminants, such as microbes and other pathogens [18]. Although processed R. glutinosa roots have been studied extensively, there have been no studies using the roots developed biotechnologically in vitro which is the basic idea of this study. A previous study showed that R. glutinosa roots developed in vitro are safer than commercial ones since they are not contaminated by pathogens and contain higher active compounds [19].
Understanding the metabolic changes during processing is of great importance for the quality control of herbal drugs. Some previous studies have reported the change in the composition of active compounds in R. glutinosa roots following the steam processing [16,20]. Two major active compounds in unprocessed R. glutinosa roots are iridoid glycoside and phenylethanoid glycoside, which can be degraded due to high temperature to producother constituents. For example, steaming process converted catalpol (iridoid glycoside) into 1,5-dialdehyde to also form other polymers Evidence-Based Complementary and Alternative Medicine [21). Verbascoside (phenylethanoid glycoside) could be isomerized by high temperatures into iso-verbascoside to make cistanoside F, hydroxytyrosol, and verbascoside by hydrolysis reaction [21]. According to these facts, both catalpol and verbascoside most probably will not be detected in processed R. glutinosa [20]. In addition, steam processing also triggers the conversion of glucose or fructose into 5-Hydroxymethyl-2-furaldehyde (5-HMF) by Maillard reaction [22]. Particularly, in processed R. glutinosa, the formation of 5-HMF is mainly through fructose mechanism, which isomerized glucose into fructose, followed by the formation of fructose dehydrate to make 5-HMF via a fructose intermediate [21]. Tis conversion was confrmed in our study, wherein unprocessed R. glutinosa roots showed a negligible amount of 5-HMF, while processed R. glutinosa roots showed a higher content of 5-HMF. Surprisingly, between the two processed R. glutinosa roots, in vitro-derivedR. glutinosa roots (PKR) showed a higher 5-HMF content (0.15%) than commercial one (0.05%). According to Chinese pharmacopoeia 2015, processed R. glutinosa roots must contain more than 0.1% of 5-HMF, which is in line with the content of PKR samples. Tis result indicates a good  In this study, we carried out an antioxidant test using the widely known DPPH method. Te DPPH antioxidant assay helps in evaluating the capacity of a test sample to scavenge free radicals [13]. Te DPPH antioxidant test on all R. glutinosa root samples indicated the presence of antioxidant properties. Surprisingly, the processed R. glutinosa root samples (PCR and PKR) showed higher antioxidant activity than the unprocessed sample (UPR). Tis result indicates the positive efect of steam processing on the antioxidant properties of R. glutinosa roots. A previous study also reported that there is a positive correlation between steam-drying time and the antioxidant activity of processed R. glutinosa roots [23]. Te increase in antioxidant activity of processed R. glutinosa roots is in accordance with their higher 5-HMF content than the unprocessed R. glutinosa roots. Previous studies have reported that the compound 5-HMF and its derivatives exhibit a good amount of antioxidant activity tested by diferent methods, such as DPPH, ferric-reducing antioxidant power (FRAP), oxygen radical absorbing capacity (ORAC), and computational approaches [24][25][26]. In addition, the antioxidant capacity of in vitroderived processed R. glutinosa roots (PKR) was higher, though not signifcantly diferent from the commercially produced ones (PCR). Tis could also be correlated with the 5-HMF content that is greater in PKR than in PCR samples. However, compared to unprocessed R. glutinosa roots, the study of antioxidant activity of processed R. glutinosa roots is still very limited and requires a more comprehensive evaluation.

Evidence-Based Complementary and Alternative Medicine
Iridoid glycosides (e.g., catalpol and harpagide) are one of the predominant compounds in fresh R. glutinosa roots, but this group of compounds exhibits a relatively low antioxidant activity [27,28]. On the other hand, the presence of antioxidant properties in unprocessed R. glutinosa roots could mainly be attributed to other bioactive compounds, such as phenolics and favonoids, both of which are perceived as plant secondary metabolites benefcial to health for their antioxidant and antimicrobial properties [9,29,30]. Based on this hypothesis, we evaluated the total phenolic and favonoid compounds in all R. glutinosa samples. Interestingly, our results showed a linear correlation between the total phenolic and favonoid levels and the antioxidant activities of all R. glutinosa extracts. Te total phenolic and favonoid content increased signifcantly from UPR to PCR and then in PKR samples. Tis high correlation may confrm that in addition to 5-HMF, phenolic and favonoid compounds are also responsible for the antioxidant properties of all R. glutinosa root samples. Previous studies on the antioxidant activity and total phenolic and favonoid content in fresh R. glutinosa roots showed similar results [30]. In addition, steam processing increased the total phenolic and favonoid content in R. glutinosa roots, as shown by its higher content in processed R. glutinosa samples (PCR and PKR) compared to unprocessed roots (UPR). Moreover, between two processed R. glutinosa roots, in vitro-derivedR. glutinosa roots (PKR) showed a higher total phenolic and favonoid content than the commercially produced samples (PCR). Tis shows that the R. glutinosa roots developed in vitro produce more phenolic and favonoid compounds indicating a good quality of raw material for medicinal use.
Te fresh roots of R. glutinosa are known for their antiinfammatory activity in traditional medicine [16,31,32]. However, to date, there have been no comprehensive studies on the anti-infammatory activities of processed R. glutinosa roots. NO exhibits a notable function in biological defense [33], but surplus levels of NO can trigger infammatory development, such as autoimmune disorders, arthritis, and rheumatoid [34]. Hence, avoiding excessive NO production is one of the main objectives of anti-infammatory treatment. Terefore, we examined whether processed R. glutinosa reduced NO accumulation and iNOS protein expression in LPS-triggered infammation. Processed R. glutinosa samples (PCR and PKR) were found to be more efective than unprocessed R. glutinosa roots in inhibiting NO production (Figure 7). Tese results strengthened other parameters (5-HMF content, antioxidant activity, total phenolic content, and total favonoid content); as a result, processed R. glutinosa roots possess better chemical properties and bioactivity than unprocessed R. glutinosa roots. Terefore, we continued our study for the anti-infammatory evaluation using only processed R. glutinosa samples. Western blot analysis showed that iNOS expression gets greatly suppressed in both processed R. glutinosa root samples (PKR and PCR). Tis indicates that processed R. glutinosa roots can reduce NO production by downregulating the iNOS expression. However, both PKR and PCR samples showed similar levels of iNOS and NO inhibition, indicating a similar antiinfammatory efect.

Conclusion
In this study, we investigated and compared the 5-Hydroxymethyl-2-furaldehyde (5-HMF) content, total favonoid and phenolic content, antioxidant activity, nitric oxide (NO) levels, and anti-infammatory activity of in vitro regenerated R. glutinosa roots (PKR) and commercially produced R. glutinosa samples (PCR). Compared to unprocessed R. glutinosa roots (UPR) and PCR, PKR samples showed a higher content of 5-Hydroxymethyl-2-furaldehyde (5-HMF), favonoids, and phenolics. Meanwhile, for antioxidant activity and NO levels, PKR and PCR samples showed similar efects, although both were signifcantly better than UPR. In addition, PKR and PCR samples also showed good anti-infammatory activity, as they exhibited a similar ability to suppress the expression of iNOS. Terefore, PKR is in line with the Chinese pharmacopeia standards in terms of its 5-HMF content and biological activities. Tis study is expected to help verify the efcacy of oriental medicines derived from plant materials [40].

Data Availability
All data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
ER and YC are cofrst authors. ER designed the study, collected and analyzed all main data, and wrote the original draft. YC conducted HPLC, antioxidant, total phenolic and favonoid, and western blot analysis. HHN took part in antiinfammatory analysis. AL supervised HPLC analysis. JHP supervised anti-infammatory analysis. YK supervised the whole research process. All authors agree to be accountable for all aspects of work, ensuring integrity and accuracy.