An Integrative Study of Scrophularia takesimensis Nakai in an Ovalbumin-Induced Murine Model of Asthma: The Effect on T Helper 2 Cell Activation

Scrophularia have traditionally been used as herbal medicines to treat neuritis, sore throats, and laryngitis. In particular, S. takesimensis, a Korean endemic species with restricted distribution on Ulleung Island, holds significant resource and genetic value. However, its pharmacological properties have not been thoroughly evaluated. Thus, we provide detailed morphological characteristics and genomic information for S. takesimensis in this study. Moreover, its pharmacological activity was evaluated in an ovalbumin-induced asthma rat model, using extracts of S. takesimensis roots (100 or 200 mg/kg). The distinguishing features of S. takesimensis from related species include the presence or absence of stem wings, leaf shape, and habitat. The chloroplast (cp) genome of this species is 152,420 bp long and exhibits a conserved quadripartite structure. A total of 114 genes were identified, which included 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The gene order, content, and orientation of the S. takesimensis cp genome was highly conserved and consistent with the general structure observed in S. buergeriana and S. ningpoensis cp genomes. Confirming the anti-inflammatory effects of S. takesimensis extract (STE) using an established mouse model of ovalbumin-induced asthma, we observed reduced asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and suppression of T helper 2 (Th2) cell. Furthermore, STE treatment reduced Th2 cell activation and differentiation. This study underscores the medicinal value of S. takesimensis. The importance of preserving S. takesimensis was revealed and crucial insights were provided for further research on its utilization as a medicinal resource.


Introduction
The genus Scrophularia L., belonging to the family Scrophulariaceae Juss., comprises more than 200 species that are widely distributed in temperate regions of the Northern Hemisphere [1][2][3].In Eastern Asia, the dried roots of Scrophularia species have traditionally been used as herbal medicines to control neuritis, sore throat, and laryngitis [4][5][6].
Pharmaceutics 2024, 16 Previous studies showed that the Scrophularia has anti-inflammatory activities and immuneenhancing effects in in vivo models, such as the ovalbumin (OVA)-induced asthma mouse, and in vitro models including the Raw 264.7,MOLT-4, and SH-SY5Y cells [7].Scrophularia is widely used in traditional medicine, but still there was a lack of evidence in clinical and preclinical studies.
In Korea, the Scrophularia genus has been recognized as having seven taxa, six species, and one variety, based on morphological characteristics [8].Among these species, S. takesimensis Nakai (Korean: Seom-Hyun-Sam), a Korean endemic species, has a restricted distribution on Ulleung Island.Given the highly limited distribution of S. takesimensis, previous studies on its population genetics, conservation status, and phylogenetic position have been carried out [9][10][11][12][13].
Determining chloroplast (cp) genome sequences is helpful for plant taxonomy, species identification, population genetics, phylogenetic analysis, and diversity and evolution studies [14,15].While several cp genomes within the Scrophularia genus have been reported [16][17][18], and despite being a valuable plant, little genomic information is available on Scrophularia.
Several studies have evaluated the biological properties of Asian Scrophularia species.They exhibit various biological properties, including neuroprotective and antioxidant activities.These properties have been linked to several active constituents, notably phenylpropanoids, iridoids, and E-p-methoxycinnamic acid [19][20][21].Additionally, they can suppress the inflammatory response in allergic disease models by regulating cytokine production [4,22].Our previous study confirmed that S. buergeriana Miq. and S. koraiensis Nakai effectively reduced airway inflammation in an OVA-induced allergic asthma model [23,24].
However, most studies on the biological properties of Scrophularia have solely focused on species that are distributed in Asia or listed in their pharmacopoeias.The pharmacological properties of the Korean endemic species, which has high resource and genetic value, remain to be evaluated.
Thus, in this integrative study, we describe the morphological and genomic characteristics of Korean endemic S. takesimensis.We also examined the effects of S. takesimensis on OVA-induced allergic airway inflammation.

Plant Materials
The plant material, S. takesimensis, was obtained from the Department of Herbal Crop Research (Eumseong-gun, Chungcheongbuk-do, Republic of Korea), originally collected from its natural habitats (Buk-myeon, Ulleung-gun, and Gyeongsangbuk-do).Accurate identification relied on protologues and other relevant taxonomic studies.A voucher specimen (2-18-0146) has been deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbariorum code: KIOM) at the Korea Institute of Oriental Medicine (Naju, Republic of Korea).

Morphological Observations
The field photographs were captured using a digital camera.Major morphological characteristics were measured using digital Vernier calipers (CD-20AX; Mitutoyo, Sakado, Japan).The leaves and flowers were examined and photographed using a stereomicroscope (SZX16; Olympus, Tokyo, Japan).

Genome Sequencing and Assembly
The total deoxyribo nucleic acid (DNA) of the tested species was extracted using a modified Cetrimonium bromide (CTAB) method [25].The library was prepared from total genomic DNA using the TruSeq DNA Nano kit (TruSeq; Illumina, San Diego, CA, USA), following the manufacturer's protocols.Short-insert paired-end sequencing libraries were established and sequenced using the NextSeq500 platform (Illumina, San Diego, CA, USA).As a result, trimmed reads (Phred quality score ≥ 20) were assembled using the CLC genome assembler (ver.4.2.1;CLC Inc., Aarhus, Denmark) with the default parameters.Based on the aligned paired-end reads, SOAPdenovo gap closure was used to fill the gaps in the sequences [26].The contigs were aligned to the National Center for Biotechnology Information non-redundant databased (NCBI nrDB) for the identification of cp contigs, and Nucmer was employed to extract these contigs from the overall contig dataset [27].The contigs were arranged based on the reference cp genome sequence of S. takesimensis (NC026202).The new cp genome sequences of S. takesimensis obtained in this study were deposited in the NCBI GenBank database under accession number OQ580987.

Genome Annotation and Comparative Analysis
Gene annotation of S. takesimensis cp genome was performed using the GeSeq [28].The protein-coding sequences underwent manual curation, were validated using Artemis [29], and were cross-verified against the NCBI protein database.tRNAs were validated using tRNAscan SE 1.21 [30].The inverted repeat (IR) region sequences were confirmed utilizing an IR finder and RepEx [31].Circular maps of the S. takesimensis cp genome were generated using OGDRAW [32].The mVISTA program was employed in Shuffle-LAGAN mode to conduct a comparative analysis of the cp genomes with S. takesimensis as the reference [33].Nucleotide variability (Pi) calculations were performed using DnaSP ver.6 [34].To ascertain precise genetic variants, the coding sequences (CDSs), introns, and intergenic spacer (IGS) regions were analyzed individually.Simple sequence repeats (SSRs) were identified using MISA ver.2.1 [35], where the minimum number of repeat parameters was set to ten, five, four, three, three, and three for mono-, di-, tri-, tetra-, penta-, and hexanucleotides, respectively.Tandem repeats with a length of ≥ 20 bp were detected using the Tandem Repeats Finder [36], applying a minimum alignment score of 50 and a maximum period size of 500; the identity of repeats was set to ≥ 90%.

Phylogenetic Analysis
For phylogenetic analysis, a total of 11 cp genomes, including nine from Scrophularia samples, and Verbascum chinense (L.) Santapau and Verbascum phoeniceum L. as outgroups, were utilized.Among these, eight cp genome sequences were obtained from the NCBI GenBank database (Supplementary Table S1).Two matrices encompassing 78 conserved protein-coding genes (CDSs) were employed.Using MAFFT ver.7.388 [37], the CDS sets underwent alignment.Subsequently, each aligned gene was extracted using Geneious software ver.2023.2.1 (https://www.geneious.com;accessed on 1 January 2023.), and the genes were organized alphabetically.The concatenated gene dataset was constructed using Geneious software ver.2023.2.1 and the CDS dataset.GBlocks ver. 5 [38] was applied to filter the alignment datasets and eliminate ambiguously aligned regions.The best-fitting model for nucleotide substitutions was determined through the Akaike information criterion in jModelTest V2. 1.10 [39], selecting the generalised time reversible + gamma distributed with invariant sites (GTR+G+I) model for maximum likelihood (ML) analysis and the GTR+G model for Bayesian inference (BI) analysis.ML analysis was executed using RaxML v. 8.0.5 [40] with 1000 bootstrap replicates, while BI analysis utilized MrBayes 3.2.2[41], involving two independent runs of four simultaneous chains for 5,000,000 generations via the Markov chain Monte Carlo algorithm.Trees were sampled every 5000 generations, discarding the first 25% as burn-ins.The trees were inferred using a 50% majority-rule consensus to estimate posterior probabilities, and the reconstructed trees were visualized using Figtree V.1.4.2 [42].

Preparation of S. takesimensis Extract (STE)
The dried and ground roots of S. takesimensis (92.5 g) were refluxed in 70% EtOH at 95 • C for 2 h.The extract, obtained by filtering through filter paper and concentrating in vacuum, weighed 53.91 g (58.28% of yield, w/w), which was stored at 4 • C for this study.

Animals
Seven-week-old female BALB/c mice (20~22 g) were acquired from Dooyeol Biotech (Seoul, Republic of Korea).The animals were maintained in a room under controlled conditions (temperature, 23 • C; relative humidity, 50%; lit electrically from 08:00-20:00; 13-18 air changes per hour).Six mice were in each cage.The mice were subjected to a weeklong quarantine and acclimation period.They were housed under standard conditions, with full access to tap water and food.They were divided into the following four groups of six mice each: saline control (normal control, NC), OVA-induced asthma group (OVA, vehicle treatment), and OVA with 100 or 200 mg/kg STE by oral administration (STE100 and STE200, respectively).Moreover, dexamethasone (DEX) was used in a positive control group that was injected as previously described [23,24].The study was approved by the Institutional Ethics Committee of Chonnam National University (CNU IACUC-YB-2022-33).All experimental design procedures are indicated in Figure 6A.

Experimental Procedures
Asthma was induced in mice using OVA (Sigma-Aldrich, St Louis, MO, USA), as previously described [43].On days 0 and 7, the mice received a brief sensitization through intraperitoneal injection of 200 µL OVA/aluminum hydroxide (50 g).On days 14, 15, 16, and 17 after the last sensitization, 25 g (50 µL) OVA was administered intranasally.Between days 11 and 17, STE (100 or 200 mg/kg) was administered once daily.On day 18, BALF from each mouse was obtained by lavage using 1 mL of Dulbecco's phosphate-buffered saline (PBS; Gibco, New York, NY, USA) after euthanizing by intravenous injection of a combination of alfaxalone (85 mg/kg) and xylazine (10 mg/kg).Cells from BALF samples (100 µL) were placed on a slide through a cytospin and stained with Diff-Quik ® reagent (Sysmex Co., Seoul, Republic of Korea) to evaluate differential cell counts.

Histology
Lung tissue samples were collected and fixed in neutral buffered formalin (10% v/v) overnight.After fixing, the tissue was embedded in paraffin and sectioned to a thickness of 4 µm for histological analysis.Sections were stained with hematoxylin and eosin (H&E) solution (Sigma-Aldrich) or periodic acid-Schiff (PAS) solution (IMEB Inc., San Marcos, TX, USA) to assess histological alterations and mucus production.Tissue slides were imaged with a panoramic DESK slide scanner (3DHISTECH, Budapest, Hungary).Each tissue section was scored on a scale of zero to four according to the extent of interstitial inflammation, alveolar wall thickening, peribronchial inflammation, and interstitial edema (zero ≤ 10%, one = up to 30%, two = up to 50%, three = up to 70%, four ≥ 70%) [44].

Flow Cytometry
Cells extracted from the lungs were stained with fluorescein isothiocyanate (FITC)conjugated anti-cluster of differentiation (CD) 4, Alexa Fluor 700-conjugated anti-CD3, and PE-conjugated anti-CD25 antibodies to examine activated T cells, as previously described [46].The number of activated T cells and Th2 cells were obtained by multiplying their frequencies by CD3+ cells.Antibodies were purchased from the following three different suppliers: BD Biosciences, eBioscience, and BioLegend.FlowJo software version 10.6 (TreeStar, Ashland, OR, USA) and an LSR Fortessa X-20 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) were used to evaluate the labelled cells.

Statistical Analysis
GraphPad Prism (version 9.3.1,GraphPad Software, San Diego, CA, USA) was used for statistical analysis.A one-way analysis of variance (ANOVA), followed by a Dunnett's test, was used to determine the statistical significance of the data.Means and standard deviations were used to express the results.Statistical significance was set at p < 0.05.

Comprehensive Comparative Analysis of Scrophularia Chloroplast Genomes
To identify repetitive sequences in the cp genomes, we studied simple sequence repeats (SSRs) and tandem repeats in the three Scrophularia cp genomes.The mononucleotide motif was the most abundant of all the cp genomes studied, followed by dinucleotide motif repeats (Figure S2A).The Scrophularia cp genomes exhibited repeat sequences in intergenic spacer (IGS) regions, with 34-38 SSRs (Figure S2B).Most tandem repeats, predominantly 21-40 bp long, were located in the LSC and SSC of the IGS region (Figure S2C,D).We also identified one-four tandem repeats of >71 bp in Scrophularia.
Sequence identities were analyzed using the mVISTA program with the S. takesimensis cp genome as a reference, revealing a well-conserved overall structure among the Scrophularia cp genomes (Figure 3).As expected, the genic regions were more conserved than the IGS regions, with the highest divergences observed in the LSC and SSC regions.dominantly 21-40 bp long, were located in the LSC and SSC of the IGS region (Figure S2C,D).We also identified one-four tandem repeats of >71 bp in Scrophularia.
Sequence identities were analyzed using the mVISTA program with the S. takesimensis cp genome as a reference, revealing a well-conserved overall structure among the Scrophularia cp genomes (Figure 3).As expected, the genic regions were more conserved than the IGS regions, with the highest divergences observed in the LSC and SSC regions.We conducted an analysis of genetic divergence utilizing three Scrophularia cp genomes and identified species-specific mutations through pairwise alignment criteria in S. takesimensis (Figure 4).Nucleotide diversity (Pi) values were determined for the three Scrophularia cp genomes, along with species-specific Pi values.Overall, the Scrophularia cp genome exhibited an average Pi value of 0.005.Divergent regions were observed in the non-coding trnH-psbA, rps18-rpl20 in the large single copy (LSC), and ndhF-rpl32 and rpl32-trnL in the short single copy (SSC).Notably, trnH-psbA displayed the highest Pi value of approximately 0.02, followed by trnH-psbA (0.033) in the LSC region.Overall, our findings indicated that the cp genomes of S. takesimensis were highly conserved in terms of genome structure and gene order compared to those of other Scrophularia species.We conducted an analysis of genetic divergence utilizing three Scrophularia cp genomes and identified species-specific mutations through pairwise alignment criteria in S. takesimensis (Figure 4).Nucleotide diversity (Pi) values were determined for the three Scrophularia cp genomes, along with species-specific Pi values.Overall, the Scrophularia cp genome exhibited an average Pi value of 0.005.Divergent regions were observed in the non-coding trnH-psbA, rps18-rpl20 in the large single copy (LSC), and ndhF-rpl32 and rpl32-trnL in the short single copy (SSC).Notably, trnH-psbA displayed the highest Pi value of approximately 0.02, followed by trnH-psbA (0.033) in the LSC region.Overall, our findings indicated that the cp genomes of S. takesimensis were highly conserved in terms of genome structure and gene order compared to those of other Scrophularia species.

Phylogenetic Relationships among Scrophularia Genus
To elucidate the phylogenetic relationships within Scrophularia, we identified 78 conserved protein-coding sequences by aligning 69,846 bp shared among all the Scrophularia samples, with Verbascum chinense and V. phoeniceum serving as outgroups (Figure 5).The

Phylogenetic Relationships among Scrophularia Genus
To elucidate the phylogenetic relationships within Scrophularia, we identified 78 conserved protein-coding sequences by aligning 69,846 bp shared among all the Scrophularia samples, with Verbascum chinense and V. phoeniceum serving as outgroups (Figure 5).The maximum likelihood (ML) and Bayesian inference (BI) topologies exhibited remarkable congruence with both the whole genome sequences and coding sequences (CDS) datasets.The phylogenetic analyses provided robust support for all lineages except one (ML = 100%, BI = 1.0).Notably, the six Scrophularia species within the tribe formed a well-clustered group.S. buergeriana and S. ningpoensis Hemsl.constituted a monophyletic group, with S. cephalantha Nakai identified as a sister group.Overall, clustering revealed a close relationship between S. buergeriana and S. ningpoensis, with S. takesimensis as their sister group, while S. dentata Royle ex Benth.showed a distinct phylogenetic position within Scrophularia.

Effects of S. takesimensis Extract (STE) on Lung Histopathological Change and Number of Inflammatory Cells in the Bronchoalveolar Lavage Fluid (BALF) of an Ovalbumin (OVA)-Induced Asthmatic Animal Model
H&E and PAS staining were performed to investigate the changes in lung tissue (Figure 6B).The results demonstrated that the OVA-induced asthmatic group exhibited a higher accumulation of immune cells than the control group.However, STE-treated and Dex-treated groups, which is the positive control group, showed a dose-dependent decrease in immune cell count, compared to that in the OVA-induced asthmatic group (Figure 6C,D).

Effects of S. takesimensis Extract (STE) on Lung Histopathological Change and Number of
Inflammatory Cells in the Bronchoalveolar Lavage Fluid (BALF) of an Ovalbumin (OVA)-Induced Asthmatic Animal Model H&E and PAS staining were performed to investigate the changes in lung tissue (Figure 6B).The results demonstrated that the OVA-induced asthmatic group exhibited a higher accumulation of immune cells than the control group.However, STE-treated and Dex-treated groups, which is the positive control group, showed a dose-dependent decrease in immune cell count, compared to that in the OVA-induced asthmatic group (Figure 6C,D).
PAS staining was used to measure mucus hypersecretion.In this study, we confirmed a significant increase in mucus production in the OVA-induced asthmatic group compared with the control group, and a decrease in mucus production was observed in the STE-treated and DEX-treated groups, compared with that in the OVA-induced asthmatic group.The STE-treated group exhibited a dose-dependent reduction in mucus production (Figure 6B).
A significant increase in the total number of cells and eosinophils was detected in the OVA-induced asthmatic group compared with the control group (p < 0.05).The STE200treated group showed a significantly reduced number of total cells, compared with the OVA-induced asthmatic model (Figure 6D-H).PAS staining was used to measure mucus hypersecretion.In this study, we confirmed a significant increase in mucus production in the OVA-induced asthmatic group compared with the control group, and a decrease in mucus production was observed in the STE-treated and DEX-treated groups, compared with that in the OVA-induced asthmatic group.The STE-treated group exhibited a dose-dependent reduction in mucus production (Figure 6B).
A significant increase in the total number of cells and eosinophils was detected in the OVA-induced asthmatic group compared with the control group (p < 0.05).The STE200treated group showed a significantly reduced number of total cells, compared with the OVA-induced asthmatic model (Figure 6D-H).

Effects of S. takesimensis Extract (STE) on Interleukin-4 (IL-4) and Immunoglobulin E (IgE) Levels in Bronchoalveolar Lavage Fluid (BALF) of Asthmatic Animal Model
IL-4 cytokine levels were measured in the BALF and showed a significant increase in the OVA-induced asthma group, compared with the control group.Decreased IL-4 levels 3.6.Effects of S. takesimensis Extract (STE) on Interleukin-4 (IL-4) and Immunoglobulin E (IgE) Levels in Bronchoalveolar Lavage Fluid (BALF) of Asthmatic Animal Model IL-4 cytokine levels were measured in the BALF and showed a significant increase in the OVA-induced asthma group, compared with the control group.Decreased IL-4 levels were detected in the STE-treated group compared with those in the OVA-induced asthma group, showcasing a particularly significant decline in the STE200 group (Figure 7A).However, the Dex-treated group was no different from the OVA-induced asthma group with regard to IL-4 levels.A significant increase in IgE production was observed in the OVA-induced asthma group, when compared with the control group (Figure 7B,C).While STE treatment influenced OVA-specific IgE levels, only the STE200-treated and Dex-treated groups demonstrated component-dependent decreased levels of OVA-specific IgE compared with the OVA-induced asthma group (Figure 7C), although there was no significant difference.
with regard to IL-4 levels.A significant increase in IgE production was observed in OVA-induced asthma group, when compared with the control group (Figure 7B,C).W STE treatment influenced OVA-specific IgE levels, only the STE200-treated and D treated groups demonstrated component-dependent decreased levels of OVA-specific compared with the OVA-induced asthma group (Figure 7C), although there was no nificant difference.

Effects of S. takesimensis Extract (STE) on CD4+ T Cell (Helper T Cell) Activation in th Lung Tissues of Asthmatic Animal Model
T cells were activated in the OVA-induced asthma model, and STE and Dex treatm significantly deactivated T cells, in comparison with the OVA-induced asthma group ( ure 7E).An increase in the number of T helper 2 (Th2) cells was also observed in the O induced asthma group.STE and Dex treatment significantly decreased the percentag Th2 cells, compared with that in the OVA-induced asthma model (Figure 7D).

Effects of S. takesimensis Extract (STE) on CD4+ T Cell (Helper T Cell) Activation in the Lung Tissues of Asthmatic Animal Model
T cells were activated in the OVA-induced asthma model, and STE and Dex treatment significantly deactivated T cells, in comparison with the OVA-induced asthma group (Figure 7E).An increase in the number of T helper 2 (Th2) cells was also observed in the OVA-induced asthma group.STE and Dex treatment significantly decreased the percentage of Th2 cells, compared with that in the OVA-induced asthma model (Figure 7D).

Discussion
Scrophularia species have been integral to traditional and folk medicine for centuries with regard to addressing neuritis, sore throats, and laryngitis [4][5][6].Scrophularia takesimensis, an endemic species that shows significant similarities to the medicinal plant S. buergeriana, has been well characterized in terms of its morphological traits and anti-inflammatory and antioxidant properties [4,[19][20][21][22].
Although the presence or absence of stem wings, leaf shape, and habitat distinguished S. takesimensis from other species in this genus, all Scrophularia species clustered into monophyletic groups.Genetically, S. takesimensis was more closely related to S. buergeriana, S. ningpoensis, S. cephalantha, and S. henryi Hemsl.from the same section (Scrophularia sect.Scrophularia).According to the Korean Herbal Pharmacopoeia, the dried roots S. buergeriana and S. ningpoensis are used as herbal medicines, known as Scrophulariae Radix [5,6].This demonstrates that, while S. takesimensis is closely related to S. buergeriana and S. ningpoensis as listed medicines, it possesses distinct features.Nevertheless, S. takesimensis has significantly similar properties to treat allergic inflammation akin to S. buergeriana.
The phylogeny of medicinal plants has proven to be a useful predictive tool for a more targeted approach [47].Thus, we expect that the two species, S. cephalantha and S. henryi clustered in the same clade as S. takesimensis of monophyly, would have a similar effect.We suggest an analysis of the inflammatory responses of S. cephalantha and S. henryi to clarify the evolutionary prediction of medicinal properties in the genus Scrophularia for future phylogeny-guided drug discovery.
Allergic asthma is a chronic respiratory disease caused by an impaired immune response.Owing to the recent spread of the coronavirus disease 2019, interest in the treatment of pulmonary disorders has rapidly increased [55].The dried roots of S. buergeriana have traditionally been used as herbal medicines to treat fever, edema, neuritis, and laryngitis [4].Similarly, S. koraiensis has protective effects on OVA-induced allergic airway inflammation by suppressing NF-κB phosphorylation and enhancing the Nrf2/HO-1 signaling pathway [23].
In this study, STE reduced eosinophilia and pro-inflammatory cytokine production, decreased IgE levels, and alleviated OVA-induced allergic airway inflammation, as supported by histological evidence of reduced airway inflammation and mucus secretion.These results suggest that STE may prove effective for suppressing the development of asthma.In the present study, we evaluated Th2 cell activation to further understand the effects of STE on Th2 cytokine regulation.STE significantly reduced the proportion of activated CD4+ T cells and Th2 cell differentiation under polarization conditions.These results suggest that STE attenuates the reduction in Th2 cytokine-mediated asthmatic responses by directly regulating the activation and differentiation of Th2 cells [46].Our results also indicate that STE has an efficacy similar to that of S. buergeriana, demonstrating the possibility of its incorporation into medicinal materials.Moreover, we underscored the possibility of using morphological and phylogenetic approaches as predictive tools for exploring new medicinal plants.

Conclusions
This study provides comprehensive data on the morphological, genomic, and biological activities of the endemic plant S. takesimensis.This endemic species also has efficacy against asthma, including asthmatic phenotypes, encompassing inflammatory cell infiltration, mucus production, and suppression of Th2 cytokines similar to S. buergeriana, which is a closely related species, demonstrating its potential for incorporation into medicinal materials.This integrative study presents the possibility of using morphological and phylogenetic approaches as useful predictive tools for exploring new medicinal plants.Consequently, this study provides insights for screening alternative medicines using predictive values from natural classification and for evaluating the biological properties of endemic species.

Figure 2 .
Figure 2. Circular gene map of the chloroplast genome from Scrophularia takesimensis.Genes drawn inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise.The darker gray in the inner circle represents GC content.* means genes containing introns.

Figure 2 .
Figure 2. Circular gene map of the chloroplast genome from Scrophularia takesimensis.Genes drawn inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise.The darker gray in the inner circle represents GC content.* means genes containing introns.

Figure 3 .
Figure 3.Comparison of Scrophularia cp genomes using mVISTA.The complete cp genomes of three Scrophularia species were compared, with S. takesimensis as a reference.Blue block: conserved genes, sky-blue block: transfer RNA (tRNA) and rRNA, red block: CNS.White represents the regions with 3. Comparison of Scrophularia cp genomes using mVISTA.The complete cp genomes of three Scrophularia species were compared, with S. takesimensis as a reference.Blue block: conserved genes, sky-blue block: transfer RNA (tRNA) and rRNA, red block: CNS.White represents the regions with sequence variation among the three Scrophularia.CNS: conserved non-coding sequences; cp: chloroplast; rRNA: ribosomal RNA; tRNA: transfer RNA.

Figure 4 .
Figure 4. Comparison of the nucleotide diversity (Pi) values among the Scrophularia.Pi value in Scrophularia chloroplast genomes and excludes regions with Pi = 0. LSC: Large single copy; IR: inverted repeat; SSC: small single copy.

Figure 4 .
Figure 4. Comparison of the nucleotide diversity (Pi) values among the Scrophularia.Pi value in Scrophularia chloroplast genomes and excludes regions with Pi = 0. LSC: Large single copy; IR: inverted repeat; SSC: small single copy.

Pharmaceutics 2024 , 16 Figure 5 .
Figure 5.A phylogenetic tree of Scrophularia representing the maximum likelihood (ML) bootstrap and Bayesian inference (BI) probability.The number of branches above or below represents the ML bootstrap and BI probability support values.The cp genomes completed in this study are indicated with bold text.BI: Bayesian inference; ML: maximum likelihood; PP: posterior probabilities.

Figure 5 .
Figure 5.A phylogenetic tree of Scrophularia representing the maximum likelihood (ML) bootstrap and Bayesian inference (BI) probability.The number of branches above or below represents the ML bootstrap and BI probability support values.The cp genomes completed in this study are indicated with bold text.BI: Bayesian inference; ML: maximum likelihood; PP: posterior probabilities.

Table 1 .
Major morphological characteristics of the Korean endemic Scrophularia takesimensis and related species, S. buergeriana and S. ningpoensis.
3.2.Chloroplast Genome Organization and Repeat Sequence of S. takesimensis S. takesimensis was sequenced at approximately 8000× coverage, resulting in the generation of 34 Gb of raw paired-end reads and 32 Gb of trimmed reads (refer to Table

Table 2 .
Features of Scrophularia takesimensis and related species' chloroplast genomes.