Full-length transcriptome profiling of Acanthopanax gracilistylus provides new insight into the kaurenoic acid biosynthesis pathway.

Acanthopanax gracilistylus is a deciduous plant in the family Araliaceae, which is commonly used in Chinese herbal medicine, as the root bark has functions of nourishing the liver and kidneys, removing dampness and expelling wind, and strengthening the bones and tendons. Kaurenoic acid (KA) is the main effective substance in the root bark of A. gracilistylus with strong anti-inflammatory effects. To elucidate the KA biosynthesis pathway, second-generation (DNA nanoball) and third-generation (Pacific Biosciences) sequencing were performed to analyze the transcriptomes of the A. gracilistylus leaves, roots, and stems. Among the total 505,880 isoforms, 408,954 were annotated by seven major databases. Sixty isoforms with complete open reading frames encoding 11 key enzymes involved in the KA biosynthesis pathway were identified. Correlation analysis between isoform expression and KA content identified a total of eight key genes. Six key enzyme genes involved in KA biosynthesis were validated by real-time quantitative polymerase chain reaction. Based on the sequence analysis, the spatial structure of ent-kaurene oxidase was modeled, which plays roles in the three continuous oxidations steps of KA biosynthesis. This study greatly enriches the transcriptome data of A. gracilistylus and facilitates further analysis of the function and regulation mechanism of key enzymes in the KA biosynthesis pathway. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01436-7.

Efficient Separation of Proteins and Polysaccharides from Dendrobium huoshanense Using Aqueous Two-Phase System with Ionic Liquids.

By applying the hydrophilic ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), and inorganic salts (K3PO4), an ionic liquid aqueous two-phase system (ILATPS) was established for the separation of Dendrobium huoshanense polysaccharides (DhPs) and proteins. The effects of inorganic salt concentration, IL quantity, crude DhPs concentration, pH value and temperature were studied to achieve the optimal condition. With the best combination of ILATPS (1.75 g K3PO4, 1.25 g [C4mim]Cl, 10 mg crude DhPs and 5.0 mL ddH2O at pH 7.0 under 25 °C), the extraction efficiency rates for DhPs and proteins were 93.4% and 90.2%, respectively. The processed DhPs retrieved from the lower salt-rich phase comprised mannose, glucose, galactose, arabinose, and galacturonic acid with a molar ratio of 185:71:1.5:1:1 and the molecular weight was 2.14 × 105 Da. This approach is fast, simple and environmentally friendly. It provides a new insight into purifying functional polysaccharides of plant origin.

Review on the Pharmacological Properties of Phillyrin.

Phillyrin is an effective lignan glycoside extracted from a traditional Chinese medicine Forsythia suspensa (Thunb.) Vahl (Oleaceae). It mainly exists in the roots, stems, leaves and fruits of the plant, with the highest content in the leaves. In terms of its medicinal application, there are a large number of experimental data proving its pharmacological effects in vitro and in animal models, such as anti-inflammatory, anti-obesity, anti-tumor, etc. Furthermore, pharmacokinetic experiments have also shown phillyrin's high effectiveness and low toxicity. Despite more than one thousand studies in the literature on phillyrin retrievable from Web of Science, PubMed, and CNKI, few reviews on its pharmacological activities have been presented conclusively. In this paper, we aimed to summarize the pharmacological and pharmacokinetic characteristics of phillyrin from the current literature, focusing on its anti-inflammatory, anti-aging, antiviral, antibacterial, hepatoprotective and anti-cancer effects, hoping to come up with new insights for its application as well as future studies.

Comparison of the Antioxidant Activities and Polysaccharide Characterization of Fresh and Dry Dendrobium officinale Kimura et Migo.

It is generally believed that fresh Dendrobium officinale (FDO) has more significant pharmacological activity than dried Dendrobium officinale (DDO); however, the difference has not been clearly shown. Our study compared their antioxidant properties both in vitro and in vivo, and the molecular weight arrangement and monosaccharide composition of the fresh Dendrobium officinale polysaccharides (FDOPs) and the dried Dendrobium officinale polysaccharides (DDOPs) were analyzed by HPLC-GPC and GC-MS. The results showed that the FDO and its polysaccharides had more significant effects on scavenging DPPH, ABTS, and hydroxyl radicals than the DDO. In addition, both the FDO and DDO significantly reduced lipid peroxidation levels and increased the SOD, T-AOC, CAT, and GSH levels in mice with acute liver damage caused by CCl4, while the FDO and its polysaccharides were more effective. Histopathological analysis further verified the protective effect of the Dendrobium polysaccharides on CCl4-induced liver injury. The determination of the polysaccharides revealed that the polysaccharide and mannose contents of the FDO were significantly higher than their dried counterparts, and the homogeneous arrangement of the polysaccharides in the FDO was degraded into three polysaccharide fragments of different molecular weights in the DDO. Overall, our data identified differences in the antioxidant activities of the FDO and DDO, as well as the reasons for these differences.

A Comprehensive Review with Updated Future Perspectives on the Ethnomedicinal and Pharmacological Aspects of Moringa oleifera.

Moringa oleifera is an ancient remedy plant, known as the miraculous plant due to its many prominent uses and significant health benefits. It is a nutrient-rich plant, with exceptional bioactive compounds, such as polyphenols that possess several medicinal properties. Many significant studies have been carried out to evaluate the ethnomedicinal and pharmacological properties of M. oleifera in various applications. Therefore, this comprehensive review compiles and summarizes important findings from recent studies on the potential properties of different parts of M. oleifera. The pharmacological properties of M. oleifera have been studied for various potential biological properties, such as cardio-protective, anti-oxidative, antiviral, antibacterial, anti-diabetic and anti-carcinogenic effects. Therefore, the potential of this plant is even more anticipated. This review also highlights the safety and toxicity effects of M. oleifera treatment at various doses, including in vitro, in vivo and clinical trials from human studies.

[Biosynthetic pathways of Polygonatum cyrtonema polysaccharide and diosgenin based on its transcriptomic data].

Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.

[Cloning and quantitative expression analysis of GMPP gene from Dendrobium huoshanense].

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.

[Simultaneous determination of five saponins in Bupleuri Radix by HPLC-DAD dual wavelength method].

Epoxy ether type and isophthalene type saponin are the main saponins of Bupleurum chinense. However,due to the difference of their UV spectrum,there is no quantitative method for simultaneous determination of these two kinds of saponins. In this paper,a dual-wavelength high performance liquid chromatography(HPLC) was developed for simultaneous determination of five saponins in epoxidized ether(saikosaponin a,c,d) and isosorbide type(saikosaponin b1,b2). The mobile phase was eluted with acetonitrile-water(0.1% phosphoric acid) gradient at a column temperature of 30 °C and a flow rate of 1.0 mL·min⁻¹. The detection wavelengths were 208 nm for saikosaponins a,c, and d, and 254 nm for saikosaponins b1 and b2. The results showed that the separation of five kinds of saikosaponin was good, with the linear range of 9.70-1 935.00(r=0.999 4),8.20-1 380.00(r=0.999 3),6.90-1 640.00(r=0.999 0),5.25-630.00(r=0.999 4), and 5.15-618.00 mg·L⁻¹(r=0.999 5), respectively. The average recoveries were 97.70%-100.2% and the RSD was less than 3%(n=6). The method is simple,rapid and reproducible. It can be used for the determination of five kinds of saikosaponins in B. chinense.

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Current and future distribution of Forsythia suspensa in China under climate change adopting the MaxEnt model.

This study evaluated the potential impact of climate change on the distribution of Forsythia suspensa, a valuable traditional Chinese medicinal plant, using the MaxEnt model integrated with Geographic Information System (GIS). By analyzing occurrence data from various databases and environmental variables including climate and soil factors, we forecasted the present and future (2050s and 2070s) habitat suitability of F. suspensa under different greenhouse gas emission scenarios (RCP8.5, RCP4.5, RCP2.6). Results indicated that the suitable habitats for F. suspensa were primarily located in North, East, Central, Northwest, and Southwest China, with a significant potential expansion of suitable habitats anticipated by the 2070s, particularly under the high emission scenario. The study identified precipitation and temperature as the primary environmental drivers impacting the distribution of F. suspensa. Furthermore, a northward shift in the centroid of suitable habitats under future climate scenarios suggested a potential migration response to global warming. This work provides crucial insights into the future conservation and cultivation strategies for F. suspensa amidst changing climatic conditions.

Quality Evaluation of Polygonatum cyrtonema Hua Based on UPLC-Q-Exactive Orbitrap MS and Electronic Sensory Techniques with Different Numbers of Steaming Cycles.

In this study, electronic sensory techniques were employed to comprehensively evaluate the organoleptic quality, chemical composition and content change rules for Polygonatum cyrtonema Hua (PCH) during the steaming process. The results were subjected to hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). These analyses revealed, from a sensory product perspective, overall differences in colour, odour and taste among the samples of PCH with different numbers of steaming cycles. Using the UPLC-Q-Exactive Orbitrap MS technique, 64 chemical components, including polysaccharides, organic acids, saponins and amino acids were detected in PCH before and after steaming. The sensory traits were then correlated with the chemical composition. From the perspectives of sensory traits, chemical composition, and multi-component index content, it was preliminarily deduced that carrying out five cycles of steaming and sun-drying was optimal, providing evidence for the quality evaluation of PCH during the steaming process.

Phillyrin: an adipose triglyceride lipase inhibitor supported by molecular docking, dynamics simulation, and pharmacological validation.

CONTEXT: Adipose triglyceride lipase (ATGL), a key enzyme responsible for lipolysis, catalyzes the first step of lipolysis and converts triglycerides to diacylglycerols and free fatty acids (FFA). Our previous work suggested that phillyrin treatment improves insulin resistance in HFD-fed mice, which was associated with ATGL inhibition. In this study, using docking simulation, we explored the binding pose of phillyrin and atglistatin (a mouse ATGL inhibitor) to ATGL in mouse. From the docking results, the interactions with Ser47 and Asp166 were speculated to have caused phillyrin to inhibit ATGL in mice. Further, molecular dynamics simulation of 100 ns and MM-GBSA were conducted for the protein-ligand complex, which indicated that the system was stable and that phillyrin displayed a better affinity to ATGL than did atglistatin throughout the simulation period. Moreover, the results of pharmacological validation were consistent with those of the in silico simulations. In summary, our study illustrates the potential of molecular docking to accurately predict the binding protein produced by AlphaFold and suggests that phillyrin is a potential small molecule that targets and inhibits ATGL enzymatic activity. METHODS: The ATGL-predicted protein structure, verified by PROCHECK, was determined using AlphaFold. Molecular docking, molecular dynamics simulation, and prime molecular mechanic-generalized born surface area were performed using LigPrep, Desmond, and prime MM-GBSA modules of Schrödinger software release 2021-2, respectively. For pharmacological validation, immunoblotting was performed to assess ATGL protein expression. The fluorescence intensity and glycerol concentration were quantified to evaluate the efficiency of phillyrin in inhibiting ATGL.

Efficacy of Forsythia suspensa (Thunb.) Vahl on mouse and rat models of inflammation-related diseases: a meta-analysis.

Objective: To evaluate the efficacy of the fruits of the medicinal plant Forsythia suspensa (Thunb.) Vahl (FS), in treating inflammation-associated diseases through a meta-analysis of animal models, and also probe deeply into the signaling pathways underlying the progression of inflammation. Materials and methods: All data analyses were performed using Review Manager 5.3 and the results are presented as flow diagrams, risk-of-bias summaries, forest plots, and funnel plots. Summary estimates were calculated using a random- or fixed-effect model, depending on the value of I2. Results: Of the 710 records identified in the initial search, 11 were selected for the final meta-analysis. Each study extracted data from the model and treatment groups for analysis, and the results showed that FS alleviated the inflammatory cytokine levels in serum; oxidant indicator: reactive oxygen species; enzymes of liver function; endotoxin and regulatory cells in blood; and improved the antioxidant enzyme superoxide dismutase. Conclusion: FS effectively reversed the change in acute or chronic inflammation indicators in animal models, and the regulation of multiple channel proteins in inflammatory signaling pathways suggests that FS is a good potential drug for inflammatory disease drug therapy.

An insight review on the neuropharmacological effects, mechanisms of action, pharmacokinetics and toxicity of mitragynine.

Mitragynine is one of the main psychoactive alkaloids in Mitragyna speciosa Korth. (kratom). It has opium-like effects by acting on µ-, δ-, and κ-opioid receptors in the brain. The compound also interacts with other receptors, such as adrenergic and serotonergic receptors and neuronal Ca2+ channels in the central nervous system to have its neuropharmacological effects. Mitragynine has the potential to treat diseases related to neurodegeneration such as Alzheimer's disease and Parkinson's disease, as its modulation on the opioid receptors has been reported extensively. This review aimed to provide an up-to-date and critical overview on the neuropharmacological effects, mechanisms of action, pharmacokinetics and safety of mitragynine as a prospective psychotropic agent. Its multiple neuropharmacological effects on the brain include antinociceptive, anti-inflammatory, antidepressant, sedative, stimulant, cognitive, and anxiolytic activities. The potential of mitragynine to manage opioid withdrawal symptoms related to opioid dependence, its pharmacokinetics and toxic effects were also discussed. The interaction of mitragynine with various receptors in the brain produce diverse neuropharmacological effects, which have beneficial properties in neurological disorders. However, further studies need to be carried out on mitragynine to uncover its complex mechanisms of action, pharmacokinetics, pharmacodynamic profiles, addictive potential, and safe dosage to prevent harmful side effects.

Protective effects of Pudilan Tablets against osteoarthritis in mice induced by monosodium iodoacetate.

Osteoarthritis (OA) is a complicated disorder that is the most prevalent chronic degenerative joint disease nowadays. Pudilan Tablets (PDL) is a prominent traditional Chinese medicine formula used in clinical settings to treat chronic inflammatory illnesses. However, there is currently minimal fundamental research on PDL in the therapy of joint diseases. As a result, this study looked at the anti-inflammatory and anti-OA properties of PDL in vitro and in vivo, as well as the mechanism of PDL in the treatment of OA. We investigated the anti-OA properties of PDL in OA mice that were generated by monosodium iodoacetate (MIA). All animals were administered PDL (2 g/kg or 4 g/kg) or the positive control drug, indomethacin (150 mg/kg), once daily for a total of 28 days starting on the day of MIA injection. The CCK-8 assay was used to test the vitality of PDL-treated RAW264.7 cells in vitro. RAW264.7 cells that had been activated with lipopolysaccharide (LPS) were used to assess the anti-inflammatory properties of PDL. In the MIA-induced OA model mice, PDL reduced pain, decreased OA-induced cartilage damages and degradation, decreased production of pro-inflammatory cytokines in serum, and suppressed IL-1ß, IL-6, and TNF-α mRNA expression levels in tibiofemoral joint. In RAW264.7 cells, PDL treatment prevented LPS-induced activation of the ERK/Akt signaling pathway and significantly decreased the levels of inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α. In conclusion, these results suggest that PDL is involved in combating the development and progression of OA, exerts a powerful anti-inflammatory effect on the knee joint, and may be a promising candidate for the treatment of OA.

Effective separation of protein from Polygonatum cyrtonema crude polysaccharide utilizing ionic liquid tetrabutylammonium bromide.

Extraction of plant polysaccharides often results in a large amount of proteins, which is hard to eliminate from the crude extract, and conventional approaches for deproteinization are time-consuming and often involve hazardous organic solvents. In this study, ionic liquid tetrabutylammonium bromide (TBABr) was used to create an ionic liquid aqueous two-phase system (ILATPS) for the separation of the polysaccharide (PcP) and protein extracted from the rhizome of Polygonatum cyrtonema. Bovine serum albumin (BSA) was first applied to assess the feasibility of the ILATPS, and MgSO4 was determined to be the most suitable inorganic salt. By adopting the Taguchi experiment with an L9 (3^4) orthogonal array, it was found that the best condition for the efficient separation of crude PcP was at 25°C, with 1.5 g of TBABr, 15 mg of PcP, and 2.0 g of MgSO4, with the extraction efficiency for the protein and polysaccharide as 98.6% and 93.5%, respectively. The purified PcP was homogeneous, and its weight average molecular weight (Mw) was 7,554 Da. Monosaccharide composition analysis indicated the PcP comprised mannose, galactose, glucose, galacturonic acid, arabinose, and rhamnose at a molar ratio of 33:13:8:3.5:2:1. This approach offers a practical tactic to purify polysaccharides of plant origin.

Phillyrin restores metabolic disorders in mice fed with high-fat diet through inhibition of interleukin-6-mediated basal lipolysis.

The function of white adipose tissue as an energy reservoir is impaired in obesity, leading to lipid spillover and ectopic lipid deposition. Adipose tissue inflammation can reduce the efficacy of lipid storage in adipocytes by augmenting basal lipolysis through producing interleukin-6 (IL-6). Therefore, pharmacological compounds targeting adipose tissue inflammation or IL-6 signaling might have the potential to combat obesity. This study aims to investigate the impact of Phillyrin, which is frequently used for treating respiratory infections in clinics in China, on obesity-related metabolic dysfunctions. Firstly, a mouse model of diet-induced obesity is used to assess the pharmacological applications of Phillyrin on obesity in vivo. Secondly, ex vivo culture of adipose tissue explants is utilized to investigate actions of Phillyrin on IL-6-linked basal lipolysis. Thirdly, a mouse model of IL-6 injection into visceral adipose tissue is explored to confirm the anti-basal lipolytic effect of Phillyrin against IL-6 in vivo. The results show that Phillyrin treatment reduces circulating level of glycerol, decreases hepatic steatosis and improves insulin sensitivity in obese mice. Meanwhile, Phillyrin attenuates obesity-related inflammation and IL-6 production in adipose tissue in obese mice. Furthermore, Phillyrin treatment results in resistance to IL-6-induced basal lipolysis in adipose tissue through suppressing expression of adipose triglyceride lipase (ATGL) both in vivo and in vitro. Collectively, these findings suggest that Phillyrin can restrain lipid efflux from inflamed adipose tissue in obesity by inhibiting IL-6-initiated basal lipolysis and ATGL expression, and thus is a potential candidate in the treatment of obesity-associated complications.

Remodeling of Macrophages in White Adipose Tissue under the Conditions of Obesity as well as Lipolysis.

Adipose tissue macrophages (ATM) are a major source of low-grade inflammation in obesity, and yet reasons driving ATM accumulation in white adipose tissue (WAT) are not fully understood. Emerging evidence suggested that ATM underwent extensive remodeling in obesity. In addition to abundance, ATM in obesity were lipid-laden and metabolically reprogrammed, which in turn was tightly related to their functional alterations and persistence in obesity. Herein, we aimed to discuss that activation of lipid sensing signaling associated with metabolic reprogramming in ATM was indispensible for their migration, retention, or proliferation in obesity. Likewise, lipolysis also induced similar but transient ATM remodeling. Therefore, we assumed that obesity might share overlapping mechanisms with lipolysis in remodeling ATM. Formation of crown-like structures (CLS) in WAT was presumably a common event initiating ATM remodeling, with a spectrum of lipid metabolites released from adipocytes being potential signaling molecules. Moreover, adipose interlerkin-6 (IL-6) exhibited homologous alterations by obesity and lipolysis. Thus, we postulated a positive feedback loop between ATM and adipocytes via IL-6 signaling backing ATM persistence by comparison of ATM remodeling under obesity and lipolysis. An elucidation of ATM persistence could help to provide novel therapeutic targets for obesity-associated inflammation.

The complete chloroplast genome of Abutilon theophrasti medic (Malvaceae).

Abutilon theophrasti Medic is a traditional Chinese medicine, which can be seen nearly everywhere in China. In order to study its complete chloroplast genome, we collected leaves and obtained chloroplast genome information through next-generation sequencing. It showed that the genome whole length is 160,331 bp, resulted from 24,578,194 raw reads with 3,669,530,829 bases in total, and the GC contents ratio is 36.90%. Besides, the large single-copy region (LSC) is 89,006 bp, the small single-copy region (SSC) 20,149 bp, and inverted repeat (IR) 25,588 bp. The chloroplast genome encodes 76 genes, which contains 38 protein genes, five rRNA genes, and 33 tRNA. By conducting phylogenetic analysis for A.theophrasti, plants from genus Gossypium demonstrated close relationship with it.

The complete chloroplast genome of Viola philippica (Violaceae).

Viola philippica, as a traditional Chinese medicine, has great value in treating various diseases. Here, we report the chloroplast genome of V. philippica and its phylogenetic feature. The complete chloroplast genome is 156,744 bp in length, assembled from 22,346,570 reads, and its GC contents ratio is 36.26%. Its long single-copy (LSC) region is 85,892 bp. The small single-copy (SSC) region covers 18,006 bp and inverted repeat (IR) is 26,423 bp. It encodes 77 genes, including 43 protein genes, 4 rRNA genes, and 30 tRNA genes. Moreover, according to the phylogenetic analysis for a total of 12 chloroplast sequences, V. philippica demonstrated close relationship within genus Viola.