An efficient flavonoid glycosyltransferase NjUGT73B1 from Nardostachys jatamansi of alpine Himalayas discovered by structure-based protein clustering.

Tilianin and linarin, two rare glycosylated flavonoids in the aromatic endangered medicinal plant Nardostachys jatamansi (D.on)DC., play an important role in the fields of medicine, cosmetics, food and dye industries. However, there remains a lack of comprehensive understanding regarding their biosynthetic pathway. In this study, the phytochemical investigation of N. jatamansi resulted in the isolation of linarin. With help of AlphaFold2 to cluster the entire glycosyltransferase family based on predicted structure similarities, we successfully identified a flavonoid glycosyltransferase NjUGT73B1, which could efficiently catalyze the glucosylation of acacetin at 7-OH to produce tilianin, also the key precursor in the biosynthesis of linarin. Additionally, NjUGT73B1 displayed a high degree of substrate promiscuity, enabling glucosylation at 7-OH of many flavonoids. Molecular modeling and site-directed mutagenesis revealed that H19, H21, H370, F126, and F127 play the crucial roles in the glycosylation ability of NjUGT73B1. Notably, comparation with the wild NjUGT73B1, mutant H19K led to a 50% increase in the activity of producing tilianin from acacetin.

Selaginellin derivatives from Selaginella tamariscina and evaluation for anti-breast cancer activity.

A phytochemical investigation of Selaginella tamariscina led to the isolation of 17 selaginellin derivatives. Their inhibitory activities against breast cancer cells were screened, and preliminary structure-activity relationships were also established. Among them, dimeric selaginellin 17 showed potential activity against MDA-MB-231 cells with an IC50 value of 3.2 ± 0.1 µM, corresponding to 4-fold higher potency than the reference compound 5-FU (IC50 14.8 ± 0.2 µM). Mechanistic studies indicated that 17 could cause G2/M phase arrest in MDA-MB-231 cells and induce apoptosis accompanied by increased ROS levels.

[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

[Main components from cultivated and wild Nardostachyos Radix et Rhizoma by LC-MS and GC-MS].

In this study, ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry(UPLC-Q-TOF-MS) and gas chromatography-mass spectrometry(GC-MS) were combined with non-targeted metabonomic analysis based on multivariate statistics analysis, and the content of five indicative components in nardosinone was determined and compared by UPLC. The main chemical components of Nardostachyos Radix et Rhizoma with imitative wild cultivation and wild Nardostachyos Radix et Rhizoma were comprehensively analyzed. The results of multivariate statistical analysis based on liquid chromatography-mass spectrometry(LC-MS) and GC-MS were consistent. G1 and G2 of the imitative wild cultivation group and G8-G19 of the wild group were clustered into category 1, while G7 of the wild group and G3-G6 of the imitative wild cultivation group were clustered into category 2. After removing the outlier data of G1, G2, and G7, G3-G6 of the imitative wild cultivation group were clustered into one category, and G8-G19 of the wild group were clustered into the other category. Twenty-six chemical components were identified according to the positive and negative ion modes detected by LC-MS. The content of five indicative components(VIP>1.5) was determined using UPLC, revealing that chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, linarin, nardosinone, and total content in the imitative wild cultivation group were 1.85, 1.52, 1.26, 0.90, 2.93, and 2.56 times those in the wild group, respectively. OPLS-DA based on GC-MS obtained 10 diffe-rential peaks. Among them, the relative content of α-humulene and aristolene in the imitative wild cultivation group were extremely significantly(P<0.01) and significantly(P<0.05) higher than that in the wild group, while the relative content of 7 components such as 5,6-epoxy-3-hydroxy-7-megastigmen-9-one, γ-eudesmol, and juniper camphor and 12-isopropyl-1,5,9-trimethyl-4,8,13-cyclotetrade-catriene-1,3-diol was extremely significantly(P<0.01) and significantly(P<0.05) lower than that in the wild group, respectively. Therefore, the main chemical components of the imitative wild cultivation group and wild group were basically the same. However, the content of non-volatile components in the imitative wild cultivation group was higher than that in the wild group, and the content of some volatile components was opposite. This study provides scientific data for the comprehensive evaluation of the quality of Nardostachyos Radix et Rhizoma with imitative wild cultivation and wild Nardostachyos Radix et Rhizoma.

Illuminating the biosynthesis pathway genes involved in bioactive specific monoterpene glycosides in Paeonia veitchii Lynch by a combination of sequencing platforms.

BACKGROUND: Paeonia veitchii Lynch, a well-known herb from the Qinghai-Tibet Plateau south of the Himalayas, can synthesize specific monoterpene glycosides (PMGs) with multiple pharmacological activities, and its rhizome has become an indispensable ingredient in many clinical drugs. However, little is known about the molecular background of P. veitchii, especially the genes involved in the biosynthetic pathway of PMGs. RESULTS: A corrective full-length transcriptome with 30,827 unigenes was generated by combining next-generation sequencing (NGS) and single-molecule real-time sequencing (SMRT) of six tissues (leaf, stem, petal, ovary, phloem and xylem). The enzymes terpene synthase (TPS), cytochrome P450 (CYP), UDP-glycosyltransferase (UGT), and BAHD acyltransferase, which participate in the biosynthesis of PMGs, were systematically characterized, and their functions related to PMG biosynthesis were analysed. With further insight into TPSs, CYPs, UGTs and BAHDs involved in PMG biosynthesis, the weighted gene coexpression network analysis (WGCNA) method was used to identify the relationships between these genes and PMGs. Finally, 8 TPSs, 22 CYPs, 7 UGTs, and 2 BAHD genes were obtained, and these putative genes were very likely to be involved in the biosynthesis of PMGs. In addition, the expression patterns of the putative genes and the accumulation of PMGs in tissues suggested that all tissues are capable of biosynthesizing PMGs and that aerial plant parts could also be used to extract PMGs. CONCLUSION: We generated a large-scale transcriptome database across the major tissues in P. veitchii, providing valuable support for further research investigating P. veitchii and understanding the genetic information of plants from the Qinghai-Tibet Plateau. TPSs, CYPs, UGTs and BAHDs further contribute to a better understanding of the biology and complexity of PMGs in P. veitchii. Our study will help reveal the mechanisms underlying the biosynthesis pathway of these specific monoterpene glycosides and aid in the comprehensive utilization of this multifunctional plant.

Metabolome and transcriptome associated analysis of sesquiterpenoid metabolism in Nardostachys jatamansi.

Background: Nardostachys jatamansi, an extremely endangered valuable plant of the alpine Himalayas, can synthesize specific sesquiterpenoids with multiple effective therapies and is widely exploited for the preparation of drugs, cosmetics and even religious functions (e.g., well-known spikenard). However, how accumulation trend of the sesquiterpenoids in tissues and the molecular mechanisms underlying the production of the active ingredients are not well understood. Methods: The single-molecule real-time (SMRT) and RNA-seq transcriptome sequencing were combined to analyse the roots, rhizomes, leaves, flowers and anthocaulus of N. jatamansi. The phytochemical analysis was performed by gas chromatography‒mass spectrometry (GC‒MS) and ultrahigh-performance liquid chromatography (UPLC). Results: A high-quality full-length reference transcriptome with 26,503 unigenes was generated for the first time. For volatile components, a total of sixty-five compounds were successfully identified, including fifty sesquiterpenoids. Their accumulation levels in five tissues were significantly varied, and most of the sesquiterpenoids were mainly enriched in roots and rhizomes. In addition, five aromatic compounds were only detected in flowers, which may help the plant attract insects for pollination. For nonvolatile ingredients, nardosinone-type sesquiterpenoids (nardosinone, kanshone C, and isonardosinone) were detected almost exclusively in roots and rhizomes. The candidate genes associated with sesquiterpenoid biosynthesis were identified by transcriptome analysis. Consistently, it was found that most biosynthesis genes were abundantly expressed in the roots and rhizomes according to the functional enrichment and expression patterns results. There was a positive correlation between the expression profile of genes related to the biosynthesis and the accumulation level of sesquiterpenoids in tissues. Gene family function analysis identified 28 NjTPSs and 43 NjCYPs that may be involved in the biosynthesis of the corresponding sesquiterpenoids. Furthermore, gene family functional analysis and gene coexpression network analysis revealed 28 NjTPSs and 43 NjCYPs associated with nardosinone-type sesquiterpenoid biosynthesis. Conclusion: Our research results reveal the framework of sesquiterpenoids accumulation and biosynthesis in plant tissues and provide valuable support for further studies to elucidate the molecular mechanisms of sesquiterpenoid regulation and accumulation in N. jatamansi and will also contribute to the comprehensive utilization of this alpine plant.

[Research progress on chemical constituents,pharmacological activities,and quality control of Patrinia villosa].

Patrinia villosa, regarding its functions in clearing heat and detoxification and eliminating carbuncles and pus, is widely used as a traditional medicinal herb that contains rich nutrition and substances such as various amino acids, vitamins, and soluble su-gar, and it is also an edible wild herb in Chinese folk tradition for 2 000 years. In 1973, Japanese scholars firstly separated three iridoids from Japanese P. villosa, and by 2021, chemical components such as flavonoids, iridoids, organic acids, triterpenoids, phenylpropanoids, and steroids have been found, which have multiple pharmacological effects, including antioxidant, antitumor, anti-diarrhea, antibacterial, sedative, and liver protection capabilities. Studies indicate that flavonoids, saponins, phenylpropanoids, and triterpenoids in P. villosa are vital substances for its pharmacological activities. However, the quality of this medicinal material cannot be controlled due to the unclear records in ancient books in the past dynasties and different drug use habits in different places, and thus its circulation is chaotic. At present, researchers have used flavonoids, organic acids, phenylpropanoids, triterpenoid saponins, and other compounds to conduct studies in this regard. Therefore, on the basis of the existing literature resources, we comprehensively summarize the chemical constituents, pharmacological activities, and quality control of P. villosa to further provide a reference for the safety and effectiveness of clinical drug use and lay a foundation for the follow-up experimental research.

[A new cyclopeptide from Selaginella tamariscina].

One new cyclopeptide was isolated from the ethyl acetate fraction of the 75% EtOH extract of Selaginella tamariscina by various column chromatography methods(HP-20, polyamide and semi-preparative HPLC). Its structure was identified as selapeptin A(1) by extensive spectroscopic analysis(HR-ESI-MS, 1 D and 2 D NMR). Compound 1 was evaluated for cytotoxic activities by MTT assay. It showed potent cytotoxic activity against B16 F10 with the inhibition rate of 51.57%±4.34% at 40 µmol·L~(-1) while had no impacts on MDA-MB-231 and MDA-MB-468 at 100 µmol·L~(-1).

[One new phenylethanoid glycoside from Forsythiae Fructus].

One new phenylethanoid glycoside was isolated from the ethyl acetate fraction of the 75% EtOH extract of Forsythiae Fructus by various column chromatographies(HP20, silica gel, ODS) and preparative HPLC.Its structure was identified as forsythiayanoside E(1) by physicochemical properties and extensive spectroscopic analysis(HR-ESI-MS, 1 D and 2 D NMR).Compound 1 was evaluated for cytotoxic activities by MTT assay and showed weak cytotoxic activity against MCF-7 and A-375 cell lines with inhibition rates of 39.85% and 43.38% at 40 µmol·L~(-1), and no cytotoxic activity against PC-3 and HepG2 cell lines at 100 µmol·L~(-1).

A new cyclic peptide from Selaginella tamariscina.

A new cyclic peptide selapeptin B (1), together with one known nor-lignan glycoside moellenoside C (2), was isolated from Selaginella tamariscina. The structures of 1 and 2 were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses and HRESIMS. Two compounds were evaluated for cytotoxic activities against B16F10, MDA-MB-231, and MDA-MB-468 cell lines by MTT assay. Compound 1 showed the potent activity against B16F10 melanoma cell lines.

Two new flavonol glycosides from Selaginella tamariscina.

Two new flavonol glycosides 3,5,7-trimethoxyflavone-4'-O-[5'''-O-p-coumaroyl-ß-D-apiofuranoyl-(1'''→2'')-ß-D-glucopyranoside] (1) and 3,5,7-trimethoxyflavone -4'-O-ß-D-glucopyranoside (2) were isolated from Selaginella tamariscina. The structures of 1 and 2 were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses and HRESIMS spectrometry. Two compounds were evaluated for cytotoxic activities against A-375, MCF-7, MDA-MB-231 and MDA-MB-468 cell lines by MTT assay. Unfortunately, two compounds displayed no cytotoxic activities.

A new diterpernoid glycoside from the fruit of Forsythia suspensa.

One new diterpernoid glycoside, lanceolatinoside A (1), together with one known iridoid glycoside, luzonoside C (2) were isolated from the fruit of Forsythia suspensa. The structure of the new compound (1) was elucidated through 1 D and 2 D NMR spectroscopic data, and HR-ESIMS. Compound 2 was identified as luzonoside C (2) on the basis of NMR spectroscopic data analyses and comparison with those reported in the literature.

[Chemical constituents of phenylethanoid glycosides from Forsythiae Fructus and their antitumor activities].

Four phenylethanoid glycosides were isolated from the 75% EtOH extract of Forsythiae Fructus by various column chromatography methods(MCI, silica gel, ODS and semi-preparative HPLC). Their structures were identified as forsythenside M(1), forsythenside K(2), forsythoside I(3) and forsythoside A(4) by physicochemical properties and extensive spectroscopic analysis(UV, 1 D and 2 D NMR, HR-ESI-MS). Among them, compound 1 was one new phenylethanoid glycoside. The in vitro cytotoxic activities of these compounds against MCF-7, A-375, SGC-7901 and B16 F10 were evaluated. The results showed that compounds 1-4 had cytotoxic activities against MCF-7, A-375, SGC-7901 and B16 F10 at 40 µmol·L~(-1).

Two new selariscinins from Selaginella tamariscina (Beauv.) Spring.

Two new selariscinins named selariscinin F (1) and selariscinin G (2), along with one known selariscinin D (3) were isolated from Selaginella tamariscina. The structures of 1-3 were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses and HRESIMS.

Current Scenario of 1,3-oxazole Derivatives for Anticancer Activity.

Cancer, which has been cursed for human beings for long time is considered as one of the leading causes of morbidity and mortality across the world. In spite of different types of treatments available, chemotherapy is still deemed as a favored treatment for the cancer. Unfortunately, many currently accessible anticancer agents have developed multidrug resistance along with fatal adverse effects. Therefore, intensive efforts have been made to seek for new active drugs with improved anticancer efficacy and reduced adverse effects. In recent years, the emergence of heterocyclic ring-containing anticancer agents has gained a great deal of attention among medicinal chemists. 1,3- oxazole is a versatile heterocyclic compound, and its derivatives possess broad-spectrum pharmacological properties, including anticancer activity against both drug-susceptible, drug-resistant and even multidrug-resistant cancer cell lines through multiple mechanisms. Thus, the 1,3-oxazole moiety is a useful template for the development of novel anticancer agents. This review will provide a comprehensive overview of the recent advances on 1,3-oxazole derivatives with potential therapeutic applications as anticancer agents, focus on the chemical structures, anticancer activity, and mechanisms of action.

Two new furofuran lignan glycoside derivatives from the fruit of Forsythia suspensa.

Phytochemical investigation of 95% ethanol extract of the fruit of Forsythia suspensa resulted in the isolation of two new furofuran lignan glycoside derivatives pinoresinoside A (1) and phillyrigeninside A (2), along with three known ones. Their structures were established based on extensive spectroscopic data analyses and comparison with literature data. Absolute configuration of 1 was determined by CD method. In addition, compounds 1 and 2 were revealed to show in vitro cytotoxicity against human tumor cell lines (SGC-7901, MCF-7 and HepG2), with IC50 values ranging from 16.77 to 37.35 µM. [Formula: see text].

Comparative Pharmacokinetic Studies of Four Ginsenosides in Rat Plasma by UPLC-MS/MS after Oral Administration of Panax quinquefolius-Acorus gramineus and Panax quinquefolius Extracts.

Panax quinquefolius (PQ) and Acorus gramineus (AG) are drug target pairs in traditional Chinese medicine (TCM), which are used to treat age-related diseases. In the present study, we simultaneously determined the contents of four main bioactive ginsenosides (Rb1, Rb2, Rd, and Re) in rat plasma using an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Plasma specimens were purified by using the solid-phase extraction procedure, and separation was performed on Waters ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 µm) in multiple reaction monitoring (MRM) mode and negative electrospray ionization (ESI) mode. The established UPLC-MS/MS method showed good linear correlation (r ≥ 0.9978), stability (-11.93 to 12.11%), precision (RSD < 14.63%), and recovery (76.43%-95.20%). The lower limit of quantification was 3.6 ng/mL for Rb1, 1.6 ng/mL for Rb2, 1.2 ng/mL for Rd, and 2.5 ng/mL for Re. This validated method was successfully employed to investigate the pharmacokinetics of the four ginsenosides in rat plasma after oral administration of PQ-AG and PQ extracts. The results revealed the pharmacokinetic profiles of PQ-AG drug pair and clarified that AG played a critical role in stimulating the absorption of active ginsenosides in PQ. Collectively, our findings provided valid and reliable evidence for the rational use of PQ-AG in clinical practice.

Dihydroisocoumarins from Radix Glycyrrhizae.

BACKGROUND: Radix Glycyrrhizae is the rhizome of Glycyrrhiza inflata Bat., Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L. The present paper describes the isolation and the structural elucidation of three new dihydroisocoumarins obtained from the 70% EtOH extract of Radix Glycyrrhizae. And the cytotoxic activities of these new compounds were also evaluated using four cell lines, subsequently. RESULTS: A pair of new dihydroisocoumarin epimers ((3R,4S)-4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (1) and ((3R,4R)-4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (2) along with a new dihydroisocoumarin (3R,4R)-4,8-dihydroxy-3,5-dimethylisochroman-1-one (3) were isolated from Radix Glycyrrhizae. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses, HR-ESI-MSand ECD calculation comparing with those of experimental CD spectra. Cytotoxic activities of the three compounds were evaluated using the HepG2, A549, LoVo and Hela cell lines, respectively. IC50 values indicated compounds 1-3 exhibited moderate or less cytotoxic activity in vitro. CONCLUSIONS: Dihydroisocoumarin is not the common components in Radix Glycyrrhizae, a series of dihydroisocoumarin were obtained in this plant could be a supplement to the chemical study of this plant.

Synthesis and in vitro evaluation of novel substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids for their anti-mycobacterial activities.

Twelve novel substituted isatin-propylene-1H-1,2,3-triazole-4-methylene-moxifloxacin hybrids 5a-l were designed, synthesized and screened for their in vitro anti-mycobacterial activities against drug-sensitive and multidrug-resistant Mycobacterium tuberculosis as well as cytotoxicity in VERO cell line. All hybrids exhibited excellent activities against two Mycobacterium tuberculosis strains with minimum inhibitory concentration in the range from 0.05 to 2.0 µg/mL. The most active hybrid 5i was 2-8 times more potent than the reference agents (moxifloxacin and rifampicin) in vitro against Mycobacterium tuberculosis H37Rv, while 2->2048 times more potent than the reference agents (moxifloxacin, rifampicin and isoniazid) in vitro against multidrug-resistant Mycobacterium tuberculosis. However, all hybrids (the 50% cytotoxic concentration/CC50: 2-32 µg/mL) were much more cytotoxic than the parent moxifloxacin (CC50: 128 µg/mL) against VERO cell line. Therefore, our further optimization will focus on their cytotoxicity reducing as well as activity enhancing. The structure-activity relationship of 1H-1,2,3-triazole-tethered isatin-fluoroquinolone hybrids was investigated, and the results could promote further development of the anti-tuberculosis properties of this kind of hybrids.

Four novel bisabolane-type sesquiterpenes from Curcuma longa.

Four novel bisabolane sesquiterpenes, turmerone A (1), turmerone B (2), turmerone C (3), and turmerone D (4), were isolated from the rhizomes of Curcuma longa. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses and HRESIMS.