[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.

[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).

[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).

Iridoid, phenylethanoid and flavonoid glycosides from Forsythia suspensa.

As part of our continuing efforts to explore bioactive compounds from natural resources, a new iridoid glycoside, adoxosidic acid-6'-oleuroperic ester (1), together with one known phenylethanoid glycoside (2) and two known flavonoid glycosides (3-4) were isolated from the fruit of Forsythia suspensa. The structure of the new compound (1) was elucidated through 1D and 2D NMR spectroscopic data and HR-ESIMS. Interestingly, compound 1 was a monoterpene ester of one iridoid glycoside. Compounds 2-4 were identified as calceolarioside A (2), kaempferol-3-O-rutinoside (3), kampferol-3-O-robinobioside (4) on the basis of NMR spectroscopic data analyses and comparison with the data reported in the literature. The antiviral activity aganisist influenza A (H5N1) virus of compound 1 was studied as well.

Two new phenolic acids from the fruits of Forsythia suspense.

Two new phenolic acids forsythiayanoside C (1) and forsythiayanoside D (2), were isolated from the fruits of Forsythia suspense (Thunb.) Vahl. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses, HRESIMS and CD spectrometry. The cytotoxic and antioxidant activities testing showed that compound 2 exhibited free radical scavenging activity.

A new cyclolignan glycoside from the tubers of Pinellia ternata.

A new 2,7'-type cyclolignan glycoside, cyclolignanyingoside A (1), together with six known compounds (2-7) were isolated from the tubers of Pinellia ternata (Thunb.) Breit. The structure of 1 was elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses, HR-ESI-MS, and CD spectrometry. The cytotoxic, antioxidant and tyrosinase-inhibiting activities of all the isolates were determined. However, all the isolates exhibited no activity on the selected cell lines (Hep-3B, Bcap-37, and MCF-7). In addition, compounds 1-3 and 7 exhibited strong 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) free radical scavenging activity, and compounds 2 and 4 showed a moderate mushroom tyrsinase inhibitory activity.

Antioxidant and anti-inflammatory active dihydrobenzofuran neolignans from the seeds of Prunus tomentosa.

Prunus tomentosa seeds were researched for antioxidant and anti-inflammatory constituents. By activity-guided fractionation of P. tomentosa seed extract, six new dihydrobenzofuran neolignans, prunustosanans AI-IV (1-4) and prunustosanansides AI and AII (5 and 6), together with 10 known compounds (7-16) were isolated from bioactive fraction. The structures were determined by spectroscopic analyses, especially NMR, HRESIMS, and CD spectra. The antioxidant activity was greatest for 5, 10, and 12 against DPPH radical and for 8, 9, and 13 against ABTS radical. Moreover, compounds 7 and 11 exhibited much stronger inhibitory activity on nitric oxide (NO) production in murine microglia BV-2 compared with positive control minocycline (IC50 = 19.7 ± 1.5 µM). The results show that P. tomentosa seeds can be regarded as a potential source of antioxidants and inflammation inhibitors.

Three new lignan glycosides from the fruits of Forsythia suspense.

Two new lignan glycosides forsythiayanoside A (1) and forsythiayanoside B (2), one new stereoisomer of lignan glycoside (+)-8-hydroxyepipinoresinol-4-O-ß-D-glucopyranoside (3), together with seven known compounds (4-10), were isolated from the fruits of Forsythia suspense (Thunb.) Vahl. Compounds 4 and 10 were isolated from this species for the first time. The structures of 1-3 were elucidated on the basis of chemical and spectral analyses, including 1D and 2D NMR data and HR-ESI-MS. The absolute configurations were determined by the circular dichroism method. All isolates were tested for their cytotoxicities against five human cancer cell lines (A549, Colo-205, Hep-3B, HL60, and KB). In particular, compound 3 showed significant cytotoxicity with IC50 values of 9.48, 7.75, 0.59, 4.06, and 38.38 µM, respectively.

Two new glycosides from the fruits of Forsythia suspense.

Two new glycosides suspensaside C (1) and 2,3,5,6-tetrahydro-jacaranone-4-O-ß-D-glucopyranoside (2), together with four known compounds suspensaside A (3), rengynic acid-1'-O-ß-D-glucopyranoside (4), forsythoside A (5), and rengynic acid (6), were isolated from the fruits of Forsythia suspense (Thunb.) Vahl. The structures of 1 and 2 were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses and HR-ESI-MS. All isolates were tested for their cytotoxicities against five human cancer cell lines (A549, Colo-205, Hep-3B, HL60, and KB). Compound 3 exhibited cytotoxicity against HL-60, Hep-3B, and A549 cancer cell lines.