Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria.

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-D-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

[Effect of polysaccharides from seeds of Vaccaria segetalis in alleviating urinary tract infection induced bladder injury by inhibiting NLRP3 inflammasome].

To study the mechanism of polysaccharides from seeds of Vaccaria segetalis( PSV) in the treatment of bacterial cystitis through the NLRP3 inflammasome pathway. The rat model of urinary tract infection was used and treated with PSV,and the urine and bladders were collected. The level of interleukin-10( IL-10) in rat urine was detected by enzyme linked immunosorbent assay( ELISA). Western blot and immunofluorescence staining were used to detect the expressions of sonic hedgehog( SHH) and NLRP3 inflammasome [NOD-like receptor thermoprotein domain 3( NLRP3),apoptosis associated speck like protein( ASC) and pro-caspase-1]. The expression of Toll-like receptor pathway was detected by RT-PCR. The death of 5637 cells induced by uropathogenic Escherichia coli( UPEC) and lactate dehydrogenase( LDH) release were evaluated using live/dead staining. The results showed that in the rat bladder,the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors were significantly up-regulated,and NLRP3 inflammasomes were significantly activated by UPEC infection. The administration with PSV could significantly increase the concentration of IL-10 in urine,inhibit the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors in bladder,and inhibit the activation of NLRP3 inflammasomes. A large number of 5637 cells were dead after UPEC infection and caused LDH production. PSV could significantly inhibit the death of 5637 cells and the release of LDH. In conclusion,PSV could inhibit the expression and activation of NLRP3 inflammasomes by inhibiting the Toll-like receptor pathway,thereby mitigating the bladder injury.

Natural cyclic polypeptides as vital phytochemical constituents from seeds of selected medicinal plants.

Cyclopolypeptides are among the most predominant biomolecules in nature, especially those derived from plant seeds. This category of compounds has gained extraordinary attention due to remarkable variety of structures and valuable biofunctions. These congeners display enormous variation in terms of both structure and function and are the most significant biomolecules due to their widespread bioproperties. The estrogenic activity, immunosuppressive activity, cytotoxicity, vasorelaxant activity, and other properties possessed by cyclic peptides from seeds of plants make these congeners attractive leads for the drug discovery process. The current study covers the important structural features, structure-activity relationship, synthesis methods, and bioproperties of plant seeds-originated bioactive peptides from Vaccaria segetalis, Linum usitatissimum, and Goniothalamus leiocarpus, which may prove vital for the development of novel therapeutics based on a peptide skeleton.

Polysaccharides extract from Vaccaria segetalis seeds inhibits kidney infection by regulating cathelicidin expression.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Chinese Pharmacopoeia, the seeds of Vaccaria segetalis, a traditional medicinal herb, can be used for treating urinary diseases. The polysaccharides extract from V. segetalis seeds (VSP) has been shown to prevent urinary tract infections (UTIs). AIM OF THE STUDY: Investigate the effects of VSP on treating kidney infection induced by uropathogenic Escherichia coli (UPEC) and the underlying mechanisms. MATERIALS AND METHODS: Both in vivo and in vitro infection models were established with the UPEC strain CFT073. After oral administration of VSP, the levels of bacterial load, cathelicidin (CRAMP), Toll-like receptors (TLRs) in the kidney were evaluated. The expression of cathelicidin (LL-37) in human renal cell carcinoma cell line (A498) was tested after the treatment of VSP. RESULTS: In the kidneys of infection models, high-titer bacteria was detected. In the kidney of rat model, the expression of CRAMP was down-regulated, no significant change was observed in the levels of TLRs. After oral administration of VSP, the bacterial load was significantly decreased in rat and mouse models, and the levels of CRAMP and TLRs were significantly up-regulated in rat model. In vitro, the expression of LL-37 was significantly inhibited by CFT073. VSP up-regulated the expression of LL-37 in A498 cells. CONCLUSIONS: The up-regulation of cathelicidin expression may contribute to the therapeutic effects of VSP on kidney infection.

Vaccaria n-Butanol Extract Lower the Production of Proinflammatory Cytokines and the Infection Risk of T. spiralis In Vivo.

INTRODUCTION: Trichinellosis is a severe zoonosis involving the activation of inflammatory cells, accompanied by the prominent expressions of proinflammatory cytokines in the host. Semen vaccariae, the seeds of Vaccaria segetalis (Neck.) Garcke. ex Asch. (Caryophyllaceae), is a famous traditional herb that is rich in vaccaria n-butanol extract (VNE). Vaccarin is one major active component of VNE, and it is reported in the treatment of stranguria disease. Hypaphorine is another main active component of VNE and has good anti-inflammatory effect, whereas the potential bioactivity of VNE in trichinellosis treatment is still unknown. MATERIALS AND METHODS: This study was designed to evaluate the potential anthelmintic and anti-inflammatory activity of VNE toward T. spiralis infection. ICR mice were used to assess the effect of VNE on repression larvae and adult worms in vivo. Immunohistochemistry analysis was performed to evaluate the expression levels of IL-1ß, IL-6, TNF-α, and COX-2. RESULTS: Our results showed that VNE could effectively depress the expressions of proinflammatory cytokines, including IL-1ß, IL-6, TNF-α, and COX-2. The adult worms were decreased by 79.53%, while the muscle larvae were diminished by 77.70% as compared to the control. CONCLUSION: These results demonstrated that VNE may be a promising therapeutic agent against the inflammation and diseases caused by T. spiralis infection.

Vaccaria hypaphorine impairs RANKL-induced osteoclastogenesis by inhibition of ERK, p38, JNK and NF-κB pathway and prevents inflammatory bone loss in mice.

Osteoclasts are sole bone-resorbing cells which exert a profound effect on skeletal metabolism. The search for medicines that affect the differentiation and function of osteoclasts is crucial in developing therapies for osteoclast-based diseases. Vaccaria hypaphorine, the main active compound of the traditionally used Chinese herb Vaccaria segetalis, has anti-inflammatory activity. The present study demonstrated for the first time that vaccaria hypaphorine could significantly inhibit the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation in vitro and alleviate lipopolysaccharide (LPS)-induced bone loss in vivo. Further study showed that vaccaria hypaphorine decreased osteoclastogenesis in a dose-dependent manner. Furthermore, vaccaria hypaphorine was confirmed to inhibit osteoclasts differentiation at early stage but not at later stage. Pit formation assay and F-actin ring staining showed that vaccaria hypaphorine inhibited the bone-resorbing activity of osteoclasts. Mechanistically, vaccaria hypaphorine impaired RANKL-induced osteoclastogenesis through reduction of extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and NF-κB p65 phosphorylation. Taken together, our results provided evidences that vaccaria hypaphorine might be considered as potential therapeutic agent for treating osteoclast-based bone loss.

Interactions of TLR4 and PPARγ, Dependent on AMPK Signalling Pathway Contribute to Anti-Inflammatory Effects of Vaccariae Hypaphorine in Endothelial Cells.

Background /Aims: Accumulating evidence indicates that endothelial inflammation is one of the critical determinants in pathogenesis of atherosclerotic cardiovascular disease. Our previous studies had demonstrated that Vaccariae prevented high glucose or oxidative stress-triggered endothelial dysfunction in vitro. Very little is known about the potential effects of hypaphorine from Vaccariae seed on inflammatory response in endothelial cells. METHODS: In the present study, we evaluated the anti-inflammatory effects of Vaccariae hypaphorine (VH) on lipopolysaccharide (LPS)-challenged endothelial EA.hy926 cells. The inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), monocyte chemoattractant protein 1 (MCP-1) and vascular cellular adhesion molecule-1 (VCAM-1) were measured by real-time PCR (RT-PCR). The expressions of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), toll-like receptor 4 (TLR4), peroxisome proliferator-activated receptor γ (PPARγ) were detected by Western blotting or immunofluorescence. RESULTS: We showed that LPS stimulated the expressions of TNF-α, IL-1ß, MCP-1, VCAM-1 and TLR4, but attenuated the phosphorylation of AMPK and ACC as well as PPARγ protein levels, which were reversed by VH pretreatment. Moreover, we observed that LPS-upregulated TLR4 protein expressions were inhibited by PPARγ agonist pioglitazone, and the downregulated PPARγ expressions in response to LPS were partially restored by knockdown of TLR4. The negative regulation loop between TLR4 and PPARγ response to LPS was modulated by AMPK agonist AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) or A769662. CONCLUSIONS: Taken together, our results suggested that VH ameliorated LPS-induced inflammatory cytokines production in endothelial cells via inhibition of TLR4 and activation of PPARγ, dependent on AMPK signalling pathway.

Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NFκB and ERK pathways in Raw 264.7 cells.

BACKGROUND: Activation of macrophage is involved in many inflammation diseases. Lipopolysaccharide (LPS) is a powerful inflammatory signal contributing to monocytes/macrophages activation associated with increased proinflammatory cytokines expressions. We recently identified that vaccarin was expected to protect endothelial cells from injury. Hypaphorine was abundantly found in vaccaria semen. However, the potential roles and underlying mechanisms of vaccaria hypaphorine on macrophage inflammation have been poorly defined. METHODS: This study was designed to determine the effects of vaccaria hypaphorine on LPS-mediated inflammation in RAW 264.7 cells. RESULTS: In this study, we demonstrated that vaccaria hypaphorine dramatically ameliorated LPS-induced nitric oxide (NO) release and productions of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1) and prostaglandin E2 (PGE2) in RAW 264.7 cells. LPS-stimulated expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were down-regulated by vaccaria hypaphorine. Furthermore, vaccaria hypaphorine retarded LPS-induced phosphorylation of ERK, nuclear factor kappa beta (NFκB), NFκB inhibitor IκBα, and IKKß. Immunofluorescence staining revealed that vaccaria hypaphorine eliminated the nuclear translocation of NFκB in LPS-treated RAW 264.7 cells. CONCLUSION: It was seen that vaccaria hypaphorine counteracted inflammation via inhibition of ERK or/and NFκB signaling pathways. Collectively, we concluded that vaccaria hypaphorine can be served as an anti-inflammatory candidate.

Metabolism and tissue distribution study of Vaccaria seeds (Wang-Bu-Liu-Xing) in benign prostatic hyperplasia model rat: toward an in-depth study for its bioactive components.

Vaccaria seeds (Wang-Bu-Liu-Xing), a well-known traditional Chinese medicine (TCM), has been used as an emperor herb of many ancient formulas to treat benign prostatic hyperplasia (BPH) in clinic. However, its metabolism and tissue distribution, especially in the target tissue, had not been investigated so far. Based on the hypothesis that the components which exert effect against BPH of Vaccaria seeds would be measureable in target tissue (prostate), in vivo metabolism and tissue distribution of Vaccaria seeds in rats were profiled using a specific and sensitive high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS). As a result, 19 major constituents in the Vaccaria seeds decoction and 19 constituents in rat plasma, feces and tissues after oral administration of Vaccaria seeds decoction were identified. Accurate mass measurement for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds. Two prototypes were detected in prostate. An in vitro metabolism analysis of them was studied after incubation with rat intestinal flora and rat liver microsome (RLM) in this paper, which is helpful for further investigation of the potential effect of these two components. The result of this study provided meaningful information for further pharmacology research on Vaccaria seeds.

Inhibitory effects of crude polysaccharides from Semen vaccariae on benign prostatic hyperplasia in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen vaccariae, the seeds of Vaccaria Segatalis, is a famous traditional herb for the treatment of stranguria disease, e.g. benign prostatic hyperplasia (BPH). The present study investigated the effect of crude polysaccharide from Semen vaccariae (SVCP) on BPH in mice in order to further understand the efficacy substance of this medicinal plant. MATERIALS AND METHODS: Sixty healthy adult male Kunming mice were randomly divided into five groups with 12 mice for each group: sham-operated group, BPH model group; BPH model group treated with Pule'an at a dose of 1.20 g/kg; BPH model group treated with SVCP at dose levels of 1.67 g/kg and 0.42 g/kg. The drugs were administered orally once a day consecutively for 12 days. The BPH in mice was created by subcutaneous injection of testosterone propionate for 5 mg/(kgd) to the uncastrated mice once a day consecutively for 12 days. The inhibitory effects on BPH of SVCP were evaluated by prostatic index, testicular index and histopathologic examination. RESULTS: Comparing with BPH model group, BPH mice fed with SVCP exhibited significant differences in both the prostatic index and testicular index. On the aspect of histopathology, the SVCP treated BPH mice exhibited similar histological aspects observed in the mice of sham group while the BPH mice exhibited typical features of prostate glandular hyperplasia. CONCLUSIONS: SVCP exhibited a significant inhibitory activity on BPH by reducing the prostatic index, testicular index, and ameliorating the pathomorphology. The results indicated that the polysaccharide should be the main efficacy substance of Semen vaccariae and may contribute, in a large part, to Semen vaccariae's traditional medicinal use for the treatment of stranguria disease.

[Study on antiangiogenesis effect of Vaccaria segetalis].

OBJECTIVE: To study the inhibiting effect of Vaccaria segetalis extracts on neovascularization. METHODS: The effect of Vaccaria segetalis extracts on the proliferation, migration in vitro and tube formation on Matrigel of endothelial cell (HMEC) in vivo were examined by MTT assay and Matrigel plug assay. RESULTS: The proliferation and migration of HMEC were inhibited significantly by Vaccaria segetalis extracts in a dose-dependent manner (IC50 = 50 microg/mL). It also inhibited angiogenesis in Matrigel plug mouse model. CONCLUSION: Vaccaria segetalis extracts can inhibit angiogenensis obviously, and it could be developed as an effective antiangiogenic drug.

Ameliorative effects of Vaccaria segetalis extract on osteopenia in ovariectomized rats.

The purpose of this study was to determine the ameliorative effects of a crude extract of Vaccaria segetalis (Neck.) Garcke (Caryophyllaceae) (VSE) on osteopenia in ovariectomized (OVX) rats over 12 weeks. Rats were divided into the sham and OVX groups. The OVX rats were allowed to lose bone for 6 weeks. At 6 weeks post-OVX, the OVX rats were divided into four groups treated with water, 17beta-estradiol (30 microg/kg, daily subcutaneous injection), or VSE (0.5 or 1.0 g/kg, daily, orally) for 6 weeks. In OVX rats, the increases of serum total cholesterol were significantly decreased by VSE or 17beta-estradiol treatment. There were decreases in bone density and calcium content, including the left femur and the fourth lumbar vertebra, when compared with the sham control rats. Treatment with 17beta-estradiol or VSE ameliorated these changes induced by OVX. In addition, ovariectomy increased urinary deoxypyridinoline (DPD) amounts (P < 0.001). The increases were suppressed by 17beta-estradiol and 0.5 or 1.0 g/kg VSE (P < 0.01, P < 0.05, P < 0.01, respectively). Our results demonstrated that VSE ameliorates ovariectomy-induced osteopenia by inhibition of bone resorption.

Cytotoxic triterpenoid saponins from Vaccaria segetalis.

By the guidance of bioassay, one new cytotoxic triterpenoid saponin, 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] quillaic acid 28-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-fucopyranosyl-(1-->4)]-beta-D-fucopyranoside (1), and five known cytotoxic triterpenoid saponins, vaccaroside E (2), vaccaroside G (3), vaccaroside B (4), segetoside H (5) and segetoside I (6), were isolated from Vaccaria segetalis. Their structures were established on the basis of ESI-MS, IR, extensive NMR ((1)H NMR, (13)C NMR, TOCSY, (1)H-(1)H COSY, DEPT, HMQC, HMBC and ROESY) analyses, chemical degradation, and by comparing with previously reported data. Compounds 1-6 showed moderate cytotoxic activities against LNcap, P-388 and A-549 cell lines with IC(50) values in the range 0.1-12.9 microM.

Comparative efficacy of five herbicides on winter cereal weeds in semi-arid region of Algeria.

Field chemical control trials on cereal weeds were conducted in the Sétif region (north-east of Algeria), using five herbicides. The most abundant weeds were Fumaria densiflora DC., Veronica cymbalaria Bodard, Vaccaria pyramidata Medik, and Papaver rhoeas L.. The herbicides applied at the end-tillering stage of wheat were bromoxynil + diclofop-methyl, flamprop-isopropyl + MCPA, 2,4-D ester, diclofop-methyl and tribenuron-methyl. Compared to the untreated control, they increased grain yield by 359, 294, 216, 174 and 417 kg ha-1, respectively. These results showed the importance of the chemical control, which is applied in the Sétif region on less than 5% of the total surface of cereals.