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.

Crude polysaccharides from the seeds of Vaccaria segetalis prevent the urinary tract infection through the stimulation of kidney innate immunity.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Vaccaria segetalis (Neck.) Garcke is used for the treatment of urinary diseases in Traditional Chinese Medicine according to the Chinese Pharmacopoeia. Crude polysaccharides and the aqueous extract from the seeds of V. segetalis (SVCP) were proved to be effective on treating benign prostatic hyperplasia. AIM OF THE STUDY: The aim of this study was to test the effects of SVCP on urinary tract infection (UTI) induced by uropathogenic Escherichia coli (UPEC) strain CFT073 in the rat model and to investigate the underlying mechanisms. MATERIALS AND METHODS: A rat UTI model was established with the infection of UPEC strain CFT073. After oral administration of SVCP, the urinalysis and histological examination were evaluated. The levels of pro-inflammatory cytokines, procalcitonin (PCT) and polymeric Ig receptor (PIGR) were used to test the effects of SVCP on host immunity. The mRNA level of PapG in CFT073 was used to test the influence of SVCP on virulence factor. The effects of SVCP on the inhibition of bacterial adhesion were evaluated with mice UTI model. RESULTS: In the rat UTI model, the levels of bacterial load, white blood cells (WBC) and red blood cells (RBC) in urine and the pathological injury in the bladder were significantly up-regulated, the expression of PIGR in kidney was down-regulated, no significant change was observed on the pro-inflammatory cytokines in urine. After oral administration of SVCP for 3 days, the levels of bacterial load, WBC and RBC in urine were significantly decreased, the pathological injury in the bladder were remarkably inhibited. The expression of IL-6, IL-8 in urine and PIGR in kidney were significantly up-regulated by SVCP (200 mg/kg). SVCP showed no effect on the concentration of PCT in serum. SVCP failed to down-regulate the mRNA level of PapG in CFT073. In the mice UTI model, pre-treatment of SVCP failed to inhibit the intracellular bacterial load in the bladder. CONCLUSIONS: The therapeutic effects of SVCP on treating UTIs might result from the up-regulation of innate immunity in the kidney. SVCP can be used as an alternative therapeutic agent for UTIs.

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.

[Effects of Auricular Acupressure on Obesity in Women with Abdominal Obesity].

PURPOSE: The purpose of this study was to examine the effects of auricular acupressure on reducing obesity in adult women with abdominal obesity. METHODS: The study design was a non-equivalent control group pretest-posttest design. Participants were 58 women, aged 20 years or older, assigned to the experimental group (n=30) or control group (n=28). Auricular acupressure using vaccaria seeds was administered to the experimental group. The sessions continued for 8 weeks. Outcome measures included body weight, abdominal circumference, and body mass index, body fat mass, body fat percentage, triglyceride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and serum glucose. RESULTS: Women in the experimental group showed significant decreases in body weight (t=6.19, p<.001), abdominal circumference (t=4.01, p<.001), and body mass index (t=2.58, p=.006) after 8 weeks compared with those in the control group. CONCLUSION: Results show that auricular acupressure using vaccaria seeds was effective in decreasing body weight, abdominal circumference, body mass index, and triglyceride levels in adult women with abdominal obesity.

Analgesic and anti-inflammatory effects of hydroalcoholic extract isolated from Semen vaccariae.

Semen vaccariae, the seeds of Vaccaria segetalis (Neck.) Garcke, is usually used as an important medication for female mammary gland diseases; it has also been used to promote lactation for centuries in China. The purpose of this work was to evaluate the analgesic and anti-inflammatory effects of hydroalcoholic extract from semen vaccariae (HESV) with oral doses of 50, 100 and 200mg/kg•bw in mice and rats. We observed that the HESV could effectively inhibit acetic acid-induced abdominal contraction and could elevate the latency time to thermal stimuli in the hot-plate test in mice. In the xylene-induced ear-swelling test in mice, HESV could suppress the ear swelling. Additionally, HESV could significantly decrease the peritoneal capillary permeability and leukocyte infiltration in mice induced by the intraperitoneal injection of acetic acid. HESV also significantly reduced paw thickness 2-4 hours after the injection of carrageenan in the carrageenan-induced rat paw edema test. This study was the first to demonstrate that the oral administration of HESV might play an important role in the process of analgesia and anti-inflammation, supporting its use for female mammary gland diseases in traditional medicine.

Three new compounds isolated from the seeds of Vaccaria segetalis.

Two new capsaicin analogs, N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide (1) and N-(3,4-dihydroxyphenethyl)-tetracosanamide (2), along with one new flavonoidal glycoside pinnatifin E (3) were isolated from the ethanolic extract of the seeds of Vaccaria segetalis. Their structures were elucidated on the basis of spectroscopic methods including 1D, 2D NMR, MS, and other spectroscopic techniques, as well as by comparison with the relevant literatures. All compounds were evaluated for their coagulation Factor Xa inhibition activities.

Identification of multiple constituents from seed of Vaccaria segetalis with an adsorbent-separation strategy based on liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

RATIONALE: Seeds of Vaccaria segetalis (Wang-Bu-Liu-Xing in Chinese) are mainly used in traditional Chinese medicine for the treatment of amenorrhea, breast infections, and edema. The study was designed to identify the components and metabolites of Wang-Bu-Liu-Xing. METHODS: A novel methodology combining an adsorbent-separation strategy with analysis by liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/QTOF-MS/MS) was established to identify the components of Wang-Bu-Liu-Xing. The adsorbent-separation technique was applied on macroporous resin (adsorbents). Different concentrations of ethanol (30%, 60%, and 95%), which covered high-to-low polarity ranges, were chosen as the elution solvent, respectively. The QTOF mass spectrometer was operated in negative ion mode with an electrospray ionization source. RESULTS: A total of 52 components were successfully identified in the Wang-Bu-Liu-Xing decoction based on the fragmentation pathways and QTOF high-accuracy mass spectral analysis. To the best of our knowledge, several new saponins were reported for the first time. A total of 20 compounds, which included 10 prototypes and 10 metabolites, were also identified in rat plasma and urine after oral administration of Wang-Bu-Liu-Xing decoction. CONCLUSIONS: An integrated adsorbent-separation strategy is powerful and reliable for global detection and identification of complex components in herbal prescriptions. The components identified in rat biofluids may also provide helpful chemical information for further pharmacology and active mechanism study on this herb.

Simultaneous determination of four major constituents of semen Vaccariae using HPLC-DAD.

A simple and reliable HPLC method was developed and validated for the simultaneous quantification of four major constituents in Semen Vaccariae. The chromatographic separation was performed on an Agilent Zorbax SB-C18 column with gradient elution using methanol and water. The calibration curves showed good linearity of R2 > 0.9999 with LOQs (S/N = 10) of 0.20-1.16 microg/mL. The precision was evaluated by intra- and inter-day assays and R.S.D. values were less than 2.09%. The recovery rates were between 97.0% and 105.0%. The developed method was applied to the quantitative analysis of Semen Vaccariae and its stir-fried products. During the stir-frying process, vaccarin degraded and yielded isovitexin-2"-O-arabinoside. The preferable stir-frying temperature is around 120 degrees C. The developed HPLC method can be applied to the quality control of crude and stir-fried Semen Vaccariae.

Application of high-speed counter-current chromatography for preparative separation of cyclic peptides from Vaccaria segetalis.

Following an initial clean-up step on silica gel, high-speed counter-current chromatography (HSCCC) was used to separate cyclic peptides from an extract of the seeds of Vaccaria segetalis. The two-phase solvent system used for HSCCC separation was composed of petroleum ether-ethyl acetate-methanol-water at an optimized volume ratio of 0.5:3.5:1:5. From 190 mg of crude extract, 38.0 mg of segetalin B and 28.5 mg of segetalin A were obtained with purities of 98.1% and 95.6% as determined by HPLC, respectively. The chemical structures of the target compounds were confirmed by high resolution electrospray ionization time of flight MS (HRESI-TOF-MS) and (1)H NMR analyses.

A new flavonoid glycoside from Vaccaria hispanica.

A profiling analysis for the methanol extract of the seeds of Vaccaria hispanica (Mill.) Rauschert was performed using a HPLC-ESI-MS technique. Five compounds were identified according to their retention times, UV spectroscopic and MS features, and by comparison with literature data. Among them, a new flavonoid glycoside, named vaccarin H, was isolated and the structure determined by spectral and chemical analysis as isovitexin 2"-O-alpha-L-arabinopyranosyl-4'-O-(6""-O-dihydroferuloyl)-3-beta-glucopyranoside.

[Determination of vaccarin in Vaccariae Semen by HPLC].

To establish a HPLC method for the determination of vaccarin in Vaccariae Semen. Analysis was carried out on an Alltima-C18 column (4.6 mm x 250 mm, 5 microm) eluted with methanol -0.3% phosphoric acid as mobile phase in gradient elution. The flow rate was 1.0 mL x min(-1) and detected wavelength was set at 280 nm. The peak areas and injection ammounts of vaccarin showed a good linear relationship in the range of 0.102-1.539 microg, R2 = 0.9997. The average recovery was 100.4%, RSD was 0.81%. The results of the assay of 10 samples showed that the contents of vaccarin varied in the range of 0.46%-0.57%. The method is simple, accurate, reproducible and specific. It can be used for the quality control of Vaccariae Semen.

[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.

Use of digitoxin and digoxin as internal standards in HPLC analysis of triterpene saponin-containing extracts.

INTRODUCTION: Saponins are widely distributed complex plant glycosides possessing a variety of structure-dependent bioactivities. Quantitation of individual saponins is difficult due to lack of available standards, mainly as a consequence of purification difficulties. Determination of total saponin content can be problematic, often relying on non-specific methods based on butanol solubility, haemolytic activity or formation of coloured derivatives. OBJECTIVE: To develop a general quantitative method based on the use of the readily available cardenolides, digitoxin (1) and digoxin (2), as internal standards in an HPLC-PAD-based analysis. METHODOLOGY: The cardenolides were run at a variety of concentrations to establish linearity and reproducibility of detector response and then evaluated as internal standards for quantitation of triterpene saponins in several plant-derived extracts by HPLC-PAD. Mixtures of saponins, largely freed from other extractables, were obtained by fractionation of total extracts on solid phase extraction columns (SPE) employing a water-methanol gradient and used for construction of calibration curves. Saponin identification and structural information was obtained via a single quadrupole mass detector using electrospray ionisation in negative ion mode (ESI(-)). RESULTS: Saponin contents in six samples from five species were determined and compared with literature results and a gravimetric method based on butanol-water partitioning. Results were generally consistent with literature reports and superior to gravimetric butanol-water partitioning. CONCLUSION: Digitoxin and digoxin are useful as internal standards in HPLC estimation of saponin content. Saponins from different species having similar structures and molecular weights afford similar calibration curves.

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.

Structure of a new cyclic nonapeptide, segetalin F, and vasorelaxant activity of segetalins from Vaccaria segetalis.

A new cyclic nonapeptide, segetalin F, has been isolated from the seeds of Vaccaria segetalis and the structure including absolute stereochemistry was elucidated by using 2D NMR and chemical means. A series of segetalins showed a vasorelaxant activity against norepinephrine (NE)-induced contractions of rat aorta.