Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1.

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.

Phytochemistry, pharmacology, and medical uses of Oldenlandia (family Rubaceae): a review.

The Oldenlandia genus comprises approximately 240 species of plants, yet only a limited number of these have been investigated for their chemical composition and medicinal properties. These species contain a wide range of compounds such as iridoids, anthraquinones, triterpenes, phytosterols, flavonoids, anthocyanidins, vitamins, essential oils, phenolic acids, and coumarins. These diverse phytochemical profiles underscore the pharmacological potential of Oldenlandia plants for various medical purposes. Among other chemical constituents, ursolic acid stands out as the most important active compound in Oldenlandia, owing to its proven anticancer, anti-inflammatory, antimicrobial, and hepatoprotective properties. The evaluation of Oldenlandia's pharmacological prospects indicates that the holistic utilization of the entire plant yields the most significant effects. Oldenlandia diffusa showcases anticancer and anti-inflammatory capabilities attributed to its varying constituents. Across a broad spectrum of pharmacological capacities, anticancer research predominates, constituting the majority of medical uses. Oldenlandia diffusa emerges as a standout for its remarkable anticancer effects against diverse malignancies. Antioxidant applications follow, with O. corymbosa demonstrating potent antioxidant properties alongside O. umbellata and O. diffusa. Subsequent priority lies in anti-inflammatory studies, wherein O. diffusa exhibits noteworthy efficacy, trailed by O. corymbosa also takes the lead in antimicrobial activity, with O. umbellata as a strong contender. Additional investigation is essential to ascertain the relative significance of these species in various pharmacological applications. This comprehensive assessment underscores the multifaceted potential of Oldenlandia as a versatile herbal resource, offering diverse pharmacological capacities. The call for sustained exploration and research remains essential to unlock the full extent of Oldenlandia's medicinal benefits.

Genomic, transcriptomic, and metabolomic analysis of Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites.

Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.

3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis.

OBJECTIVE: To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). METHODS: The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. RESULTS: Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. CONCLUSIONS: ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.

Chemical constituents from Oldenlandia diffusa and their cytotoxic effects on human cancer cell lines.

Oldenlandia diffusa is an important Chinese traditional medicine with various biological activities such as anti-tumor, anti-inflammatory, anti-oxidant, antibacterial, neuroprotective and hepatoprotective effects. During our course of finding novel compounds from O. diffusa, two new alternariol derivatives named 9-O-(trans-p-coumaroyl)-alternariol (1), 9-O-(trans-caffeoyl)-alternariol (2), together with six known compounds (3-8) were isolated. Their structures were established on the basis of spectroscopic and physicochemical analysis. All isolates were evaluated for in vitro cytotoxic activities on MCF-7, HepG2, A549 and A2780 cancer cells. As a result, new compounds 1 and 2 exhibited potent cytotoxic activities on A2780 cancer cells with IC50 values of 3.1 and 9.4 µM, respectively. And a conclusion was deduced that the p-coumaroyl or caffeoyl moiety could greatly increased the cytotoxic activity of alternariol on cancer cells.

Diffusosides C and D, two new iridoid glucosides from Oldenlandia diffusa.

During our continual investigations on Oldenlandia diffusa (Willd.) Roxb., two new iridoid glucosides, diffusosides C (1) and D (2), were isolated and their structures were elucidated through cumulative analysis of NMR and HRESIMS spectroscopic data as well as computational studies. Both isolated compounds displayed no obvious neuroprotective activity on H2O2-induced injury PC12 cells at 50 µM in vitro.

Interaction of cyclotide Kalata B1 protein with model cellular membranes of varied electrostatics.

A uni-molecular layer of lipids at air-water interface mimicking one of the leaflets of the cellular membrane provides a simple model to understand the interaction of any foreign molecules with the membrane. Here, the interactions of protein Kalata B1 (KB1) of cyclotide family with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG), and 1,2-distearoyl-sn-glycero-3-ethylphosphocholine chloride salt (DSEPC) have been investigated. The addition of KB1 induces a change in pressure of the lipid monolayers. The characteristic time of the change in pressure is found to be dependent on the electrostatic nature of the lipid. Even though the protein is weakly surface active, it is capable of modifying the phase behavior and elastic properties of lipid monolayers with differences in their strength and nature making the layers more floppy. The KB1-lipid interaction has been quantified by calculating the excess Gibb's free energy of interaction and the 1-anilino-8-naphthalenesulfonate (ANS) binding studies. The interaction with zwitterionic DPPC and negatively charged DPPG lipids are found to be thermodynamically favorable whereas the protein shows a weaker response to positively charged DSEPC lipid. Therefore, the long ranged electrostatic is the initial driving force for the KB1 to recognize and subsequently attach to a cellular membrane. Thereafter, the hydrophobic region of the protein may penetrate into the hydrophobic core of the membrane via specific amino acid residues.

Scutellaria barbata D.Don and Oldenlandia diffusa (Willd.) Roxb crude extracts inhibit hepatitis-B-virus-associated hepatocellular carcinoma growth through regulating circRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. AIM OF THE STUDY: The aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. MATERIALS AND METHODS: We determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet-visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. RESULTS: The extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. CONCLUSIONS: The extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.

Promotion of Cell Death in Cisplatin-Resistant Ovarian Cancer Cells through KDM1B-DCLRE1B Modulation.

Ovarian cancer is the gynecological malignancy with the poorest prognosis, in part due to its high incidence of recurrence. Platinum agents are widely used as a first-line treatment against ovarian cancer. Recurrent tumors, however, frequently demonstrate acquired chemo-resistance to platinum agent toxicity. To improve chemo-sensitivity, combination chemotherapy regimens have been investigated. This study examined anti-tumor effects and molecular mechanisms of cytotoxicity of Oldenlandia diffusa (OD) extracts on ovarian cancer cells, in particular, cells resistant to cisplatin. Six ovarian cancer cells including A2780 and cisplatin-resistant A2780 (A2780cis) as representative cell models were used. OD was extracted with water (WOD) or 50% methanol (MOD). MOD significantly induced cell death in both cisplatin-sensitive cells and cisplatin-resistant cells. The combination treatment of MOD with cisplatin reduced viability in A2780cis cells more effectively than treatment with cisplatin alone. MOD in A2780cis cells resulted in downregulation of the epigenetic modulator KDM1B and the DNA repair gene DCLRE1B. Transcriptional suppression of KDM1B and DCLRE1B induced cisplatin sensitivity. Knockdown of KDM1B led to downregulation of DCLRE1B expression, suggesting that DCLRE1B was a KDM1B downstream target. Taken together, OD extract effectively promoted cell death in cisplatin-resistant ovarian cancer cells under cisplatin treatment through modulating KDM1B and DCLRE1B.

Chemical constituents of Oldenlandia pinifolia and their antiproliferative activities.

This study describes the chemical constituents of Oldenlandia pinifolia (Wall. Ex G. Don) Kuntze (synonym Hedyotis pinifolia Wall. Ex G. Don) and discusses their anti-proliferative activities. Thirteen compounds were isolated from the n-hexane, ethyl acetate and n-butanol extracts of whole plants O. pinifolia by chromatography method. Their structures were elucidated using MS and NMR analysis and compared with reported data. They are three anthraquinones, a carotenoid, two triterpenes, four iridoid glycosides and three flavonoid glycosides. Among them, 2-methyl-1,4,6-trihydroxy-anthraquinone is a new one, and three compounds were found for the first time in this genus. MTT assay resulted that the n-butanol extract and four isolated compounds inhibited the proliferation of chronic myelogenous leukaemia cells. The results from Hoechst 33343 staining and caspase 3-inducing exhibited that those four tested compounds induced apoptosis and activated caspase 3 (p < 0.05). One of them, isorhamnetin-3-O-ß-rutinoside showed the most activity with IC50 value of 394.68 ± 25.12 µM.

Ethanol Extract of Oldenlandia diffusa Herba Attenuates Scopolamine-Induced Cognitive Impairments in Mice via Activation of BDNF, P-CREB and Inhibition of Acetylcholinesterase.

Though Oldenlandia diffusa Herba (ODH) has been known to exhibit anti-cancer and anti-inflammatory effects, its anti-amnestic effect has never been reported so far. The aim of this present study was to elucidate the anti-amnestic effect of ODH. ODH pretreatment significantly reduced escape latency of scopolamine treated Institute of Cancer Research (ICR) mice compared to untreated control groups in a Morris water maze test. Similarly, the passive avoidance test showed that ODH treatment recovered the scopolamine induced amnesia in the ICR mouse model. Concentration of Ach in brains of ODH treated mice was increased compared to that of scopolamine treated mice. In addition, activity of acetylcholinesterase (AChE) was notably decreased by ODH. The protein expression of brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding protein (p-CREB) (Ser133) was increased in ODH pretreated group compared to control group. Consistently, immunohistochemistry (IHC) revealed the elevated expression of brain-derived neurotrophic factor (BDNF) and p-CREB in brains of ODH treated mice compared to the control group. Overall, these findings suggest that ODH has anti-amnestic potential via activation of BDNF and p-CREB and inhibition of AChE in mice with scopolamine induced amnesia.

Anti-plasmodial and anti-inflammatory activities of cyclotide-rich extract and fraction of Oldenlandia affinis (R. & S.) D.C. (Rubiaceae).

BACKGROUND: Oldenlandia affinis, commonly called 'kalata-kalata', a versatile plant used locally to treat malaria fever in some parts of sub-Saharan Africa was investigated for anti-plasmodial and anti-inflammatory activities. OBJECTIVE: The study was designed to evaluate the antiplasmodial as well as anti-inflammatory activities of whole extract and cyclotide-rich fraction of Oldenlandia affinis. METHOD: The dichloromethane-methanol extract (ODE) of the plant, O. affinis was investigated for suppressive and curative antiplasmodial activities against Plasmodium berghei in mice. ODE and the cyclotide-rich fraction (CRF) was investigated for chronic and acute anti-inflammatory activities in rat models of inflammation. Inhibition of pro-inflammatory mediators was studied in RAW264.7 macrophages. RESULTS: ODE exhibited significant (p<0.05) reduction in mean parasitaemia in both the suppressive and curative models of Plasmodium berghei infection in mice.Administration of ODE(100, 200, or 400 mg/kg) and CRF (100, 200, or 400 mg/kg) produced significant inhibition of rodent models of acute and chronic inflammation . This observation is supported by the significant (P<0.05) inhibition of pro-inflammatory mediators, inducible nitric oxide (iNO) and tumour necrosis factor-alpha (TNF-α), and the reactive radical scavenging activities in RAW264.7 macrophages. CONCLUSION: These findings could explain, at least in part, the successes reported in the use of the herb, Oldenlandia affinis in the traditional treatment of malaria fever.

Oldenlandia diffusa suppresses metastatic potential through inhibiting matrix metalloproteinase-9 and intercellular adhesion molecule-1 expression via p38 and ERK1/2 MAPK pathways and induces apoptosis in human breast cancer MCF-7 cells.

ETHNOPHARMACOLOGY RELEVANCE: Oldenlandia diffusa (OD) has long been known as an apoptotic inducer in breast tumors in ethnomedicine. AIM OF THE STUDY: To scientifically confirm the anti-breast cancer effects of water, methanol (MeOH) and butanol (BuOH) extracts of O. diffusa on cell apoptosis, matrix metalloproteinases (MMPs), intercellular adhesion molecule (ICAM)-1 and intracellular signaling in MCF-7 breast cancer cells. MATERIALS AND METHODS: MeOH extracts (MOD) and BuOH extracts (BOD) were prepared and examined for their ability to inhibit phorbol myristate acetate (PMA)-induced matrix metalloproteinase (MMP)-9 and intercellular adhesion molecule (ICAM)-1 expressions in MCF-7 human breast cancer cells. Additionally, transwell migration, invasion and transcriptional activity were assessed. Results of immunofluorescence confocal microscopy for translocation of NF-κB and p-ERK and p-p38 were also checked. Finally, apoptotic signals including processed caspase-8, caspase-7, poly ADP-ribose polymerase, Bax and Bcl-2 were examined. RESULTS: MOD and BOD specifically inhibited PMA-induced MMP-9 expression as well as invasive and migration potential via ICAM-1. The inhibitory activity was also based on the suppressed transcriptional activity in MCF-7 breast cancer cells. Results of immunofluorescence confocal microscopy showed that translocation of NF-κB decreased upon BOD and MOD treatments, with a decreased level of p-ERK and p-p38 phosphorylation. In addition, treatment of MCF-7 cells with MOD and BOD activated apoptosis-linked proteins including enzymatically active forms of processed caspase-8, caspase-7 and poly ADP-ribose polymerase, together with increased expression of mitochondrial apoptotic protein, Bax and decreased expression of Bcl-2. CONCLUSION: The results indicate that OD as an anti-metastatic agent suppresses the metastatic response by targeting p-ERK, p-38 and NF-κB, thus reducing the invasion capacity of MCF-7 breast cancer cells through inhibition of MMP-9 and ICAM-1 expression and plays an important role in the regulation of breast cancer cell apoptosis.

Oldenlandia diffusa Extract Inhibits Biofilm Formation by Haemophilus influenzae Clinical Isolates.

Oldenlandia diffusa has been empirically used as a therapeutic adjunct for the treatment of respiratory infections. To establish the basic evidence of its clinical usefulness, antimicrobial and biofilm inhibitory activities of an O. diffusa extract were examined against clinical isolates of Haemophilus influenzae, a major causative pathogen of respiratory and sensory organ infections. No significant growth inhibitory activity was observed during incubation for more than 6 h after the extract addition into a culture of H. influenzae. On the other hand, biofilm formation by H. influenzae, evaluated by a crystal violet method, was significantly and dose-dependently inhibited by the O. diffusa extract. Furthermore, the mRNA level of the biofilm-associated gene luxS of H. influenzae significantly decreased soon after the extract addition, and the suppressive effect continued for at least 2 h. At 2 h after the addition of the O. diffusa extract, the autoinducer in the culture supernatant was also significantly reduced by the O. diffusa extract in a dose-dependent manner. These results revealed that O. diffusa extract shows inhibitory activity against luxS-dependent biofilm formation but has no antimicrobial activity against planktonic cells of H. influenzae. Thus, O. diffusa extract might be useful as an adjunctive therapy for the treatment of respiratory infections caused by H. influenzae.

The N-terminal pro-domain of the kalata B1 cyclotide precursor is intrinsically unstructured.

Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins.

In-Silico Template Selection of In-Vitro Evolved Kalata B1 of Oldenlandia Affinis for Scaffolding Peptide-Based Drug Design.

Structural stability of Oldenlandia affinis cyclotide, kalata B1 of native (1NB1) and two mutants 2F2I ([P20D, V21K] kB1) and 2F2J ([W19K, P20N, V21K] kB1) was investigated. Single model analysis showed high number of intra-molecular interactions followed by more proportion of beta sheet contents in [P20D, V21K] kB1 as compared to that of native and the other mutant of kalata B1. Further, the modern conformational sampling approach, an alternate to classical molecular dynamics was introduced, which revealed that the [P20D, V21K] kB1 was identified as structurally stable one, substantiated by various structural events viz., root mean square deviation, root mean square fluctuation, and angular deviation by Ramachandran plot. Moreover, the statistically validated contours of polar surface area, hydrogen bond distribution and the distance of disulfide bridges also supported the priority of [P20D, V21K] kB1 with respect to stability. From this work, it is proposed that the [P20D, V21K] kB1 (2F2I) could be the best template for scaffolding peptide based drug design.

[UPLC Fingerprint of Oldenlandia corymbosa].

OBJECTIVE: To establish the UPLC fingerprint of Oldenlandia corymbosa from different regions and to distinguish it from Oldenlandia diffusa. METHODS: UPLC procedure was performed on an ACQUITY UPLC BEH C18 (50 mm x 2. 1 mm, 1. 7 µm) column and eluted with a mobile phase consisted of methanol-l % acetic acid at a flow rate of 0. 2 mL/min. The column temperature was 30 °C . The detection wavelength was 254 nm. A matrix was constructed for similarity evaluation, cluster analysis and principle component analysis. RESULTS: The collected samples had a good similarity. A specificity fingerprint chromatogram was produced and 15 common peaks were designated. Samples were divided into four groups. CONCLUSION: It is a reliable and available method for specific identification of Oldenlandia corymbosa and for distinguishing Oldenlandia corynbosa and Oldenlandia diffusa.

[Study on effect of total flavonoids of Oldenlendia difflusa on ulcerative colitis and its immunological mechanism].

OBJECTIVE: To observe the effect of total flavonoids of Oldenlendia difflusa (FOD) on NF-kappaB and IL-8, TNF-alpha, IL-10 expressions of ulcerative colitis (UC) model rats, and explore its immunological mechanism of anti-UC. METHOD: Sixty Kunming male mice with the average weight of (20 +/- 2) g were randomly divided into six groups. The control group (cont) was orally administered with distilled water. Whereas the remaining five groups were fed with 4% dextran sulphate sodium (DSS) solution for seven days to induce acute UC, and orally administered with the following drugs: distilled water (for the DSS group), SASP at dose of 500 mg x kg(-1) x d(-1) for the DSS + SASP group, FOD at dose of 60 mg x kg(-1) x d(-1) for the DSS + FOD-H group, FOD at dose of 40 mg x kg(-1) x d(-1) for the DSS + FOD-M group, and FOD at dose of 26.7 mg x kg(-1) x d(-1) for the DSS + FOD-L group. During the modeling and drug administration, the mice were scored for DAI. Seven days later, the mice were put to death, and their colonic tissue samples were collected to evaluate colonic mucosal lesions. The NF-kappaB p65, IL-8, TNF-alpha, IL-10 expressions were tested by immunohistochemical staining and ELISA. RESULT: Seven-day feeding with 4% DSS solution could successfully induce acute UC in mice. Compared with the cont group, the DSS group showed significantly higher DAI and colonic mucosal lesions, remarkable increase in NF-kappaB p65, IL-8, TNF-alpha expression in colonic tissues, and notable decrease in IL-10 expression (P < 0.05). FOD could prevent acute UC in mice included by DSS. Seven-day administration of 60 mg x kg(-1) x d(-1) or 40 mg x kg(-1) x d(-1) FOD could completely or partially resist the above mentioned changes caused by DSS. Compared with the DSS group, the DSS + FOD-H group and the DSS + FOD-M group showed reduction in colonic mucosal lesions, down-regulation in IL-8, TNF-alpha and NF-kappaB p65 expressions and up-regulation in IL-10 expression (P < 0.05). CONCLUSION: FOD could significantly resist UC in mice. Its mechanism may be related to the inhibition of NF-kappaB p65 activation, the reduction of IL-8 and TNF-alpha expressions and the increase in the anti-inflammatory factor IL-10.

[Study on Chemical Constituents of Oldenlandia diffusa].

OBJECTIVE: To study the chemical constituents of Oldenlandia diffusa. METHODS: The chemical constituents were isolated and purified by silica gel and Sephadex LH-20 column chromatography as well as recrystallization method, and their structures were elucidated on the basis of physical and spectral analyses. RESULTS: Nine compounds were isolated and their structures were identified as quercetin (1), kaempferol (2), scopoletin (3), 2-hydroxy-3-methylanthraquinone (4), 2-hydroxy-l-methoxyanthraquinone (5), α-linolenic acid (6), vanillic acid (7), p-hydroxyphenylethanol (8) and, ß-sitosterol (9). CONCLUSION: Compound 6 is obtained from this genus for the first time. Compounds 7 and 8 are obtained from this plant for the first time.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design.

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.