In silico and in vivo study of anti-inflammatory activity of Morinda longissima (Rubiaceae) extract and phytochemicals for treatment of inflammation-mediated diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the plant Morinda longissima Y.Z.Ruan (Rubiaceae) is used by ethnic people in Vietnam for the treatment of liver diseases and hepatitis. AIM OF THE STUDY: The study was designed to assess the efficacy of the 95% ethanolic extract of Morinda longissima roots (MLE) in experimental immune inflammation. The phytochemical variation of root extract and the chemical structures of natural compounds were also investigated using HPLC-DAD-HR-MS analysis. MATERIALS AND METHODS: Three different doses (100, 200, and 300 mg/kg b.w.) of MLE were chosen to determine anti-inflammatory activity. The mice were given orally extracts and monitored their behavior and mortality for 14 days to evaluate acute toxicity. The volume of the paw and the histopathological evaluation were carried out. The polyphenolic phytoconstituents of MLE extract were identified using LC/MS analysis. The anti-inflammatory efficacy in silico and molecular docking simulations of these natural products were evaluated based on their cyclooxygenase (COX)-1 and 2 inhibitory effects. RESULTS: This investigation showed the 95% ethanolic extract of Morinda longissima roots was found non-toxic up to 2000 mg/kg dose level in an acute study, neither showed mortality nor treatment-related signs of toxicity in mice. Eight anthraquinones and anthraquinone glycosides of Morinda longissima roots were identified by HPLC-DAD-HR-MS analysis. In the in vivo experiments, MLE was found to possess powerful anti-inflammatory activities in comparison with diclofenac sodium. The highest anti-inflammatory activity of MLE in mice was observed at a dose of 300 mg/kg body weight. The in silico analysis showed that seven out the eight anthraquinones and anthraquinone glycosides possess a selectivity index RCOX-2/COX-1 lower than 1, indicating that these compounds are selective against the COX-2 enzyme in the following the order: rubiadin-3-methyl ether < morindone morindone-6-methyl ether < morindone-5-methyl ether < damnacanthol < rubiadin < damnacanthol-3-O-ß-primeveroside. The natural compounds with the best selectivity against the COX-2 enzyme are quercetin (9), rubiadin-3-methyl ether (7), and morindone (4), with RCOX2/COX1 ratios of 0.02, 0.03, and 0.19, respectively. When combined with the COX-2 protein in the MD research, quercetin and rubiadin-3-methyl ether greatly stabilized the backbone proteins and ligands. CONCLUSION: In conclusion, the anthraquinones and ethanolic extract of Morinda longissima roots may help fight COX-2 inflammation. To develop novel treatments for inflammatory disorders linked to this one, these chemicals should be investigated more in the future.

Betulinic and ursolic acids from Nauclea latifolia roots mediate their antimalarial activities through docking with PfEMP-1 and PfPKG proteins.

BACKGROUND: Chemotherapies target the PfEMP-1 and PfPKG proteins in Plasmodium falciparum, the parasite that causes malaria, in an effort to prevent the disease's high fatality rate. This work identified the phytochemical components of Nauclea latifolia roots and docked the chemical compounds against target proteins, and examined the in vivo antiplasmodial effect of the roots on Plasmodium berghei-infected mice. METHODS: Standard protocols were followed for the collection of the plant's roots, cleaning, and drying of the roots, extraction and fraction preparation, assessment of the in vivo antiplasmodial activity, retrieval of the PfEMP-1 and PfPKG proteins, GCMS, ADME, and docking studies, chromatographic techniques were employed to separate the residual fraction's components, and the Swis-ADME program made it possible to estimate the drug's likeness and pharmacokinetic properties. The Auto Dock Vina 4.2 tool was utilized for molecular docking analysis. RESULTS: The residual fraction showed the best therapeutic response when compared favorably to amodiaquine (80.5%) and artesunate (85.1%). It also considerably reduced the number of parasites, with the % growth inhibition of the parasite at 42.8% (D2) and 83.4% (D5). Following purification, 25 compounds were isolated and characterized with GCMS. Based on their low molecular weights, non-permeation of the blood-brain barrier, non-inhibition of metabolizing enzymes, and non-violation of Lipinski's criteria, betulinic and ursolic acids were superior to chloroquine as the best phytochemicals. Hence, they are lead compounds. CONCLUSION: In addition to identifying the bioactive compounds, ADME, and docking data of the lead compounds as candidates for rational drug design processes as observed against Plasmodium falciparum target proteins (PfEMP-1 and PfPKG), which are implicated in the pathogenesis of malaria, the study has validated that the residual fraction of N. latifolia roots has the best antiplasmodial therapeutic index.

Previously undescribed tetrahydroanthraquinones from Prismatomeris tetrandra (roxb.) K. Schum with antitumor cell proliferation activities.

To find structurally previously undescribed compounds with pharmacological effects from Prismatomeris tetrandra (Roxb.) K. Schum (Rubiaceae), thirteen undescribed tetrahydroanthraquinones (1⎼13) named prisconnatanones J⎼V and seven known anthraquinones (14⎼20) were isolated and characterized. The structures of these compounds were elucidated by detailed spectroscopic analyses, and their absolute configurations were established by modified Mosher's method and ECD calculations. The antitumor cell proliferative activities of prisconnatanones J⎼V were determined. Among them, prisconnatanones J possessed high antitumor cell proliferation in HGC27 cells (IC50, 0.792 µM) by blocking HGC27 cells in the S phase and significantly inducing apoptosis in HGC27 cells. Prisconnatanone J has no cytotoxicity to normal gastric cells line (GES-1) at 10 µM and showed a considerable selectivity for HGC27 cells. Prisconnatanone J can potentially inhibit tumor cell proliferation and should be further investigated.

Two new indole alkaloids from Nauclea officinalis and their evaluation for cytotoxic activities.

Two new indole alkaloids, naucleamide H (1) and (±)-19-O-butylangustoline (8), along with seven known alkaloids, 3,14-dihydroangustine (2), (-)-naucleofficine D (3a), (+)-naucleofficine D (3b), nauclefine (4), angustidine (5),19-O-ethylangustoline (6) and angustine (7) were isolated from the water extract of Nauclea officinalis. The structures of these compounds were established by spectroscopic analysis. Among them, the cytotoxicity of 1, 2, 6 and 8 were evaluated against six human cancer cell lines (HepG-2, SKOV3, HeLa, SGC 7901, MCF-7 and KB) in vitro for the first time with 5-fluorouracil as a positive control drug. The new compound 1 had a strong inhibitory effect on the proliferation of HepG-2 with an IC50 value of 19.59 µg/mL. The new compound 8 had a strong inhibitory effect on HepG-2, SKOV3, HeLa, MCF-7 and KB, IC50 value was 5.530, 23.11, 31.30, 32.42 and 37.26 µg/mL, respectively.

Two new anthraquinone derivatives from Saprosma crassipes H. S. Lo.

Two new anthraquinone derivatives sapranquinones A and B (1 and 2) together with two known biogenetically related anthraquinone derivatives (3 and 4) were isolated from the stems of Saprosma crassipes H. S. Lo. The structures of these compounds were elucidated using comprehensive spectroscopic methods. Compounds 1-4 were evaluated for their antibacterial activities and compounds 1 and 3 had a broad spectrum antibacterial activity against Staphylococcus albus, Escherichia coli, Bacillus cereus, Micrococcus tetragenus, and Micrococcus luteus with MIC values ranging from 1.25 to 5 µg/mL.

Toxicological effects of aqueous extract of Genipa americana L. leaves on adult zebrafish (Danio rerio): Chemical profile, histopathological effects and lack of genotoxicity.

Genipa americana is a native plant of Brazil with potential applications in folk medicine. Whereas most of the phytochemical and pharmacological studies on this plant have focused on its fruits, the crude extracts of its leaves contain chemical metabolites that may have toxicity to organisms, which have yet to be investigated. This study aimed to determine the main groups of secondary metabolites in the aqueous extract of the leaves of G. americana by phytochemistry and qualitative HPLC, and to evaluate the possible toxicological effects and histopathological changes caused by this extract in zebrafish (Danio rerio) adults, through micronucleus test, nuclear abnormalities and histopathological analyses of gills and liver. While three metabolites of high intensity (phenolic compounds, flavonoids and triterpenes) were found in the phytochemical evaluation, the HPLC showed results compatible with flavonoids and iridoids, all belonging to common classes for this species and the Rubiaceae family. The acute toxicity test did not induce mortality or genotoxicity in zebrafish, but after exposure for 96 h, it was possible to observe injuries to the fish gill tissue, such as lamellar fusion, vasodilation and telangiectasia; in the liver, necrosis was visualized at 40 mg/L, and at higher concentrations (80 and 100 mg/L) induced sinusoidal widening was identified. In conclusion, the results demonstrated the toxic potential of this plant for aquatic species.

Natural blue.

A food dye from a South American fruit has become a test case for the ethical development of natural resources.

Anti-inflammatory iridoid glycosides from Paederia scandens (Lour.) Merrill.

Eight previously undescribed iridoid glycosides together with 20 known congeners were isolated from the aerial parts of Paederia scandens (Lour.) Merrill (Rubiaceae). Their structures incorporating absolute configurations were elucidated based on the comprehensive analyses of NMR data, HR-ESI-MS spectrometry, and ECD data. The potential anti-inflammatory activities of the isolated iridoids were evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages. Compound 6 significantly inhibited the production of nitric oxide with an IC50 value of 15.30 µM. The results of immunoblotting, qPCR, and immunofluorescence staining assays revealed that compound 6 exhibited anti-inflammatory activity by suppressing nuclear translocation of NF-κB and reducing the expression of COX-2, iNOS, IL-1ß, and IL-6. These results provide a basis for further development and utilization of P. scandens as a natural source of potential anti-inflammatory agents.

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae).

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC50 s) of 0.57 and 1.26 µg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC50 s of 2.68 and 1.85 µg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside (1) with IC50 s of 0.33 and 1.30 µM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid (2), ursolic acid (3), quafrinoic acid (4), quinovic acid (5), quinovic acid 3-O-ß-D-fucopyranoside (6), oleanolic acid (7), oleanolic acid 3-acetate (8), friedelin (9), ß-sitosterol (10a), stigmasterol (10b) and stigmasterol 3-O-ß-D-glucopyranoside (11). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside (1) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.

Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) extracts: promising prospects for anticancer and antibacterial potential through in vitro and in silico studies.

Neolamarckia cadamba is an Indian traditional medicinal plant having various therapeutic potentials. In the present study, we did solvent-based extraction of Neolamarckia cadamba leaves. The extracted samples were screened against liver cancer cell line (HepG2) and bacteria (Escherichia coli). MTT cytotoxic assay was performed for in vitro analysis of extracted samples against the HepG2 cell lines and the normal human prostate PNT2 cell line. Chloroform extract of Neolamarckia cadamba leaves showed better activity with IC50 value 69 µg/ml. DH5α strain of Escherichia coli (E. coli) was cultured in Luria Bertani (LB) broth media and minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) were calculated. Solvent extract chloroform showed better activity in MTT analysis and antibacterial screening and it was taken for characterization of phytocomposition by Fourier transform infrared (FTIR) and gas chromatography mass spectrometry (GC-MS). The identified phytoconstituents were docked with potential targets of liver cancer and E. coli. The phytochemical 1-(5-Hydroxy-6-hydroxymethyl-tetrahydropyran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione shows highest docking score against the targets PDGFRA (PDB ID: 6JOL) and Beta-ketoacyl synthase 1(PDB ID: 1FJ4) and their stability was further confirmed by molecular dynamics simulation studies.

Sono-Maceration - a rapid and inexpensive method for the isolation of ursolic acid from Neolamarckia cadamba leaves.

This article records for the first time the isolation of Ursolic acid from the leaves of Neolamarckia cadamba (Roxb.) Bosser (Family: Rubiaceae) using ultrasonic waves. This bioactive triterpenic acid was isolated without its isomer, oleanolic acid, in a very convenient way with good yield. The structure was identified by means of one dimensional Nuclear Magnetic Resonance (NMR) spectroscopic techniques like 1H NMR, 13C NMR, distortionless enhancement by polarization transfer (DEPT) and two dimensional NMR spectroscopic method for example, heteronuclear single quantum coherence (HSQC). It was also assayed for antidiabetic and antioxidant potencies. About 71.5 mg of pure ursolic acid was isolated from 2.6 grams of ethyl acetate soluble fraction using sono-maceration as an extraction technique.

Recurvataside, a new saponin from aerial parts of Mussaenda recurvata.

A new glycoside, recurvataside (1) and six known compounds, quinovic acid (2), quinovic acid 28-O-ß-D-glucopyranosyl ester (3), 3-O-ß-D-glucopyranosylquinovic acid (4), 3-O-ß-D-glucopyranosylquinovic acid 28-O-ß-D-glucopyranosyl ester (5), pomolic acid (6), and ursolic acid (7) were isolated from aerial parts of Mussaenda recurvata. The structure of compound 1 was identified from its spectroscopic data and by comparison with the literature. Recurvataside represents the first occurrence of δ-oleanolic acid saponin bearing two D-glucose units at C-3 and C-28 in nature. This is the first time δ-oleanane-type saponin reported in the genus Mussaenda. Compounds 1-7 were evaluated the cytotoxicity against two cancer cell lines MCF-7 and HepG2. Among them, only compound 7 exhibited moderate activity against MCF-7 and HepG2 cell lines with IC50 value of 16.97 ± 1.55 and 20.28 ± 1.00 µM, respectively. Compounds 1-7 were also tested for their inhibitory NO production in LPS-stimulated RAW264.7 cells. Compounds 3, 5, and 7 showed significant reduction of nitrite accumulation in LPS-stimulated RAW 264.7 cells with the IC50 values of 8.81 ± 0.48, 13.42 ± 0.84, and 18.37 ± 0.67 µM, respectively.

Lasibidoupins A and B, two new compounds from the stems of Lasianthus bidoupensis V.S. Dang & Naiki.

From the Lasianthus bidoupensis stems, two new compounds, including one new 9,10-anthraquinone, lasibidoupin A (1), and one new 6,7-benzocoumarin, lasibidoupin B (2), together with one known compound, 11-O-methyldamnacanthol (3) were isolated using chromatographic method. Their structures were determined by extensive HRMS, and NMR assignments. Compound 3 was reported for the first time from this species. New compounds (1 & 2) were tested for the cytotoxicity against three human cancer cell lines (MCF-7, HeLa, and NCI-H460) by SRB assay. As results, 1 & 2 exhibited significant cytotoxic activity against all cancer cell lines (IC50 ranged from 0.058 ± 0.003 to 0.177 ± 0.014 µM).

New cytochalasans from an endophytic Xylaria species associated with Costa Rican Palicourea elata (Rubiaceae).

Four new leucine-derived cytochalasans, possessing a 5,6,5,8-ring (1) and a 5,6,11-ring core (2-4), were isolated from a cultivated endophytic fungus Xylaria sp. strain WH2D4 (Xylariaceae). This fungus was isolated from leaves of the neotropical tree species Palicourea elata (Sw.) Borhidi (Rubiaceae) collected in Costa Rica. The chemical structures were determined by employing IR, MS as well as 1D- and 2D-NMR experiments. The stereochemistry at C-15 of compound 4 was determined by quantum calculations. The isolated compounds did not affect germination and growth of Trichoderma reesei and the opportunistic human fungal pathogen T. longibrachiatum.

Strategy for the quality control of herbal preparations made of Sarcocephalus latifolius: Development and validation of a UHPLC-PDA method for quantification of angustoline and strictosamide and chemical profiling using LC-QToF.

INTRODUCTION: Sarcocephalus latifolius is one of the most used plants in West African traditional medicine to treat malaria. OBJECTIVE: The aim is to establish a strategy to control the quality of herbal preparations made from S. latifolius. METHOD: A UHPLC-PDA method was developed for the determination and quantification of the two main bioactive compounds (angustoline and strictosamide) in various parts of the plant. Additionally, an LC-QToF with electrospray ionization method is described for the identification and confirmation of compounds in samples of different parts of the plant. RESULTS: With the UHPLC-PDA method, separation was achieved within 5 min using a C18 column stationary phase at a temperature of 45°C and a gradient system with a mobile phase of water and acetonitrile, both containing 0.1% formic acid. The method was validated for linearity, accuracy, precision (repeatability and intermediate precision), limit of detection (LOD), and limit of quantification (LOQ). The LOD and LOQ of angustoline were found to be 0.3 and 0.8 µg/ml, respectively, and those of strictosamide were found to be 0.1 and 0.3 µg/ml, respectively. Using the LC-QToF method, 90 secondary metabolites, including four isolated compounds from the plant's roots, were identified from leaf, bark, and root samples of S. latifolius. CONCLUSION: This work is the first to propose a strategy to control the quality of herbal preparations made from S. latifolius. The developed method allows the quantification of the main bioactive compounds and the established chemical profile allows to distinguish the plant from any other species.

Screening for Cyclotides in Sri Lankan Medicinal Plants: Discovery, Characterization, and Bioactivity Screening of Cyclotides from Geophila repens.

Cyclotides are an intriguing class of structurally stable circular miniproteins of plant origin with numerous potential pharmaceutical and agricultural applications. To investigate the occurrence of cyclotides in Sri Lankan flora, 50 medicinal plants were screened, leading to the identification of a suite of new cyclotides from Geophila repens of the family Rubiaceae. Cycloviolacin O2-like (cyO2-like) gere 1 and the known cyclotide kalata B7 (kB7) were among the cyclotides characterized at the peptide and/or transcript level together with several putative enzymes, likely involved in cyclotide biosynthesis. Five of the most abundant cyclotides were isolated, sequenced, structurally characterized, and screened in antimicrobial and cytotoxicity assays. All gere cyclotides showed cytotoxicity (IC50 of 2.0-10.2 µM), but only gere 1 inhibited standard microbial strains at a minimum inhibitory concentration of 4-16 µM. As shown by immunohistochemistry, large quantities of the cyclotides were localized in the epidermis of the leaves and petioles of G. repens. Taken together with the cytotoxicity and membrane permeabilizing activities, this implicates gere cyclotides as potential plant defense molecules. The presence of cyO2-like gere 1 in a plant in the Rubiaceae supports the notion that phylogenetically distant plants may have coevolved to express similar cytotoxic cyclotides for a specific functional role, most likely involving host defense.

Phytochemistry Meets Geochemistry­Blumenol C Sulfate: A New Megastigmane Sulfate from Palicourea luxurians (Rubiaceae: Palicoureeae)

There is a previously neglected influence of geochemical conditions on plant phytochemistry. In particular, high concentrations of dissolved salts can affect their biosynthesis of natural products. Detoxification is most likely an important aspect for the plant, but additional natural products can also give it an expanded range of bioactivities. During the phytochemical analysis a Palicourea luxurians plant collected in a sulfate-rich environment (near the Río Sucio, Costa Rica) showed an interesting natural product in this regard. The structure of this compound was determined using spectroscopic and computational methods (NMR, MS, UV, IR, CD, optical rotation, quantum chemical calculations) and resulted in a megastigmane sulfate ester possessing a ß-ionone core structure, namely blumenol C sulfate (1, C13H22O5S). The levels of sulfur and sulfate ions in the leaves of the plant were determined using elemental analysis and compared to the corresponding levels in comparable plant leaves from a less sulfate-rich environments. The analyses show the leaves from which we isolated blumenol C sulfate (1) to contain 35% more sulfur and 80% more sulfate than the other samples. Antimicrobial and antioxidant activities of compound 1 were tested against Escherichia coli, E. coli ampR and Bacillus subtilis as well as measured using complementary in vitro FRAP and ATBS assays, respectively. These bioactivities are comparable to those determined for structurally related megastigmanes. The sulfur and sulfate content of the plant leaves from the sulfate-rich environment was significantly higher than that of the other plants. Against this background of salt stress, we discuss a possible biosynthesis of blumenol C sulfate (1). Furthermore, there appears to be no benefit for the plant in terms of extended bioactivities. Hence, the formation of blumenol C sulfate (1) probably primarily serves the plant detoxification process.

Yellow Twig (Nauclea orientalis) from Thailand: Strictosamide as the Key Alkaloid of This Plant Species

Comprehensive phytochemical examination from different perspectives using preparative and analytical chromatographic techniques combined with spectroscopic/spectrometric methods of the so-called "yellow twig" Nauclea orientalis (L.) L. (Rubiaceae) led to the identification of 13 tryptamine-derived (=monoterpene-indole) alkaloids. The identified alkaloids comprise strictosamide and four of its glucosidic derivatives, three oxindole derivatives, and five yellow-colored angustine-type aglycones. Qualitative and quantitative HPLC analyses showed the enrichment of strictosamide in all studied organs. Based on these results, we performed metabolomic analyses of monoterpene-indole alkaloids and made a 1H NMR in vitro monitoring of enzymatic deglucosylation of strictosamide. A comparison of the stability of strictosamide and its enantiomer vincoside lactam by theoretical calculations was also performed revealing a slightly higher stability of vincoside lactam. Additionally, we conducted two different anti-feedant assays of strictosamide using larvae of the polyphageous moth Spodoptera littoralis Boisduval. The obtained results indicate that generally two different biosynthetic pathways are most likely responsible for the overall alkaloid composition in this plant. Strictosamide is the key compound in the broader pathway and most likely the source of the identified angustine-type aglycones, which may contribute significantly to the yellow color of the wood. Its cross-organ accumulation makes it likely that strictosamide is not only important as a reservoir for the further biosynthesis, but also acts in the plants' defense strategy.

Structurally Diverse Metabolites from the Ophiorrhiza japonica Bl. and Their Antioxidant Activities In Vitro and PPARα Agonistic Activities In Silico.

Ophiorrhiza japonica Bl. is a traditional Chinese materia medica widely used to treat several diseases. Chemical and pharmacological studies on O. japonica have been carried out; however, neither of them has been fully explored. In this study, an array of compounds was isolated from the title plant, including a new anthraquinone, ophiorrhizaquinone A (1), three alkaloids 2-4 and seven other compounds 5-11 with diverse structural types. Additionally, compounds 2, 5, 7, 8, 10 and 11 were isolated from the genus of Ophiorrhiza for the first time. Antioxidant bioassays in vitro using DPPH and ABTS were performed, and the results showed that compound 3 exhibited modest antioxidant activity with IC50 values of 0.0321 mg/mL and 0.0319 mg/mL, respectively. An in silico study of PPARα agonistic activities of compounds 2 and 3 was conducted by molecular docking experiments, revealing that both of them occupied the active site of PPARα via hydrogen bonds and hydrophobic interactions effectively. This study enriched both the phytochemical and pharmacological profiles of O. japonica.

Recent studies on chemical constituents of Ophiorrhiza plants.

Ophiorrhiza plants (Family Rubiaceae) are known to produce diverse monoterpenoid indole alkaloids including camptothecin with potent antitumor activity. This review contains a summary of recent chemical studies reported over the past 10 years regarding alkaloids (monoterpenoid indole and tetrahydroisoquinoline alkaloids, and cyclopeptide) in Ophiorrhiza plants. In addition, the alkaloid biosynthetic pathways based on their reported structures were proposed.