Anti-angiogenic activity of Morinda citrifolia extracts and its chemical constituents

Morinda citrifolia L. has been used for the treatment of a wide variety of diseases, including cancer. This study was undertaken to evaluate the anti-angiogenic effect of M. citrifolia fruits and leaves. Anti-angiogenic activity was evaluated in?vivo using the chick chorioallantoic membrane assay. Bioactivity-guided fractionation and isolation were performed to identify the active constituent, and high-performance liquid chromatography analysis was then used to quantify the amount of this active constituent in the active extracts and fraction. The methanol extracts of fruits and leaves of M. citrifolia and the subsequent chloroform fraction of the fruit methanolic extract were found to have potential anti-angiogenic activity and were more potent compared to suramin. Scopoletin was identified as one of the chemical constituents that may be partly responsible for the anti-angiogenic activity of M. citrifolia fruits. The present findings further support the use of M. citrifolia in cancer or other pathological conditions related to angiogenesis.     

Cytotoxic,antibacterial and antioxidant activities of extracts of the bark of Melia azedarach (China Berry)

Nature provides a variety of drugs and medicinal agents derived from plants. This study was conducted to determine antimicrobial, antioxidant and cytotoxic activities of extracts of Melia azedarach bark with methanol/water (9:1 v/v), chloroform, butanol, hexane, water and ethyl acetate. For the determination of the antimicrobial activities, the agar well diffusion method was employed. Cytotoxicity was studied by brine shrimp lethality assay; antioxidant activities were measured using 1,1-diphenyl-2-picrylhydrazyl. The chloroform extract was active against Enterobacter aerogenes and Proteus mirabilis, the ethyl acetate extract had highest antibacterial spectrum against Pseudomonas aeruginosa, the n-hexane extract had highest inhibition against E. aerogenes, the aqueous extract showed highest activities against P. mirabilis, the butanol fraction showed highest activities against E. aerogenes and the methanolic extract was highly active against P. mirabilis.     

Preparative isolation and purification of harpagoside and angroside C from the root of Scrophularia ningpoensis Hemsley by high‐speed counter‐current chromatography

In this study, the bioactive component harpagoside and angroside C in the root of Scrophularia ningpoensis Hemsley was simultaneously separated by high‐speed counter‐current chromatography (HSCCC). A two‐phase solvent system containing chloroform/n‐butanol/methanol/water (4:1:3:2, v/v/v/v) was selected following consideration of the partition coefficient of the target compound. The crude extract (200 mg) was loaded onto a 280‐mL HSCCC column and yielded 22 mg harpagoside and 31 mg angroside C with the purity of higher than 98 and 98.5%, respectively. It is feasible to isolate active compounds harpagoside and angroside C from S. ningpoensis using HSCCC.     

Complementary and Comparative Study on Hypoglycemic and Antihyperglycemic Activity of Various Extracts of Eugenia jambolana Seed, Momordica charantia Fruits, Gymnema sylvestre, and Trigonella foenum graecum Seeds in Rats

In present study, we investigated hypoglycemic and antihyperglycemic potential of five extracts (water, ethanol, methanol, hexane, and chloroform) of four plants (i.e., seeds of Eugenia jambolana, fruits of Momordica charantia, leaves of Gymnema sylvestre, and seeds of Trigonella foenum graecum) alone and/or in combination with glimepiride in rats. Ethanol extract of E. jambolana, water extract of M. charantia, ethanol extract of G. sylvestre, and water extract of T. graecum exhibited highest hypoglycemic and antihyperglycemic activity (most active) in rats among all the extracts, while hexane extracts exhibited least activities. Most active extracts were further studied to dose-dependent (200, 100, and 50 mg/kg body weight (bw)) hypoglycemic and antihyperglycemic effects alone and in combination with glimepiride (20, 10, and 5 mg/kg bw). The combination of most active extracts (200 mg/kg bw) and lower dose of glimepiride (5 mg/kg bw) showed safer and potent hypoglycemic as well as antihyperglycemic activities without creating severe hypoglycemia in normal rats, while higher doses (200 mg/kg bw of most active extracts, and 10 and 20 mg/kg bw of glimepiride) were generated lethal hypoglycemia in normal rats. From this study, it may be concluded that the ethanol extract of E. jambolana seeds, water extract of M. charantia fruits, ethanol extract of G. sylvestre leaves, and water extract of T. graecum seeds have higher hypoglycemic and antihyperglycemic potential and may use as complementary medicine to treat the diabetic population by significantly reducing dose of standard drugs.     

Anti-fertility effects of fractions from Carica papaya (Pawpaw) Linn. methanol root extract in male Wistar rats

The methanol root extracts of Carica papaya (Pawpaw) are used in eastern Nigeria for the treatment of malaria, hepatitis and jaundice. The aim of this study was to investigate the effects of the fractions isolated from C. papaya methanol root extract on fertility in male Wistar rats using sperm counts, percentage defective sperm cells (morphology), biochemical and hormonal assays as biomarkers. The roots of C. papaya were extracted using 80% methanol for 72 h. Oral acute toxicity study was done with the crude extract for 24 h. The extract was fractionated by column chromatography using petroleum ether, chloroform, ethyl acetate and methanol. The petroleum ether fraction was further fractionated on preparative TLC using ethyl acetate–methanol solvent systems to isolate CPFE 1, CPFE 2 and CPFM 1. The 3 fractions (75 mg/kg) were used to treat male Wistar rats orally for 60 days. Animals were euthanized and testes collected, homogenized and used for sperm count and motility. Plasma and serum were used to assay biochemical parameters including aspartate aminotransferase (AST), blood urea nitrogen (BUN), total bilirubin (TB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), triglycerides, hormones (LH and FSH). Histopathological study of the testes, kidney, heart and liver were conducted. Acute toxicity result showed that C. papaya root extract produced no mortalities at the dose of 2000 mg/kg but induced CNS-related symptoms as well as diuresis. The fractions significantly (P < 0.01) produced decreases in sperm counts and increased the percentage of defective sperm cells. There were significant (P < 0.05) increases in aspartate aminotransferase (AST) and blood urea nitrogen (BUN). Histopathological studies showed mild kidney and cardiac hyperaemia, slight hepatic degeneration and severe necrosis of the germinal epithelium of the testes. This study calls for some level of caution in the use of these roots and its extracts/fractions in traditional medicine for treating diseases. On the other hand, it could be a good source of drug for birth control.     

Studies on the polymerization of methyl methacrylate activated by azobisisobutyramidine

Kinetic studies on methyl methacrylate polymerization were carried out with watersoluble 2,2′-azobisisobutyramidine (ABA). The rate of polymerization was proportional to the square root of the initiator concentration in the solvents chloroform, methanol, and dimethyl sulfoxide (DMSO), which confirms the bimolecular nature of the termination reaction. The monomer exponent was unity in chloroform but in methanol and DMSO the rate of polymerization passed through a maximum when plotted against the monmer concentration. This behavior in methanol has been attributed to be due to the enhanced rate of production of radical with increasing proportion of methanol. The rate of decomposition of the ABA has been observed to be faster in methanol than in chloroform. The situation becomes more complicated with DMSO, which was found to reduce the value of δ = (2k_t)^1/2/k_p in methyl methacrylate polymerization. The rate of polymerization was observed to be highly dependent on the nature of the solvent, the rate increasing with increased electrophilicity of the solvent. The dependence of R_p on the solvent has been explained in the light of the stabilization of the transition state due to increased solvation of the basic amidine group of the initiator with the increased electrophilicity of the solvent.     

Bioactivity-guided isolation of ethyl-p-methoxycinnamate, an anti-inflammatory constituent, from Kaempferia galanga L. extracts

This study evaluated the anti-inflammatory effect of Kaempferia galanga (KG) using an activity-guided approach. KG rhizomes were serially extracted with petroleum ether, chloroform, methanol and water. These extracts (2 g/kg each) were tested for their ability to inhibit carrageenan-induced rat paw edema. The chloroform extract was found to exert the highest inhibition (42.9%) compared to control (p < 0.001), hence it was further fractionated by washing serially with hexane, hexane-chloroform (1:1) and chloroform. The chloroform fraction (1 g/kg) showed the highest inhibitory effect (51.9%, (p < 0.001), on carrageenan-induced edema. This chloroform fraction was further fractionated with hexane-chloroform (1:3) and chloroform, and of the two fractions, the hexane-chloroform sub-fraction was the most effective in inhibiting edema (53.7%, p < 0.001). GC-MS analysis of the active sub-fraction identified ethyl-p-methoxycinnamate (EPMC) as the major component, which was re-crystallized. EPMC dose-dependently inhibited carrageenan-induced edema with an MIC of 100 mg/kg. Moreover, in an in vitro study, EPMC non-selectively inhibited the activities of cyclooxygenases 1 and 2, with IC?? values of 1.12 μM and 0.83 μM respectively. These results validate the anti-inflammatory activity of KG which may be exerted by the inhibition of cyclooxygenases 1 and 2. EPMC isolated from this plant may be the active anti-inflammatory agent.     

Antitumor evaluation and multiple analysis on different extracted fractions of the root of Cynanchum auriculatum Royle ex Wight

The root of Cynanchum auriculatum (C. auriculatum ) Royle ex Wight has been shown to possess various pharmacological effects and has recently attracted much attention with respect to its potential role in antitumor activity. The C‐21 steroidal glycosides are commonly accepted as the major active ingredients of C. auriculatum . In this study, the antitumor abilities of different extracted fractions of the root bark and the root tuber of C. auriculatum were investigated by using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay in human cancer cell lines HepG2 and SMMC‐7721. The results showed that the chloroform and ethyl acetate fractions of the root tuber suppressed tumor cell growth strongly. To identify and characterize the chemical constituents of different active fractions, an ultra high performance liquid chromatography with triple‐quadrupole tandem mass spectrometry method was developed for the simultaneous quantitation of eight C‐21 steroidal glycosides. The analysis revealed that the C‐21 steroidal glycosides were concentrated in the chloroform and ethyl acetate fractions, and the total contents of different fractions in the root tuber were significantly higher than those of corresponding ones in the root bark. Furthermore, the C‐21 steroidal glycosides based on different types of aglucones were prone in different medicinal parts of C. auriculatum .     

Bioassay‐guided Isolation and Antioxidant Activity of Sulfur‐containing Compounds from Clinacanthus nutans

Clinacanthus nutans has been used in traditional herbal medicine for cancer prevention, but the specific bioactive compounds responsible for the observed activities have not been explored. Different polar solvents such as methanol, chloroform, ethyl acetate, and hexane were used for the extraction. The extracts, fractions, and isolated compounds were subjected to DPPH and ferric reducing antioxidant potential (FRAP) assays. Methanol extracts show significant free‐radical scavenging activity of 69.09% in DPPH and 56.49% FRAP. Purification of MeOH extracts afforded the fraction FB28 and two new sulfur‐containing compounds, named clinamide D and E ( 1 , 2 ). Compound ( 1 ) proved to be more active with an IC₅₀ value for DPPH radical scavenging of 118.27 ± 0.01 µg/mL and reduction of Fe³⁺–TPTZ complex of 386.24 ± 0.02, higher than that of the standard ascorbic acid. Sulfur‐containing compounds isolated from C. nutans is a potential natural antioxidant.     

Application of off‐line two‐dimensional high‐performance countercurrent chromatography on the chloroform‐soluble extract of Cuscuta auralis seeds

In this study, the chloroform‐soluble extract of Cuscuta auralis was separated successfully using off‐line two‐dimensional high‐performance countercurrent chromatography, yielding a γ‐pyrone, two alkaloids, a flavonoid, and four lignans. The first‐dimensional countercurrent separation using a methylene chloride/methanol/water (11:6:5, v/v/v) system yielded three subfractions (fractions I–III). The second‐dimensional countercurrent separations, conducted on fractions I–III using n‐hexane/ethyl acetate/methanol/water/acetic acid (5:5:5:5:0, 3:7:3:7:0, and 1:9:1:9:0.01, v/v/v/v/v) systems, gave maltol ( 1 ), (−)‐(13S)‐cuscutamine ( 2 ), (+)‐(13R)‐cuscutamine ( 3 ), (+)‐pinoresinol ( 4 ), (+)‐epipinoresinol ( 5 ), kaempferol ( 6 ), piperitol ( 7 ), and (9R)‐hydroxy‐d ‐sesamin ( 8 ). To the best of our knowledge, maltol was identified for the first time in Cuscuta species. Furthermore, this report details the first full assignment of spectroscopic data of two cuscutamine epimers, (−)‐(13S)‐cuscutamine and (+)‐(13R)‐cuscutamine.     

Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots

The aim of this study was to explore the extract/fractions and compounds of Diospyros lotus against various Gram-positive and Gram-negative bacteria strain. The results showed marked susceptibility of extract and its fractions against test pathogens. Among them, chloroform fraction was most dominant and effective against all tested bacteria. The chloroform fraction was subjected to column chromatography which led to the isolation of lupeol (1), 7-methyljuglone (2), β-sitosterol (3), stigmasterol (4), betulinic acid (5), diospyrin (6) and 8-hydroxyisodiospyrin (7). Among the isolated compounds, betulinic acid (5) showed significant activity against most of the tested pathogen. In conclusion, our study validated the traditional uses of the plant in the treatment of infectious diseases which was also strongly supported by the isolated compound, betulinic acid (5).     

Phytochemical investigation and antioxidant activities of methanol extract,methanol fractions and essential oil of Dillenia suffruticosa leaves

Oxidative stress has been known as a key factor of many disorders affecting human beings. Reactive oxygen species (ROS) attack vital biomolecules, weakening their functioning, thus exacerbating diseases. To attenuate oxidative stress-associated diseases a novel approaches of antioxidant therapies have been anticipated. Antioxidants have the potential to inhibit the propagation and formation of ROS. Dillenia suffruticosa is a medicinal plant, used by the local people for the treatment of various ailments. The study aimed to evaluate the phytochemical screening, antioxidative activity, total phenolic and flavonoid contents of methanol extract, fractions and essential oil of D. suffruticosa. Furthermore, the analysis of phytochemicals was done using gas chromatography and mass spectrometry (GCMS). The result showed the existence of alkaloids, anthraquinones, flavonoids, phytosterol, saponins, tannins, triterpenoids and steroids in the methanol extract and fractions of D. suffruticosa. The butanol fraction and methanol extract showed high phenolic (379.00 ± 9.25 and 277.00 ± 3.50 mg/g) and flavonoid values (74.44 ± 2.18 and 34.83 ± 0.71 mg/g) as compared to ethyl acetate, n-hexane and chloroform fractions. The scavenging capacity of butanol fraction and methanol extract was also higher than other fractions. GCMS analysis indicated the presences of various compounds in methanol extract, fractions and essential oil including methyl glycolate, lauryl acetate, phenol, 2,4-bis (1,1-dimethylethyl), 9,12-octadecadienoic acid, hexadecanoic acid, methyl ester, methyl stearate, phenol, benzyl alcohol, 3-hexen-1-ol, acetate and phytol. Thus, methanol extract, fractions and essential oil of D. suffruticosa leaves mainly contain vital phytochemical and shows good antioxidant activity.     

A convenient separation strategy for fungal anthraquinones by centrifugal partition chromatography

As recently shown, some fungal pigments exhibit significant photoactivity turning them into promising agents for the photodynamic treatment of microbial infections or malignant diseases. In the present study, a separation strategy for fungal anthraquinones was developed based on centrifugal partition chromatography. A suitable method was explored employing a methanolic extract of the fruiting bodies of Cortinarius sanguineus (Agaricales, Basidiomycota). An excellent fractionation was achieved using a biphasic solvent system comprising chloroform/ethyl acetate/methanol/water/acetic acid (3:1:3:2:1, v/v/v/v/v) operating in ascending mode. Experiments on an analytical scale with extracts of closely related Cortinarius species exhibited broad applicability of the devised system. Up to six pigments could be purified directly from the crude extract. Preparative-scale fractionation of the methanol extracts of C. malicorius and C. sanguineus demonstrated that up-scaling was possible without compromising selectivity.     

New butyrolactone and other metabolites from the bark of Endlicheria arenosa against of the phytopathogen Colletotrichum tamarilloi

In this work, screening of Lauraceae species for their antifungal activity against Collectotrichum tamarilloi was carried out and the ethanol extract derived from the bark of Endlicheria arenosa was found to be the best candidate. From the ethanolic extract of the bark of E. arenosa, the hexane and chloroform fractions were found to be active, from these five fatty acids were identified and two lactones were isolated. The most active fatty acid was the dodecanoic acid with a minimal inhibitory concentration (MIC) of 78.0 μM. The butyrolactone 3R,4R-licunolide A, it has not previously reported, and licunolide B show both the lowest MIC (55.3 μM). This is the first report of compounds of natural origin as growth inhibitors of C. tamarilloi.     

Antihyperglycemic effect of orthosiphon stamineus benth leaves extract and its bioassay-guided fractions

Preliminary investigations were carried out to evaluate the antidiabetic effects of the leaves of O. stamineus extracted serially with solvents of increasing polarity (petroleum ether, chloroform, methanol and water); bioassay-guided purification of plant extracts using the subcutaneous glucose tolerance test (SbGTT) was also carried out. Only the chloroform extract, given at 1 g/kg body weight (b.w.), significantly reduced (P < 0.05) the blood glucose level of rats loaded subcutaneously with 150 mg/kg (b.w.) glucose. The active chloroform extract of?O. stamineus was separated into five fractions using a dry flash column chromatography method. Out of the five fractions tested, only chloroform fraction 2 (C?2), at the dose of 1 g/kg (b.w.) significantly inhibited (P < 0.05) blood glucose levels in SbGTT. Active C?2 was split into two sub-fractions C?2-A and C?2-B, using a dry flash column chromatography method. The activities C?2-A and C?2-B were investigated using SbGTT, and the active sub-fraction was then further studied for anti-diabetic effects in a streptozotocin-induced diabetic rat model. The results clearly indicate that C?2-B fraction exhibited a blood glucose lowering effect in fasted treated normal rats after glucose-loading of 150 mg/kg (b.w.). In the acute streptozotocin-induced diabetic rat model, C?2-B did not exhibit a hypoglycemic effect on blood glucose levels up to 7 hours after treatment. Thus, it appears that C?2-B functions similarly to metformin, which has no hypoglycemic effect but demonstrates an antihyperglycemic effect only in normogycemic models. The effect of C?2-B may have no direct stimulatory effects on insulin secretion or on blood glucose levels in diabetic animal models. Verification of the active compound(s) within the active fraction (C?2-B) indicated the presence of terpenoids and, flavonoids, including sinensitin.     

Enthalpies of solution of polyvinylacetate in chloroform,acetone, and methanol using titration calorimetry

Heats of mixing of the solvents chloroform, methanol, and acetone with their solutions in polyvinylacetate have been measured at 25°C using a titration calorimeter. Titration of pure solvent to solutions as well as titration of solutions to the pure solvent or to diluted solutions have been performed. The second method, called reverse titration, proved to be more sensitive than normal dilution titration at sufficiently low polymer concentration. Equations describing the relative sensitivity of both titration methods have been developed. The concentration range covered by the experiments is restricted to volume fractions of polyvinylacetate not higher than 0.1–0.15 due to the high viscosities of concentrated solutions. The interaction parameterX _H of the Flory-Huggins theory has been determined from the calorimetric data.X _H is positive for methanol solutions and negative for chloroform and acetone solutions reflecting endothermic and exothermic heat effects respectively.     

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.     

Antiangiogenic Activity and Bioassay‐guided Isolation of Aqueous Extract of Orthosiphon stamineus

Orthosiphon stamineus Benth. (Family: Lamiaceae), a famous traditional medicinal plant in tropical and sub‐tropical region, is being used for treating various ailments including inflammatory conditions. Since anti‐inflammation and antiangiogenesis involve cyclooxygenase‐II pathway, the present study is aimed to carry out antiangiogenic studies and bioassay‐guided isolation on aqueous extract of the plant. After antiangiogenic activity studies on the extract, fractions were prepared by partitioning the extract with hexane, chloroform and ethyl acetate, which were then evaluated for antiangiogenic activity. The most active, hexane fraction (HF) upon activity‐guided isolation gave three compounds, 1 (betulinic acid), 2 (oleanolic acid) and 3 (ursolic acid). The total contents of these three compounds were determined in the extract and its fractions using HPLC. The extract and HF exhibited 20% and 80% antiangiogenic activity, respectively, whereas, chloroform and ethyl acetate fractions were inactive. The three isolated compounds exhibited 100% antiangiogenic activity. Median effective concentrations (EC_50s) of HF, 1, 2 and 3 were found to be 84, 32, 34 and 38 μg/mL, respectively. The results of this study indicate that hexane fraction and the isolated compounds have promising antiangiogenic activity. Additionally, the isolated compounds may be sued as analytical markers to standardize extracts and formulations prepared from O. stamineus.     

Determination of Visnagin inAmmi visnagaHairy Root Cultures Using Solid-Phase Extraction and High-Performance Liquid Chromatography

A high-performance liquid chromatographic method was developed for separation of the furochromone fraction and for determination of visnagin inAmmi visnagahairy root cultures. Lyophilized samples were extracted with chloroform:methanol (1:1, v/v) and purified on solid-phase extraction cartridges. HPLC analyses were performed on a Eurospher 100-C₈Knauer column and the mobile phase was 29:28:526:417 (v/v/v/v) acetonitrile:tetrahydrofuran:30 mM citric acid (pH 3.0):methanol. Quercetin was used as internal standard. Peaks were identified by addition of authentic standards and/or by diode-array detection.     

Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata

The chloroform fraction of methanol (MeOH) extract of the aerial parts of Launaea mucronata was in vitro investigated for cytotoxicity against HCT116, HepG2 and MCF-7 cell lines, and resulted with IC₅₀ = 20.0, 18.6 and 14.30 μg/mL, respectively. The chloroform fraction of the MeOH extract was subjected to further fractionation, which led to the isolation of two new coumarin compounds (6-isobutyl coumarin and 6-isobutyl-7-methyl- coumarin). The structures of the new compounds were elucidated by high field 1D and 2D NMR and ESI-MS spectroscopies. Low polar fractions revealed the isolation of other known triterpene compounds which were identified according to its spectral data and comparison with the literature. New coumarin compounds show high cytotoxicity against MCF-7, HCT116 and HepG2 cell lines.