Vacuum Liquid Chromatography Research Articles

Phytochemical and Biological Characterization of the Fractions of the Aqueous and Ethanolic Extracts of Parthenium hysterophorus

In this study, the fractions of the aqueous (AE) and ethanolic (EE) crude extracts of Parthenium hysterophorus were evaluated for their phytochemical composition, cytotoxic, and antioxidant activity. The two extracts were subjected to a fractionation by vacuum liquid chromatography, obtaining seven fractions for each extract. These fractions were evaluated for the presence of phenolic compounds by reverse phase high performance liquid chromatography coupled to mass spectrometer (RP-HPLC-MS) analysis. Their cytotoxic activity was tested with a hemolysis assay. The antioxidant activity was evaluated with the Trolox equivalent antioxidant capacity (TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and hydroxyl radical (–OH) scavenging assays. In addition, the effect of the fractions on the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), from human erythrocytes, was evaluated. The phytochemical screening by RP-HPLC-MS mainly showed the presence of flavonoids and hydroxycinnamic acids. The hemolysis assay exhibited a low cytotoxic activity by the fractions of the AE, but the fractions of the EE exhibited a hemolytic effect. The fractions of the AE and EE showed significant antioxidant activity to inhibit radicals in the three radical scavenging assays. Moreover, only some fractions of the AE showed a significant increase in the activity of the SOD enzyme, while the activity of CAT exhibited a significant increase by the fractions of the two extracts. The fractions of the AE and EE of P. hysterophorus have phytochemicals with antioxidant activity to inhibit radicals and increase the activity of in vitro antioxidant enzymes.

ISOLATION, IDENTIFICATION AND BIOASSAY OF FLAVONOIDS FROM Bouea macrophylla GRIFF.

Bouea macrophylla Griff., a species belonging to the Anacardiaceae family is a flowering plant native to Southeast Asia and also known as kundang, kundang daun besar, and setar in Malaysia. The fruit can be eaten raw or as a pickle, while the young leaves can be consumed as salads. It has been claimed to be able to accelerate wound healing, prevent cancer, reduce the risk of stroke, and be good for blood circulation. The previous study on the plant from the same genus, known as B. oppositofolia has shown the presence of various. The present study was designed to isolate and elucidate flavonoids from this plant. The twig extract of kundang was purified by using several chromatographic techniques including Vacuum Liquid Chromatography (VLC), Column Chromatography (CC), and preparative-Thin Layer Chromatography (pTLC). The structures of isolated compounds were characterized by using spectroscopic methods including Nuclear Magnetic Resonance (NMR), infrared (IR), and ultraviolet (UV) spectral data, as well as comparison with the data reported in the literature. Five flavonoids were isolated and purified from the twigs of B. macrophylla which includes one flavanonol known as garbanzol; one flavonol which is resokaempferol; one flavandiol characterized as catechin; and two flavandiol known as mollisacacidin and guibourtacacidin. The results of the glucose uptake experiment indicated that the extract and compounds tested affected the glucose uptake rate of the insulin-resistant C2C12 cell line as compared to the standard. This is the first report describing the elucidation of the stated compounds from B. macrophylla as well as its glucose uptake study.

Active Blood Pressure Lowering Fractions from the Aqueous Extract of the Leaves of Phyllanthus amarus Schum and Thonn (Euphorbiaceae)

Background: The blood pressure-lowering effffect of aqueous leaves extract of Phyllanthus amarus has been reported in earlier work in normotensive albino rabbits. The effffects of organic solvent fractions were evaluated in Wistar rats. Methods: The aqueous fraction obtained from the crude extract of the leaves was evaluated for blood pressure-lowering effffect in anaesthetized normotensive Wistar rats, at the graded doses of 2.5-40 mg/kg after which it was subjected to vacuum liquid chromatography (VLC) using combinations of chloroform, ethyl acetate, methanol and water. This yielded eleven fractions which were bulked into six samples (A-F) after thin layer chromatograph analysis, and samples A, B and C were evaluated for activities on the blood pressure of hypertensive Wistar rats at the graded doses of 5 to 20 mg/kg. Fraction “C” showed the highest blood pressure lowering effffect and was further subjected to column chromatographic and VLC analysis, to obtain fraction (BVLC2) which was evaluated for possible blood pressure lowering effffect in hypertensive Wistar rats. Results: The aqueous fraction caused a dose de pendent decrease in the blood pressure of normotensive Wistar rats, decreasing the mean arterial blood pressure (MAP) from a basal level of 104.58 ± 8.29 mmHg to 45.22 ± 6.71 mmHg (p<0.0001; 66.4%) at the dose of 40 mg/kg. Fraction “C” caused signifificant decrease in the MAPfrom 116.99 ± 10.28 mmHg to 68.33 ± 6.78 mmHg (p < 0.001; 0.01%) at the dose of 20 mg/kg, while the fraction BVLC2 decreased the MAPfrom 146.11 ± 8.29 mmHg to 79.33 ± 6.18 mmHg (***p< 0.001) at the dose of 10 mg/kg, indicating a trend of increasing potency along the course of purifification. Conclusion: The aqueous fraction of the leaves of Phyllanthus amarus yielded column chromatographic sample (BVLC2), that might possibly serve as a lead sample from which pure active hypotensive constituent(s) could be isolated. 

Metabolites of Colletrotricum species, an endophytic fungus isolated from Vernonia amygdalina Del possess antimicrobial and antioxidant activities

The endophytic fungus, colletrotricum species, isolated from the leaves of Vernonia amygdalina was investigated for its chemical constituents and biological activity. The pure fungus was grown using solid fermentation on rice medium and the metabolites were extracted using ethyl acetate. Further purification of the extract was carried out using vacuum liquid chromatography (VLC) and gel chromatography on Sephadex LH-20. The chemical constituents were detected by dereplication using HPLC-DAD. Antimicrobial assay of the Sephadex fractions EC3-1 and EC3-3 were evaluated using agar well diffusion assay against seven pathogenic microorganisms (Staphylococcus aureus, Escherichia coli, Salmonella typhi, Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans). EC3-3 was also subjected to antioxidant assay using DPPH free radical scavenging model. HPLC-DAD analysis of the VLC fractions revealed the presence of compounds like Hydroxybenzaldehyde, Cladosporin, Desmethyldichlorodiaportin and p-Hydroxybenzoic acid. EC3-1 and EC3-3 showed mild antimicrobial activity against S. aureus, S. typhi and A. niger strains, with minimal inhibitory concentrations ranging from 0.19 to 0.46 mg/mL. EC3-3 was found to exhibit very high ability to scavenge DPPH radicals with IC50 of 3.16 µg/mL compared to Ascorbic acid (IC50 of 50.72 µg/mL). HPLC analysis of EC3-3 revealed the presence of Palitantin, which has been shown to possess antimicrobial and antioxidant activity, and two unidentified major peaks. The endophytic fungus, Colletotrichum species isolated from Vernonia amydalina produced bioactive metabolites which exhibited antimicrobial and antioxidant activities thus projecting Vernonia amydalina as a potential source of fungal endophytes that can be further studied for generation of novel bioactive compounds.

The inhibitory effect of α-amylase enzyme from fraction and isolate of mareme leaves (Glochidion arborescens Blume.)

Mareme leaves (Glochidion arborescens Blume) is a plant that has potential as an antidiabetic agent. A previous study reported that ethanol extract of Mareme leaves exhibits active inhibitory activities of α-amylase enzyme. This study aimed to determine the α-amylase enzyme inhibitory activities of fraction and isolate from Mareme leaves. The ethanol extract of Mareme leaves was fractionated by using vacuum liquid chromatography. Then, it was isolated and purified using a chromatotron, a radial chromatographic method. The fraction and isolate were assayed for α-amylase enzyme inhibitory activities. The result indicated that Mareme leaves could inhibit an active α-amylase enzyme inhibitory activity with an IC50 value of 28.262 ppm. Mareme leaves isolate has a very active inhibition (IC50 value = 21.024 ppm) comparable with acarbose, a positive control (IC50 value = 19.486 ppm). In addition, the statistical analysis using the t-test also resulted in a significant difference in average inhibition of α-amylase enzyme between fraction and isolate of Mareme leaves. The study concludes that the fraction and isolate of Mareme leaves are potential inhibitors of α-amylase enzyme in reducing calorie intake. As such, this plant extract has been proven that it could be a potential antidiabetic or anti-obesity agent to prevent or treat chronic diseases.

Detection, isolation and identification of more bioactive compounds from Fusarium equiseti, an endophytic isolated from Ocimum gratissimum

Endophytes have continued to gain fame due to their ability to produce an array of secondary metabolites within the host system with huge untapped pharmacological potentials. This study was carried out to further identify and isolate novel therapeutic compounds from Fusarium equiseti, an Endophytic fungus isolated from leaves of Ocimum gratissimum. Endophytic fungal isolation, fungal fermentation, and extraction of secondary metabolites were carried out using standard laboratory methods. The crude extracts of Fusarium equiseti were subjected to further chromatographic techniques using vacuum liquid chromatography, Sephadex LH 20 and semipreparative HPLC for isolation of bioactive compounds. The fractions and the isolated compounds obtained were further subjected to high performance liquid chromatography-Diode Array Detector (HPLC-DAD). The analytical HPLC led to the further detection of many bioactive compounds namely: Enniatin A, Aureonitol, Serasinoside H1, Altenusin, Aplysinamisin, benzylnitril, ruspolinone and Orientin. Semipreparative HPLC led to the isolation of 6 pure compounds of which two were identified as benzylnitril, and ruspolinone. The remaining four were not identified due to lack of library hits. The detected and the isolated compounds have been previously shown to exhibit a wide array of biological activities including antiviral, antifungal, hepatoprotection, antibacterial, anticancer, cytotoxic, and antioxidant properties. The unidentified compounds may hold enormous potential as new bioactive lead compounds for development into novel therapeutic agents. Therefore, the Endophytic fungus, Fusarium equiseti should be harnessed for its potential pharmacological, pharmaceutical, agricultural and industrial applications.

Antimalarial Activity of Extract and Fractions of Sauropus androgynus (L.) Merr.

Sauropus androgynus (L.) Merr., in the Indonesian local name known as “Katuk,” is a tropical shrub plant of the family Euphorbiaceae. Based on genus and chemotaxonomic approaches, as well as in vitro testing of Plasmodium falciparum, leaves of S. androgynus are presumed to have an active compound content as an antimalarial. The current study aims to investigate the antimalarial activity of 96% ethanol extract and fractions of S. androgynus leaves. The ethanolic extract was fractionated using the vacuum liquid chromatography (VLC) method with three solvents of different polarities (n-hexane, chloroform, and 96% ethanol). The fraction obtained was then evaluated for antimalarial activity against P. falciparum 3D7 strain. The ethanolic extract was evaluated for antimalarial suppressive and prophylactic activity against P. berghei-infected mice, as well as inhibitory activity against the heme detoxification process in vitro. Fractionation of ethanolic extract resulted in seven combined fractions, with the most active fraction being FV (50% inhibitory concentration (IC50) = 2.042 µg/mL). The ethanolic extract showed good parasitic suppressive (therapeutic) activity with a median effective dose (ED50) value of 15.35 mg/kg body weight. In a prophylactic test, ethanolic extract showed parasite growth inhibitory activity of 67.74 ± 9.21% after the administration of 400 mg/kg body weight for 4 days before infection, and 65.30 ± 10.44% after the administration of 200 mg/kg body weight for 8 consecutive days (4 days before and after infection). The ethanolic extract also showed an effect in inhibiting the formation of β-hematin of about 26.87–79.36% at a concentration of 0.1–4 mg/mL and an IC50 value of 0.479 mg/mL. The S. androgynus leaves were shown to have antimalarial activity in vitro and in vivo, where ethanolic extract were more active compared with the fraction obtained. The antimalarial properties of the extract showed a higher suppressive activity than prophylactic activity.

Extract and Fractions from Soil Bacteria (Streptomyces canus ATCC 12647) Possess Antimicrobial and Anti-Oxidative Potential in vitro

Streptomyces species are the most prolific producers of antibiotics within the group actinobacteria. The in-vitro antimicrobial and antioxidant activities of the methanol (MeOH) extract and vacuum liquid chromatography (VLC) fractions of a soil bacteria Streptomyces canus ATCC 12647 were evaluated. Agar well diffusion method was used for the antimicrobial assay, while phosphomolybdate and DPPH radical scavenging methods were used for the antioxidant assay. The antimicrobial assay showed remarkable activities against Bacillus subtilis, Staphylococcus aureus, and Candida albicans. Also, the extract and fractions showed good in-vitro antioxidant activities in both models. Our results showed that extract and VLC fractions from bacterial isolate had good antimicrobial and antioxidant activities.

Identifikasi Senyawa Fraksi Larut n-Heksana Rimpang Temu Mangga (Curcuma mangga Val) Menggunakan LC-MS/MS dan Review Potensinya sebagai Antibakteri

Plants are like the pharmaceutical industry that provides products that have the potential as active medicinal ingredients. In order to search for compounds useful for health, the purpose of this study was to identify secondary metabolites of Curcuma mango (temu mango) growing in the experimental garden of Biopharmaca LPPM IPB and review its activity as antibacterial. Identification begins with phytochemical screening, then separation and purification by chromatographic techniques. Intersection mango simplicia was extracted with methanol, and the crude extract of methanol was partitioned with n-hexane, ethyl acetate, and methanol, respectively. The n-hexane soluble fraction was carried out by phytochemical tests, fractionation by vacuum liquid chromatography (KCV) and radial (KR), and the selected fractions was analyzed by LC-MS/MS. The results of the phytochemical test of the soluble n-hexane fraction showed a positive presence of alkaloids and terpenoids through successive tests with Mayer, Wagner, Dragendorff (alkaloid), and Lieberman-Burchard reagents. Fractionation of the n-hexane soluble fraction with KCV resulted in 9 fractions (H1-H9) and the H5 fraction showed good separation between spots in the n-hexane: ethyl acetate eluent mixture (97:3). Purification of H5 with KR obtained 7 fractions (H5.1-H5.7). Based on LC-MS/MS, the H5.2 fraction was dominated by the terpenoid group, and the results of the literature review showed the contribution of terpenoids as antibacterial compounds

Acute toxicity and anticancer potential of knobweed (Hyptis capitata) ethanolic leaf extract and fraction

Ethanolic leaf extract of Hyptis capitata Jacq. showed a toxic effect on Artemia salina larvae1. The extract was simplified through fractionation using vacuum liquid chromatography to trace compounds with biological activity. The F1, F2, F3 and F4 fractions were used in the acute toxicity test on A. salina larvae. F1 showed the highest toxicity value of 196.77±2.7 µg/ml. As an initial screening for its anticancer potential, ethanolic leaf extract and F1 fraction were subjected to cytotoxicity test on T47D cells to obtain an IC50 value of 45.32±2.0 and >1000 µg/mL. The LC50 of the F1 fraction and its cytotoxicity level decreased compared to the ethanolic leaf extract. Based on GC-MS analysis, the ethanolic leaf extract and the F1 fraction contained neophytadiene, hexadecanoic acid, methyl ester, hexadecanoic acid, ethyl ester and heptadecanoic acid, 16-methyl-, methyl ester. Meanwhile, ?-sitosterol, docosanoic acid, ethyl ester and squalene are only found in the ethanolic leaf extract. The compounds contained in ethanolic leaf extract work synergistically. This implies the ethanolic leaf extract has the potential to be developed as an anticancer candidate.

Isolation of secondary metabolite compounds of Coffee Benalu leaves (Loranthus parasiticus (L.) Merr.) and its antibacterial activity test

Coffee parasite leaves (Loranthus parasiticus (L.) Merr) is one of the plants that can be used as a medicinal material commonly obtained in various subtropical regions or tropical regions. This study aims to isolation of secondary metabolite compounds from methanol extract of coffee leaves and antibacterial activity by of disc diffusion and microdilution methods against S. aureus bacteria, S. mutans, and S. viridians. The result of antibacterial activity against S. aureus bacteria, S. mutans, and S. viridians respectively is 7.5 mm; 7.9 mm; and 8 mm which indicates that the ability to inhibit the growth of methanol extract bacteria 1% coffee leaves belongs to the medium category. The results of the microdilution method in determining the value of KHM against bacteria S. Aureus, S. Mutans, and S. Viridians are equal to the KHM value of 5000 μg/mL. Meanwhile, the MBC value for S. aureus was >5000 g/Ml, for S. mutans was >5000 g/mL and for S. viridians it was 5000 g/mL, indicating that the methanol extract of the coffee parasite leaves was only an inhibitor. Isolation of secondary metabolite compounds is carried out by fractionation using Liquid Vacuum Chromatography and Gravitational Column Chromatography which are further characterized using the GC-MS instrument. The results of the isolation of secondary metabolites from the methanol extract of the leaves of the coffee parasite (Loranthus parasiticus (L.) Merr.) showed that the leaves of the coffee parasite contained any 9 compounds.

Active Antialopecia Chemical Identification of Merremia peltata Leaves and Computational Study toward Androgen Receptor Using Molecular Docking and Molecular Dynamic Simulation

Alopecia is a health condition in which the hair loses its function in some or all of the body. Alopecia occurs due to various genetic, environmental, and nutritional factors. One of the methods developed to treat alopecia is through inhibition of the enzyme 5-α-reductase, which converts testosterone into its more potent metabolite, dihydrotestosterone (DHT). In ethnomedicine, the leaves of Merremia peltata are used by the people of Sulawesi as a remedy for baldness. Therefore, in this study, an in vivo study was conducted on rabbits to investigate the antialopecia activity of the ethanolic extract of M. peltata leaves. The purified M. peltata leaf extract was fractionated using vacuum liquid chromatography with several solvents to produce fractions F1–F5. Each fraction was then retested in vivo in rabbits, and its content was then analyzed by LC-MS. An in silico study was then carried out using minoxidil as a comparison ligand; 17 compounds derived from M. peltata leaves were identified as antialopecia compounds through prediction of molecular interactions and molecular dynamics simulation and prediction of absorption, distribution, metabolism, excretion, and toxicology (ADME-Tox). The assay results showed that fractions F2 and F3 had a better effect on hair growth compared to the positive control, and the test compound obtained from the LC-MS analysis, bufotalinin, had a strong binding energy to the receptor in the molecular docking interaction study: −5.99 kcal/mol compared to −4.8 kcal/mol for minoxidil. Molecular dynamics simulation analysis with complex stability parameters based on solvent-accessible surface area (SASA), principal component analysis (PCA), root mean square deviation (RMSD), and root mean square fluctuation (RMSF) showed that bufotalinin has good affinity for androgen receptors. ADME-Tox prediction for bufotalinin showed good results for the parameters of skin permeability, absorption, and distribution. Therefore, bufotalinin, a steroid compound, is a potential androgen receptor antagonist and could be useful in the treatment of alopecia.

ISOLASI DAN KARAKTERISASI SENYAWA FENOLIK DARI DAUN PUTAT (Planchonia valida Blume) (ISOLATION AND CHARACTERIZATION OF PHENOLIC COMPOUND FROM PUTAT LEAVES (Planchonia valida Blume))

Gallic acid have been isolated from the leaves of the Putat plant (Planchonia valida Blume). This study aimed to isolate and characterize isolates from Putat leaves by phytochemical and FTIR spectrophotometry. The research procedures included extraction by maceration, partitioning by fractionation, vacuum liquid chromatography (KCV), gravity column chromatography (KKG), thin layer chromatography (TLC), and FTIR analysis. A total of 1400 grams of putat leaf powder was macerated to produce 159.243 grams (11.38%) of methanol extract. A total of 101 grams of methanol extract was dechlorophyllated to obtain 21.430 grams (21.21%) of dechlorophyllated extract. The dechlorophyllated extract was partitioned gradually with n-hexane and ethyl acetate to produce n-hexane fractions of 0.341 grams (1.591%), and ethyl acetate of 10.143 grams (47.33%), and methanol of 9.170 grams (42.79%). A phytochemical test showed that in the dechlorophyllated extract, the methanol and ethyl acetate fractions contained alkaloids, phenolics, flavonoids, and terpenoids while the n-hexane fraction contained alkaloids and flavonoids. A total of 7 grams of ethyl acetate fraction was fractionated by vacuum liquid chromatography to produce fractions B1 to B8. The B4 fraction was continued to the separation step by gravity column chromatography so that the fractions S1 to S9 were obtained. The S4 fraction was continued to the separation stage using preparative thin layer chromatography (TLC) to produce isolates S4.1 to S4.5. Isolate S4.1 was tested for purity by two-dimensional TLC showed that the isolate was not pure. The results of the phytochemical test with 5% FeCl3 spray reagent indicated a positive phenolic compound which was strengthened by FTIR characterization of the presence of –OH, -CH aromatic, -CH aliphatic, C=C, C=O and C-O goups. Based on these results, it was concluded that the isolate from the dechlorophyllated extract of Putat leaves belonged to phenolic compounds.

Phytochemical Screening and Antibacterial Activity of the Vacuum Liquid Chromatography Fractions of Mentha pulegium Growing in Iraq

Mentha pulegium, a species of Lamiaceae family, is flowering plant native to Europe, North Africa and the Middle East. Crushed M. pulegium leaves exhibit a very strong fragrance like spearmint. The objective of the study was to investigate the phytochemical constituents and antibacterial activity of M. pulegium stems and leaves vacuum liquid chromatography (VLC) fractions. The powdered leaves and stems were macerated with absolute methanol then filtered using Buchner system. The filtrate was evaporated, then submitted to VLC using different solvents, and their fractions were collected. Phytochemical screening tests were done to investigate the chemical composition of methanol extract. The antibacterial effect of both methanol crude and the five extracts was studied on gram positive (S. aureus and S. haemolyticus) and gram negative (P. aeruginosa and K. pneumoniae) bacteria using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The results indicate the presence of flavonoids, saponins, tannins in plant aerial parts with the presence of alkaloids but in a lesser degree. The result of antibacterial assay showed that MICs of methanol fraction were approximately 900 µg/ml for gram positive bacteria (S. aureus and S. haemolyticus), 225 µg/mL for P. aeruginosa and 450 µg/mL for K. pneumonia. While the best MBCs of the five fractions along with MOHM extract gave, the bactericidal effect in concentrations 450 μg/mL for P. aeruginosa. Our study is the first report on phytochemical screening and antibacterial activity of VLC fractions of Mentha pulegium.

GC-MS Analysis and Antimicrobial Activity of the Extract and Fractions of Bacillus subtilis subsp. subtilis 168 Isolated from a River Bank in Nigeria

The menace of drug resistant pathogens is increasing and their level of evading conventional antimicrobials is rising. It is therefore important to discover new antimicrobials to counter the current challenges. Our preliminary investigation identified Bacillus subtilis subsp. subtilis 168 isolated from soil sample sourced from a river bank in Abuja, Nigeria, as the most potent antibiotic-producing bacteria among the other identified producers. The current study screened for the antimicrobial activity of the extract and fractions of the isolate by broth microdilution method. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the ratio of the MBC/MIC were determined. All the tested pathogens were susceptible to the ethyl-acetate extract (MIC between 28.70 mg/ml and 57.40 mg/ml). The extract displayed bactericidal activity against all tested pathogens (MBC/MIC between 1.00 and 2.00) while Proteus mirabilis was least susceptible. The extract was purified by vacuum liquid chromatography and the fractions challenged with pathogenic strains. The fraction E was the most potent (MIC between 0.09 mg/ml and 0.75 mg/ml) and also bactericidal against all the test microbes (MBC/MIC between 2.00 and 2.11). GC-MS analysis of the purified sub fraction obtained from fraction E identified 13 compounds with different Retention time and peak areas. Among these were three major compounds which include: (i) bis(2-ethylhexyl) phthalate (ii) 1,4-epoxynaphthalene-1(2H)-methanol, 4,5,7-tris(1,1-dimethylethyl)-3,4-dihydro- (iii) D:B-Friedo-B':A'-neogammacer-5-en-3-ol, (3.beta.)-. Our findings suggest that Bacillus subtilis subsp. subtilis 168 isolated locally could serve as a valuable source of lead compounds for pharmaceutical and biotechnological purposes.

Antimicrobial Activity of β-Sitosterol Isolated from Kalanchoe tomentosa Leaves Against Staphylococcus aureus and Klebsiella pneumonia.

<b>Background and Objective:</b> <i>Kalanchoe tomentosa</i> is identified and their different characteristics regarding the antibacterial and antioxidant properties have a vast effect. Fresh <i>K. tomentosa</i> leaves obtained from Bandung, Indonesia was extracted using n-hexane followed by serial dichloromethane maceration. <b>Materials and Methods:</b> N-hexane and ethyl acetate were used to separate the dichloromethane extract using vacuum liquid chromatography and the isolated compounds were recrystallized with n-hexane. <b>Results:</b> About 37 mg of dichloromethane extract was obtained from the extraction process. Recrystallized compound isolates were identified as stigmast-5-en-3-ol or β-sitosterol. Both dichloromethane extract and β-sitosterol isolated compounds showed strong bacteriostatic activity against <i>S. aureus</i> with MIC = 15.63 and 7.81 μg mL<sup></sup><sup>1</sup> and<i> K. pneumonia</i> with MIC = 7.81 and 31.25 μg mL<sup></sup><sup>1</sup>, respectively. However, only dichloromethane extract exhibited a bactericidal effect (7.81 μg mL<sup></sup><sup>1</sup>). <b>Conclusion:</b> The pure β-sitosterol compound was isolated from<i> K. tomentosa</i> dichloromethane extract. Both the dichloromethane extract and the isolated β-sitosterol compound had antibacterial effects against <i>S. aureus</i> and <i>K. pneumonia.</i&gt.

Immunomodulatory effects of cyclotides isolated from Viola odorata in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis

BackgroundMultiple sclerosis (MS) is a demyelinating disease of the central nervous system that causes chronic inflammation. Cyclotides are small plant proteins with a wide range of biological activity, making them a target for researchers to investigate. This study was conducted to investigate the possible effects of cyclotide-rich fractions from Viola odorata as an immunomodulatory agent in an experimental autoimmune encephalomyelitis (EAE) model of MS. MethodsAt room temperature, the plant materials were subjected to maceration in methanol: dichloromethane (1:1; v/v) for 3 days. The extraction was repeated 3 times, and the final concentrated extract was partitioned 3 times by 1/2 volume of double-distilled water. The aqueous phases were separated and freeze-dried. Finally, the crude extract was fractionated by C18 silicagel using vacuum liquid chromatography, with mobile phases of 30%, 50% and 80% of ethanol: water, respectively. The 50%, and 80% fractions were analyzed by HPLC and MALDI-TOF analysis and administrated intraperitoneally to forty-five female C57BL/6 EAE-induced mice, at 5, 25, and 50 mg/kg doses. After 28 days, the animals were evaluated using EAE clinical scoring which was done every 3 days, cytokine levels, and myelination level. ResultsThe results confirmed the presence of cyclotides in V. odorata based on their retention time and the composition of mobile phase in HPLC and the molecular weight of the peaks in MALDI-TOF analysis. It was observed that cyclotides, especially in the 80% fraction group at the dose of 50 mg/kg significantly reduced the clinical scores, inflammation, and demyelination in EAE mice compared with the normal saline group (P<0.05), and the results of this group were comparable with fingolimod (P>0.05). ConclusionIt could be concluded that V. odorata is a rich source of cyclotides which they could be extracted by an easily available process and also, they could be used as immunomodulatory agents in MS, with similar effects to fingolimod.

Coumarins from the Roots of Angelica archangelica and Antibacterial Activity Against Methicillin Resistant Staphylococcus aureus

Angelica archangelica (Fam. Apiaceae) roots were extracted sequentially with n-hexane, dichloromethane (DCM) and methanol (MeOH) using a Soxhlet apparatus. The n-hexane and DCM extracts were fractionated by Vacuum Liquid Chromatography (VLC) over silica gel followed by Preparative Thin Layer Chromatography (PTLC) to yield two coumarins, osthol (1) and osthenol (2). However, the methanol extract was fractionated by solid-phase extraction using C18 cartridge followed by analysis of components by analytical High Performance Liquid Chromatography (HPLC) and purification by preparative High Performance Liquid Chromatography (HPLC) to give two coumarins, bergapten (3) and heraclenol (4) along with sucrose (5). Compounds were identified by a series of 1D (1H and 13C) and 2D (COSY, HSQC and HMBC) NMR spectroscopy and mass spectrometric analysis. The crude extracts (n-hexane, DCM, and methanol) and compound 1, were screened for antimicrobial activity against Gram-positive bacteria (Methicillin resistant Staphylococcus aureus, MRSA and Micrococcus luteus), Gram-negative bacteria, Pseudomonas aeruginosa, and a fungus, Candida albicans. The antimicrobial assays showed that the crude DCM and n-hexane extracts, alongside compound 1, largely inhibited the growth of the microorganisms at high concentrations. Compound 1, which was further tested for antibacterial activity against a series of clinical isolates of methicillin resistance Staphylococcus aureus, showed moderate level of activity against only one strain. Dhaka Univ. J. Pharm. Sci. 20(3): 275-281, 2022 (June) Centennial Special Issue

The Antioxidant and Hematopoietic Effects of the Methanolic Extract Fractions of Ocimum basilicum in Acetaminophen-Induced Albino Rats

This study investigated the antioxidant properties of the fractions of the methanolic extract of Ocimum basilicum (OB) and their effects on the hematological parameters of acetaminophen-induced albino rats. Using vacuum liquid chromatography, the methanolic extract of OB was separated into four fractions (i, ii, iii, and iv) and studied using the DPPH and FRAP assays. Selected fractions ii and iv with the highest antioxidant activities for the animal study. Forty-two albino rats were divided into seven treatment groups (GP) of six animals each, subdivided into three replicates with two rats each. For seven days, rats in GP1 and GP2 were administered water and silymarin (1000mg/kg), gave those in GP4 and GP5 500mg/kg and 1000mg/kg of fraction ii, respectively, while gave those in GP6 and GP7 500mg/kg and 1000mg/kg of fraction iv respectively. Rats in GP3 were administered 750mg/kg of acetaminophen (per os) on day eight only. The results showed that, in comparison with the control groups (1, 2, and 3), rats in GRP4 showed increased (P&lt;0.05) hemoglobin concentration and total WBC count while GRP5 had increased (P&lt;0.05) PCV and MCV. Rats in GRP 6 showed increased (P&lt;0.05) MCV and MCHC compared to the control group, while those in GRP7 showed decreased (P&lt;0.05) PCV and MCV compared with the positive control. It was concluded that the free radical scavenging activity of methanolic extract fractions of OB increased in a dose-dependent manner and high doses (1000mg/kg) of fraction ii showed increased hematopoietic activity.

In vitro effect of three tropical plants on adult Haemonchus placei, an haematophagous nematode from cattle

BackgroundVernonia amygdalina (leaf), Garcinia kola (seed), and Leucaena leucocephala (seed) are three well-known tropical plants used in African ethnomedicine to reduce parasitic worm burdens and are potential sources of alternative solution for controlling parasitic helminths infection in grazing livestock. This study investigated extracts from these plants for anthelmintic activity against adult Haemonchus placei, an haematophagous nematode from cattle abomasa. Powdered plant materials were macerated in acetone and the crude acetone extracts evaluated for anthelmintic activity using H. placei adult worm motility assay. Afterwards, fresh sample of V. amygdalina was macerated successively in chloroform and acetone and the extracts evaluated for anthelmintic activity. The chloroform extract was subjected to phytochemical and FT-IR analyses and fractionated by vacuum liquid chromatography. Anthelmintic data were fitted to a nonlinear regression equation (Log [extract or fraction] vs. lethality; variable slope) to produce best-fit sigmoidal curves and LC50 values computed with associated uncertainty.ResultsOf the three tropical plants, only V. amygdalina was active against adult H. placei with best-fit LC50 of 6.51 mg/mL (95% CI: 5.32–7.75). Evaluation of the two extracts obtained by successive maceration showed that chloroform extract (LC50, 2.46 mg/mL, 95% CI: 1.87–3.28) was 11 times as potent as acetone extract (LC50, 27.01 mg/mL, 95% CI: 21.32–48.57) (α < 0.0001). Chromatographic fractionation of the chloroform extract yielded four fractions (FA-FD) with FB (LC50, 2.38 mg/mL, 95% CI: 1.76–3.28) 2.19 times as potent as FC (LC50, 5.21 mg/mL, 95% CI: 4.40–5.79) against H. placei, while FA and FD were inactive. Phytochemical evaluation of the chloroform extract revealed the presence of saponins, steroids, terpenoids, cardiac glycosides, and the absence of tannins, flavonoids, alkaloids, and anthraquinones. FT-IR structural analysis of chloroform extract indicated the presence of key functional groups which are chemical fragments/ structural motifs known to be present in the two major classes of bioactive compounds (sesquiterpene lactones and steroid glucosides) reportedly to be found in V. amygdalina.ConclusionsThe findings showed that chloroform extract of V. amygdalina leaf possessed relatively good anthelmintic activity against adult H. placei. This could be indicative of its potential usefulness as an anthelmintic phytomedicine to control gastrointestinal nematodes infection in cattle.Key highlightsExtracts of three different plant materials (one leaf, two seeds) were tested against adult Haemonchus placei in vitro;Chloroform extract of Vernonia amygdalina was 11 times as potent as acetone extract;Fractionation of the chloroform extract yielded a bioactive fraction responsible for about 90% of the total lethal effect of the chloroform extract.Bioprocessing of V. amygdalina leaf could produce phytomedicines for organic livestock farming.Graphical abstract