POTENTIAL ANTIDIABETIC ACTIVITIES OF FRACTIONS FROM PURIFIED EXTRACT OF LAWSONIA INERMIS LEAVES IN ALLOXAN–INDUCED DIABETIC MICE

Objective: This research was conducted to determine the potential antidiabetic activity fractions of purified extract Lawsonia inermis leaves in mice (Mus musculus) and identification of the compound. Methods: The method included maceration, purification using ethanol and distilled water was followed by liquid-liquid extraction using ethyl acetate and magnesium sulfate as drying agents. Furthermore, the extract was analyzed using thin layer chromatography (TLC) for testing the purified extract. Fractionation using vacuum liquid chromatography, antidiabetic activity test of fractions at dose 100 mg/kgBW with alloxan induced and compound identification by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) using HPLC connected to a Q-TOF spectrometer equipped with an ESI source, with Phenomenon column C8, and methanol with 0.3% formic acid as solvent. Results: The results showed that from the purification step of L. inermis leaves by vacuum liquid chromatography method, 7 fractions were obtained, i.e. A-G fractions. While the antidiabetic activity of fractions shown by decreasing blood sugar level in mice on the 15th day were 64, 75, 73, 73, 57, 45 and 67%, respectively. The identified compounds from each fraction were the ester groups namely 12-hydroxy-methyl abietate, 9,12-octadecadienoic acid (Z,Z)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl ester, dehydromorroniaglycone, and (E)-hexadecyl-ferulate; the steroid group namely siraitic acid E; phenylpropanoid groups namely umbelliferone and bletilol C, and the alkaloid groups namely moupinamide and valine. Conclusion: L. inermis leaves had activity in lowering blood sugar levels. LC-MS/MS analysis revealed the presence of ester groups, steroid groups, phenylpropanoid groups and alkaloid groups. The presence of these compounds mostly contribute to antidiabetic activity.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.

Isolation of the antidiarrhoeal tiliroside and its derivative from Waltheria indica leaf extract

The antidiarrhoeal effect of Waltheria indica methanol extract and fractions have been reported earlier but, the present work examined the intestinal relaxant effects of two flavonoid-phenyl propanoids isolated from the methanol extract. The active aqueous fraction was subjected to vacuum liquid chromatography using dichloromethane with increasing concentration of ethyl acetate, and that of methanol and water successively. The ten (10) fractions obtained were combined to give seven (7). The fraction 2 (C, D) was subjected to preparative thin layer chromatography on silica gel GF254 (10-40μm) using CHCl3-CH3OH (8:2) to obtain compound coded F2. Fraction 4 (F) was subjected to column chromatography using silica gel (60-120μm mesh) and eluted with dichloromethane with increasing concentrations of methanol. Fractions 9-28 were combined and subjected to column chromatography using chloroform with increasing concentration of methanol. The fractions 1-16 of these were purified on Sephadex LH-20 to obtain compound BAA. The identities of the two compounds were established using spectroscopic methods. The antidiarrheal effect of compound F2 was evaluated on mice using charcoal transit (100,200, 400mg/kg), castor oil (40, 60 mg/kg) while the two compounds were examined for their inhibitory effects on Ach-induced ileum contraction. The effects of the compounds were compared with loperamide (3mg/kg) and atropine (80μg). Compounds F2 and BAA were identified as tiliroside and 3’’’, 5’’’-dimethoxy tiliroside respectively. Tiliroside inhibited the charcoal transition in the animals in a dose dependent pattern with 400mg/ mL eliciting 63.41% inhibition compared to 59.23% produced by loperamide. The compound also elicited significantly (P<0.05) prolonged onset of stooling and reduced the number and weight of stools produced lower than the control. The two compounds drastically inhibited the Ach-induced contractions of the ileum. The compound, tiliroside at 10mg, completely abolished the contraction by Ach unlike 3’’’, 5’’’-dimethoxy tiliroside which reduced the contraction to 1.92% at 20mg. The identified compounds seem to be responsible for the ethnomedicinal use of the plant in treating diarrhea.

Bioassay-guided isolation of cytotoxic constituents of Aframomum melegueta K.Schum. seeds

The seeds of Aframomum melegueta are used extensively in the Nigerian ethnomedicine for the management of cancer. This study therefore aimed at isolating and characterizing its cytotoxic constituents. Methanol extract of the seed was obtained through cold maceration, and it was further partitioned into n – hexane, dichloromethane and ethylacetate. The most active fraction was purified on repeated chromatographic techniques, using vacuum liquid chromatography (VLC), column (CC), and high-performance liquid chromatography (HPLC). The extract, purified fractions and isolated compounds were tested for their cytotoxic activities against the human Rhabdomyosarcoma (RD) and breast (MCF-7) cancer cell lines, using MTT assay. The crude extract and n-hexane fraction were found to be selectively cytotoxic to the cancer cell lines. Bioassay-guided fractionation of the n-hexane fraction led to the isolation of three compounds, which were identified as 6-shogaoal, 6-paradol, and 1-dehydro-6-gingerdione. 6-shogaoal demonstrated the highest cytotoxicity with CC50 values of 0.11 ± 0.02 and 0.25 ± 0.05ìg/mL against RD and MCF-7 cell lines respectively, and these were higher in activity when compared with cyclophosphamide (CC50 = 1.98 ± 0.15 and 0.71 ± 0.7ìg/mL). The presented data validates the ethnomedicinal use of A. melegueta in the treatment of cancer and is also indicative of the potential of 6-shogaol as an anticancer agent.

Senyawa Steroid dari Cocor Bebek (Kalanchoe tomentosa) sebagai Antibakteri Pseudomonas aeruginosa

<p>Plak adalah pembentukan komunitas bakteri yang terorganisir pada permukaan gigi yang berupa lapisan tipis tidak berwarna. Pseudomonas aeruginosa adalah salah satu bakteri yang sangat berperan pada pembentukan plak. Metabolit sekunder yang terkandung dalam tanaman Kalanchoe tomentosa dapat digunakan sebagai antibakteri. Tahapan penelitian diawali dengan maserasi menggunakan pelarut n-heksan dan diklorometana. Ekstrak diklorometana dipisahkan menggunakan Kromatografi Cair Vakum (KCV) dengan n-heksana dan etil asetat sebagai pelarut bergradien selanjutnya direkristalisasi dengan n-heksan, hasil rekristalisasi didapatkan isolat 1. Isolat 1 dikarakterisasi dengan spektroskopi infra-red (IR), ultraviolet (UV) dan nuclear magnetic resonance hydrogen (¹H NMR) serta dibandingkan dengan literatur. Isolat 1 diketahui merupakan stigmast-5-en-3ꞵ-ol atau ꞵ-sitosterol. Pada ekstrak diklorometana dan senyawa ꞵ-sitosterol kemudian ditentukan aktivitas antibakteri secara mikrodilusi didapatkan hasil bahwa ekstrak diklorometana dan senyawa ꞵ-sitosterol bukan hanya menunjukan sifat bakteriostatik tetapi juga bakterisid kuat terhadap bakteri Pseudomonas aeruginosadengan nilai Konsentrasi Hambat Minimum (KHM) &lt;100 µg/mL.</p><p><strong>Steroid Compounds from Cocor Bebek (Kalanchoe tomentosa) as Antibacterial Pseudomonas aeruginosa. </strong>Plaque is a thin, colorless layer that adheres tightly to the surface of the teeth and contains a collection of bacteria. One of the bacteria that can form dental plaque is Porphyromonas aeruginosa. Kalanchoe tomentosa contain secondary metabolite which can be used as antibacterial. Stages of the study begin with maceration using n-hexane and dichloromethane. Dichloromethane extract was separated using Vacuum Liquid Chromatography (KCV) using n-hexane and ethyl acetate solvents and then recrystallized with n-hexane, the recrystallization results were obtained by isolate 1. Isolate 1 was marked by IR spectroscopy, and ¹HNMR and compared with literature. Isolate 1 is known as stigmast-5-en-3ꞵ-ol or ꞵ-sitosterol. In dichloromethane extracts and ꞵ-sitosterol is determined by microdilution by microdilution. The results showed that dichloromethane extract and ꞵ-sitosterol compound not only showed strong bacteriostatic but also bacterisid activity against P. aeruginosa with Minimum Inhibiroty Concentration (MIC) &lt;100 μg / mL.</p>

Potency of Dioscorea sansibarensis (Dioscoreaceae) Leaf Eluates on Callosobruchus chinensis, Linnaeus 1758 (Coleoptera: Bruchidae) in the Protection of Phaseolus vulgaris (Fabaceae)

Post-harvest losses of stored Phaseolus vulgaris to the bean weevil Callosobruchus chinensis have reached levels of significant concern. Governments and health organisations propose the discovery of reliable, healthy and biodegradable pesticides with higher selectivity and a broad spectrum. This study presents investigations on the activities of Dioscorea sansibarensis leaf extracts on the mortality of C. chinensis and reduction of their egg-laying ability. Laboratory experiments under Completely Randomized Design (CRD) were carried out to determine the mortality and anti-oviposition activity of the vacuum liquid chromatography (VLC) eluates of n-hexane (HE), chloroform (CE), ethyl acetate (EE), n-butanol (BE) and methanol (BE) at different concentrations. Bioassay data were subjected to nonparametric statistical analysis and a generalized linear model at p = 0.05. Statistical results showed that the VLC eluates had a mortality activity of 88.01% (R2 = 0.8801). Treatment by 0.025 g of HE and 0.075 g of CE had 9.60 and 11.50, respectively at p = 0.181. These mortality records were high as to 0.1 g of ME, 0.05 g of EE and 0.075 g of BE that recorded 8.55, 8.45 and 7.80, respectively. Treatments by 0.05 g of CE, 0.025 g of HE, 0.05 g of EE and the positive control recorded mortality of 10.50, 9.60, 8.45 and 8.35, respectively. The highest mortality was observed in the treatment by 0.075 g of HE and 0.1 g of HE with 12.85 and 13.70, respectively at p = 0.377. The VLC eluates had an anti-oviposition activity of 24.98% (R2 = 0.2498) on the C. chinensis. The generalized linear model reported Wald Chi-Square values of 4.363; p = 0.037, 0.711; p = 0.399, 9.125; p = 0.003, 4.363; p = 0.037 on the treatment by 0.025 g of CE, 0.05 g of EE, 0.075 g of BE and 0.1 g of ME, respectively. At p = 0.051, 0.1 g of CE and the positive control attained oviposition of 89.25 and 96.75 respectively. The study presents the first documentation of the lethal activity of D. sansibarensis on the C. chinensis pulse beetle. This could help in the development of Integrated Pest Management (IPM) and could help in the elimination or suppression of the infestation. Keywords: anti-oviposition, Callosobruchus chinensis, Dioscorea sansibarensis leaf, mortality, Phaseolus vulgaris, vacuum liquid chromatography (VLC) eluates.

Antimicrobial Evaluation of Extracts and Fractions of Daniella oliveri Leaf on Selected Clinical Enteropathogens

Background: Diarrhoea is one of the major health threats to the populace in the tropics, and also one of the killer diseases in children under 5 years of age. Antimicrobial resistance and its spread pose serious public health threats, hence the need for development of safer and more effective antibacterial agents. Daniella oliveri Hutch and Dalz is used in ethnomedicine for the treatment of diarrhoea and other gastrointestinal disturbances. Objective: To investigate the antimicrobial activity of Daniella oliveri leaves on diarrhoeal pathogens. Methods: Successive plant extraction was carried out with hexane, ethylacetate and methanol using soxhlet apparatus. Methanol extract was fractionated using vacuum liquid chromatography (VLC). Phytochemical screening was done using standard chemical assays. Antibiogram of test isolates was carried out using disc diffusion assay. Antimicrobial activity of plant extract and fractions against strains of diarrhoeagenic Escherichia coli, Salmonella cholerae-suis, Shigella dysenteriae, Proteus mirabilis and Acinetobacter baumannii was determined by agar well diffusion and MIC by agar dilution methods. Kill-kinetics study was carried out using viable count technique. Results: Terpenoids, steroids and anthraquinones were detected in fractions of D. oliveri. Antibiogram assay showed that 66% of isolates were MDR. Extract and fractions produced appreciable zones of inhibition on all challenge organisms except Acinetobacter baumannii. MICs ranged between 6.25-25 mg/ml. Kill kinetics studies showed total kill on susceptible pathogens after 24 hours. Conclusion: This research has shown D. oliveri is a promising drug candidate for the management and treatment of diarrhoea.

Acetylcholinesterase inhibitory activity of extract and fractions from the root of Rauvolfia serpentina(L.) Bth.ex Kurz

Objectives Alzheimer’s disease (AD) is a degenerative brain disease characterized by confusion, behavior changes, decline in memory and cognitive skills. One of the strategies in the treatment of AD is to use acetylcholinesterase (AChE) inhibitors. The current study aims to determine the AChE inhibitory activities of the extract and fractions of the root of Rauvolfia serpentina. Methods Extraction was carried out by maceration method using ethanol, followed by liquid–liquid partition using n-hexane, ethyl acetate and n-butanol. Further fractionation was conducted by using vacuum liquid chromatography (VLC). The AChE inhibitory assays were performed by using Ellmann’s method. Phytochemical screening was carried out by TLC method. Results The ethanolic extract of R. serpentina showed inhibition against AChE enzyme with an IC50 value of 7.46 μg/mL. The extract and fractions showed higher inhibition against butyrylcholinesterase (BChE) compared to AChE. Amongst three fractions obtained, the n-butanol fraction showed the strongest inhibition with an IC50 value of 5.99 μg/mL against AChE. VLC fractionation of the n-butanol fraction yielded 13 subfractions (VLC 1–VLC 13). Four out of 13 subfractions gave more than 80% inhibition against AChE, namely subfractions 4–7, with IC50 values ranging from 4.87 to 47.22 μg/mL. The phytochemical screening of these subfractions suggested the presence of alkaloids. Conclusions The ethanolic extract, as well as fractions of R. serpentina root, are potential for AChE inhibitor. The alkaloid compound may be responsible for this activity.

Specialty Dihydrobenzoxanthone’s Artocarpus Purified By Vacuum Liquid Chromatography (VLC)

One family of plants that are source of bioactive chemicals is the Moraceae.Artocarpus is the main genus of the Moraceae. Several species of the genus Artocarpus have been isolated their secondary metabolites. The main fractions obtained from the VLC are analyzed again by TLC. Fractions that have same spots (Rf) pooled. Purification process on main factions are done repeatedly by radial chromatography. Flavonoid is the most found from Artocarpus plant. Dihydrobenzoxanthone is one of flavonoid derivatives which is successfully isolated from Artocarpus. Dihydrobenzoxanthone is only formed from the flavone with ring B which isoxygenated with pattern of 2', 4' and 5'. Students can be learned dihydrobenzoxanthone’s Artocarpus by laboratory activities.

Antibacterial Potential by Rupture Membrane and Antioxidant Capacity of Purified Phenolic Fractions of Persea americana Leaf Extract

The present research focused on evaluating the antibacterial effect and the mechanism of action of partially purified fractions of an extract of Persea americana. Furthermore, both its antioxidant capacity and composition were evaluated. The extract was fractionated by vacuum liquid chromatography. The antimicrobial effect against Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 15442), and Salmonella choleraesuis (ATCC 1070) was analyzed by microdilution and the mechanism of action by the Sytox green method. The antioxidant capacity was determined by DPPH, FRAP, and ABTS techniques and the composition by Rp-HPLC-MS. All fractions showed a concentration-dependent antibacterial effect. Fractions F3, F4, and F5 (1000 µg/mL) showed a better antibacterial effect than the extract against the bacteria mentioned. The F3 fraction showed inhibition of 95.43 ± 3.04% on S. aureus, F4 showed 93.30 ± 0.52% on E. coli, and F5 showed 88.63 ± 1.15% on S. choleraesuis and 86.46 ± 3.20% on P. aeruginosa. The most susceptible strain to the treatment with the extract was S. aureus. Therefore, in this strain, the bacterial membrane damage induced by the extract and fractions was evidenced by light fluorescence microscopy. Furthermore, the extract had better antioxidant action than each fraction. Finally, sinensitin was detected in F3 and cinnamoyl glucose, caffeoyl tartaric acid, and cyanidin 3-O-(6′′-malonyl-3′′-glucosyl-glucoside) were detected in F4; esculin and kaempferide, detected in F5, could be associated with the antibacterial and antioxidant effect.