Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis.

Globally, traditional knowledge systems are a powerhouse of information which can revolutionise the world, if decoded accurately and logically. Plant-based ethno-traditional and folklore curatives/medicines has a firm basis in the psyche of the common masses of West Bengal and Holarrhena pubescens is a representative example of it. This article communication on depicting the anthelmintic efficacy of ethanolic extract and Ethyl acetate fraction of the stem bark of Holarrhena pubescens against the cestode Raillietina spp. through efficacy studies, ultra-structural observations, histochemical and biochemical analysis on some tegumental enzymes i.e., Acid Phosphatase (AcPase), Alkaline Phosphatase (AlkPase), Adenosine Triphosphatase (ATPase) and 5'-Nucleotidase (5'-Nu) along with Gray Level Co-occurrence Matrix (GLCM) analysis of histochemical study. Praziquantel was used as the reference drug. Investigations revealed 10mg/ml dosage of crude extract was the most efficacious dose and amongst the fractions the ethyl acetate fraction showed the most anthelmintic property. Ultrastructural studies through Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) clearly depicted the damage in head, sucker, proglottids, proximal and distal cytoplasm (DC), microtriches (MT), basal lamina (BL), nuclear membrane (NM), and, nucleolus (NL) in the treated worms. Histochemical studies revealed decrease in staining intensity for all the tegumental enzymes in the treated worms compared to control. The GLCM analysis strongly supported the result of histochemical studies. Biochemical studies revealed marked reduction in enzyme activity in the treated worms with maximum reduction in the activity of 5'- Nu (77.8%) followed by ATPase (63.17%).

MicroRNAs from Holarrhena pubescens stems: Identification by small RNA Sequencing and their Potential Contribution to Human Gene Targets.

Holarrhena pubescens is an effective medicinal plant from the Apocynaceae family, widely distributed over the Indian subcontinent and extensively used by Ayurveda and ethno-medicine systems without apparent side effects. We postulated that miRNAs, endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level, may, after ingestion into the human body, contribute to the medicinal properties of plants of this species by inducing regulated human gene expression to modulate. However, knowledge is scarce about miRNA in Holarrhena. In addition, to test the hypothesis on the potential pharmacological properties of miRNA, we performed a high-throughput sequencing analysis using the Next Generation Sequencing Illumina platform; 42,755,236 raw reads have been generated from H. pubescens stems from a library of small RNA isolated, identifying 687 known and 50 new miRNAs led. The novel H. pubescens miRNAs were predicted to regulate specific human genes, and subsequent annotations of gene functions suggested a possible role in various biological processes and signaling pathways, such as Wnt, MAPK, PI3K-Akt, and AMPK signaling pathways and endocytosis. The association of these putative targets with many diseases, including cancer, congenital malformations, nervous system disorders, and cystic fibrosis, has been demonstrated. The top hub proteins STAT3, MDM2, GSK3B, NANOG, IGF1, PRKCA, SNAP25, SRSF1, HTT, and SNCA show their interaction with human diseases, including cancer and cystic fibrosis. To our knowledge, this is the first report of uncovering H. pubescens miRNAs based on high-throughput sequencing and bioinformatics analysis. This study has provided new insight into a potential cross-species control of human gene expression. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for the beneficial properties of this valuable species.

Holanamine, a Steroidal Alkaloid from the Bark of Holarrhena pubescens Wall. ex G. Don Inhibits the Growth of Leishmania donovani by Targeting DNA Topoisomerase 1B.

Leishmaniasis is a group of neglected tropical diseases (NTDs) caused by about 20 species of obligate intracellular protozoan parasites of the genus Leishmania, which occurs in cutaneous, mucocutaneous, and visceral forms. Many researchers have sought to utilize natural products for novel and effective treatments to combat many infectious diseases, including leishmaniasis. Holarrhena pubescens Wall. ex G. Don (Apocynaceae) bark is a rich source of bioactive steroidal alkaloids. The total alkaloidal extract (IC50 6.12 ± 0.117 µg/mL), and the isolated alkaloid, holanamine, showed significant antileishmanial activity (IC50 2.66 ± 0.112 µM against AG83 and 3.80 ± 0.126 µM against BHU-575) against the Leishmania donovani parasite, better than miltefosine (IC50 19.61 ± 0.093 µM against AG83 and 23.20 ± 0.094 µM against BHU-575). Holanamine inhibited the L. donovani topoisomerase 1B (LdToP1B) in a non-competitive manner (IC50 2.81 ± 0.105 µM), indicating that it interacts with the free enzyme and enzyme-DNA complex without inhibiting human topoisomerase. Hydrogen bonding and hydrophobic interactions of holanamine with the N-terminal and hinge region of the large subunit of LTop1B is responsible for its potent antileishmanial activity, as shown by docking studies. Treatment with holanamine causes apoptotic-like cell death by generating cellular and mitochondrial reactive oxygen species, disrupting the mitochondrial membrane potential and inducing ultrastructural alterations in the promastigotes. Holanamine effectively clears intracellular amastigotes but minimally affects host macrophages with no significant cytotoxicity in HEK 293 and L929 cell lines. Thus, our studies show that holanamine can further be used to develop effective antileishmanial agents against evolving drug-resistant parasites.

Hydro-ethanol extract of Holarrhena floribunda stem bark exhibits anti-anaphylactic and anti-oedematogenic effects in murine models of acute inflammation.

BACKGROUND: Holarrhena floribunda (G.Don) T.Durand & Schinz stem bark has anecdotal use in Ghanaian folk medicine for the management of inflammatory conditions. This study was conducted to investigate the in vivo anti-inflammatory activity of the bark extract using models of acute inflammation in male Sprague Dawley rats, C57BL/6 mice and ICR mice. METHODS: A 70% hydro-ethanol extract of the stem bark (HFE) was evaluated at doses of 5-500 mg/kg bw. Local anaphylaxis was modelled by the pinnal cutaneous anaphylactic test. Systemic anaphylaxis or sepsis were modeled by compound 48/80 or lipopolysaccharide, respectively. Clonidine-induced catalepsy was used to investigate the effect on histamine signaling. Anti-oedematogenic effect was assessed by induction with carrageenan. Effects on mediators of biphasic acute inflammation were studied using histamine and serotonin (early phase) or prostaglandin E2 (late phase). RESULTS: HFE demonstrated anti-inflammatory and/or anti-oedematogenic activity comparable to standard doses of aspirin and diclofenac (inhibitors of cyclooxygenases-1 and -2), chlorpheniramine (histamine H1-receptor antagonist), dexamethasone (glucocorticoid receptor agonist), granisetron (serotonin receptor antagonist) and sodium cromoglycate (inhibitor of mast cell degranulation). All observed HFE bioactivities increased with dose. CONCLUSIONS: The data provide evidence that the extract of H. floribunda stem bark has anti-anaphylactic and anti-oedematogenic effects; by interfering with signalling or metabolism of histamine, serotonin and prostaglandin E2 which mediate the progression of inflammation. The anti-inflammatory and antihistaminic activities of HFE may be relevant in the context of the management of COVID-19.

In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies.

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein-protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

The Alkaloid-Enriched Fraction of Pachysandra terminalis (Buxaceae) Shows Prominent Activity against Trypanosoma brucei rhodesiense.

In the course of our studies on antiprotozoal natural products and following our recent discovery that certain aminosteroids and aminocycloartanoid compounds from Holarrhena africana A. DC. (Apocynaceae) and Buxus sempervirens L. (Buxaceae), respectively, are strong and selective antitrypanosomal agents, we have extended these studies to another plant, related to the latter-namely, Pachysandra terminalis Sieb. and Zucc. (Buxaceae). This species is known to contain aminosteroids similar to those of Holarrhena and structurally related to the aminocycloartanoids of Buxus. The dicholoromethane extract obtained from aerial parts of P. terminalis and, in particular, its alkaloid fraction obtained by acid-base partitioning showed prominent activity against Trypanosoma brucei rhodesiense (Tbr). Activity-guided fractionation along with extended UHPLC-(+)ESI QTOF MS analyses coupled with partial least squares (PLS) regression modelling relating the analytical profiles of various fractions with their bioactivity against Tbr highlighted eighteen constituents likely responsible for the antitrypanosomal activity. Detailed analysis of their (+)ESI mass spectral fragmentation allowed identification of four known constituents of P. terminalis as well as structural characterization of ten further amino-/amidosteroids not previously reported from this plant.

Induction of apoptosis in human bladder cancer cells by triterpenoids isolated from Holarrhena antidysenterica through differential reactive oxygen species generation.

A novel triterpenoid, holarol(1),3ß-lup-20(31)-en-3,29,30-triol along with one seco-triterpenoid, dihydrocanaric acid(2) and one known pentacyclic triterpenoid, betulin(3) have been isolated from Holarrhena antidysenterica (L.)Wall. (Family: Apocynaceae). The structures of the compounds were elucidated by extensive IR, 1D, 2D NMR and mass spectrometric analysis. The optimised geometry of (1) was calculated by density-functional theory (DFT) using M06-2X hybrid functional and 6-31 G(D) basis set. The compounds showed differential cytotoxic activities in the cell lines-HeLa, EAC, Raji and T24. Seco-triterpenoid (2) showed highest sensitivity (IC50: 1.710 µg/mL) against the bladder cancer cell line T24 followed by (1) (IC50 9.698 µg/mL) and (3) (IC50 11.769 µg/mL). Compound (1) showed highest reactive oxygen species (ROS) generation in T24 cell line followed by (3) and (2) resulting in induction of apoptosis through activation of caspase, cleavage of PARP and reduction of Bcl-2/Bax ratio. Thus compounds (1), (2) along with (3) could be potent anticancer agents.

An Insight into the Mechanism of Holamine- and Funtumine-Induced Cell Death in Cancer Cells.

Holamine and funtumine, steroidal alkaloids with strong and diverse pharmacological activities are commonly found in the Apocynaceae family of Holarrhena. The selective anti-proliferative and cell cycle arrest effects of holamine and funtumine on cancer cells have been previously reported. The present study evaluated the anti-proliferative mechanism of action of these two steroidal alkaloids on cancer cell lines (HT-29, MCF-7 and HeLa) by exploring the mitochondrial depolarization effects, reactive oxygen species (ROS) induction, apoptosis, F-actin perturbation, and inhibition of topoisomerase-I. The apoptosis-inducing effects of the compounds were studied by flow cytometry using the APOPercentageTM dye and Caspase-3/7 Glo assay kit. The two compounds showed a significantly greater cytotoxicity in cancer cells compared to non-cancer (normal) fibroblasts. The observed antiproliferative effects of the two alkaloids presumably are facilitated through the stimulation of apoptosis. The apoptotic effect was elicited through the modulation of mitochondrial function, elevated ROS production, and caspase-3/7 activation. Both compounds also induced F-actin disorganization and inhibited topoisomerase-I activity. Although holamine and funtumine appear to have translational potential for the development of novel anticancer agents, further mechanistic and molecular studies are recommended to fully understand their anticancer effects.

Metabolic Diversity and Therapeutic Potential of Holarrhena pubescens: An Important Ethnomedicinal Plant.

Holarrhena pubescens is an important medicinal plant of the Apocynaceae family that is widely distributed over the Indian subcontinent. The plant is extensively used in Ayurveda and other traditional medicinal systems without obvious adverse effects. Beside notable progress in the biological and phytochemical evaluation of this plant over the past few years, comprehensive reviews of H. pubescens are limited in scope. It has economic importance due to the extensive use of seeds as an antidiabetic. Furthermore, the plant is extensively reported in traditional uses among the natives of Asia and Africa, while scientifical validation for various ailments has not been studied either in vitro or in vivo. This review aims to summarize information on the pharmacology, traditional uses, active constituents, safety and toxicity of H. pubescens. Chemical analysis of H. pubescens extracts revealed the presence of several bioactive compounds, such as conessine, isoconnessine, conessimine, conimine, conessidine, conkurchicine, holarrhimine, conarrhimine, mokluangin A-D and antidysentericine. Overall, this review covers the ethnopharmacology, phytochemical composition, and pharmacological potential of H. pubescens, with a critical discussion of its toxicity, biological activities (in vitro and in vivo), the mechanism of action, as well as suggestions for further basic and clinical research.

Cytotoxic and cell cycle arrest properties of two steroidal alkaloids isolated from Holarrhena floribunda (G. Don) T. Durand & Schinz leaves.

BACKGROUND: The plant Holarrhena floribunda (H. floribunda; G. Don) is indigenous to sub-Saharan Africa and is traditionally used to treat several ailments. The present study was carried out to isolate and characterize bioactive compounds with anti-proliferative activity present in H. floribunda extracts. METHODS: Compounds were isolated from H. floribunda using the bioassay-guided fractionation technique of repeated column chromatography and the step-wise application of the MTT reduction assay to assess antiproliferative bioactivity. The structures of the compounds were identified mainly using NMR. The effects of the isolated compounds on the viability, cell cycle and proliferation of human cancer cell lines (MCF-7, HeLa and HT-29) as well as the non-cancerous human fibroblast cell line (KMST-6) were investigated. RESULTS: Bioassay-guided fractionation yielded two steroidal alkaloids: holamine (1) and funtumine (2). The MTT reduction assay shows that both compounds exhibited selective dose-dependent cytotoxicity against the cancer cell lines studied. The isolated compounds induced cell cycle arrest at the G0/G1 and G2/M phases in the cancer cell lines with significant reduction in DNA synthesis. The results obtained show that the cancer cells (MCF-7, HeLa and HT-29) used in this study were more sensitive to the isolated compounds compared to the noncancerous fibroblast cells (KMST-6). CONCLUSION: The ability of the isolated compounds to cause cell cycle arrest and reduce DNA synthesis raises hopes for their possible development and use as potent anticancer drugs. However, more mechanistic studies need to be done for complete validation of the efficacy of the two compounds.

Antimalarial and cytotoxic activities of pregnene-type steroidal alkaloids from Holarrhena pubescens roots.

The phytochemical investigation of an alkaloidal extract of Holarrhena pubescens roots led to the isolation and identification of a new pregnene-type alkaloid, mokluangin D (1), together with nine known steroidal alkaloids (2-10). The structure of the new metabolite was determined on the basis of spectroscopic analyses including 1D- and 2D-NMR spectroscopy and mass spectrometry. Compounds 3 and 4 showed potent antimalarial activity against Plasmodium falciparum K1 stain with IC50 values of 1.2 and 2.0 µM, respectively, and showed weak cytotoxic activity against the NCI-H187 cell line with IC50 values of 27.7 and 30.6 µM, respectively. The substituent groups at C-3 and the carbonyl group at C-18 are important for the activity against the P. falciparum K1 stain.

Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection.

BACKGROUND: This study aimed to evaluate the efficacy of combinations of steroidal alkaloids and conessine from the Thai medicinal plant Holarrhena antidysenterica with antibiotics against Pseudomonas aeruginosa strains possessing different efflux-pump-mediated multidrug-resistant (MDR) phenotypes in a Galleria mellonella infection model. METHODS: P. aeruginosa strains with defined mutations that result in the overexpression of the MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux pumps, and a strain with all three of these pumps deleted, were used. In vitro, the effect of combinations of steroidal alkaloids and conessine with antibiotics was compared with antibiotic treatment alone via MIC determination and time-kill assays. Efficacy of combinations of the steroidal alkaloids and conessine with levofloxacin were compared with monotherapies against infections in G. mellonella larvae by measuring larval mortality and bacterial burden. RESULTS: Combination therapies of conessine or steroidal alkaloids with levofloxacin enhanced bacterial inhibition in vitro and restored antibiotic efficacy in vivo compared to the constituent monotherapies. Neither conessine nor the steroidal alkaloids induced any detectable toxicity in G. mellonella larvae. The enhanced efficacy of the combination treatments was most pronounced with conessine and correlated with reduced larval burden of infecting P. aeruginosa. Notably, the enhanced efficacy of conessine/levofloxacin combinations was only detected in the parent strain and strains that overexpressed the MexAB-OprM or MexEF-OprN efflux systems. CONCLUSIONS: Steroidal alkaloids from Holarrhena antidysenterica, and particularly the principal active ingredient conessine, restored levofloxacin efficacy against resistant P. aeruginosa strains possessing efflux-mediated MDR phenotypes. The compounds should be investigated further as a potential novel therapy.

Green synthesis of silver nanoparticles using Holarrhena antidysenterica (L.) Wall.bark extract and their larvicidal activity against dengue and filariasis vectors.

The present study was carried out to evaluate the larvicidal potential of methanol, hexane, acetone, chloroform, and aqueous bark extracts of Holarrhena antidysenterica (L.) Wall. and silver nanoparticles (AgNPs) synthesized using aqueous bark extract against the third instar larvae of Aedes aegypti L. and Culex quinquefasciatus Say. AgNPs were prepared by adding 10 ml of aqueous bark extract in 90 ml of 1 mM silver nitrate (AgNO3) solution. After 5 min of mixing, a change in color from yellow to dark brown occurred indicating the synthesis of AgNPs. Their further characterization was done through ultraviolet-visible spectroscopy (UV-Vis), X-ray diffraction analysis (XRD), field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FT-IR). UV-Vis spectrum of synthesized AgNPs showed a maximum absorption peak at 420 nm wavelength. Crystalline nature of AgNPs was confirmed by the presence of characteristic Bragg reflection peaks in XRD pattern. TEM images have shown that most of the AgNPs were spherical in shape with an average size of 32 nm. FT-IR spectrum of AgNPs showed prominent absorbance peaks at 1012.2 (C-O) and 3439.44 cm-1 (O-H) which represent the major constituents of phenolics, terpenoids, and flavonoids compounds. LC-MS analysis of the bark extract confirmed the presence of carbonyl and hydroxyl functional groups which were directly correlated with FT-IR results. These AgNPs were assayed against different mosquito vectors, and the maximum mortality was recorded against the larvae of A. aegypti with LC50 and LC90 values being 5.53 and 12.01 ppm, respectively. For C. quinquefasciatus, LC50 and LC90 values were 9.3 and 19.24 ppm, respectively, after 72 h of exposure. Bark extracts prepared in different solvents such as methanol, chloroform, hexane, acetone, and water showed moderate larvicidal activity against A. aegypti their respective LC50 values being 71.74, 94.25, 102.25, 618.82, and 353.65 ppm and LC90 values being 217.36, 222.24, 277.82, 1056.36, and 609.37 ppm. For C. quinquefasciatus, their LC50 values were 69.43, 112.39, 73.73, 597.74, and 334.75 ppm and LC90 values of 170.58, 299.76, 227.48, 1576.98, and 861.45 ppm, respectively, after 72 h of treatment. AgNPs proved to be nontoxic against the non-target aquatic organism, Mesocyclops thermocyclopoides Harada when exposed for 24, 48, and 72 h. The results showed that bark extract-derived AgNPs have extremely high larvicidal potential compared to other organic solvents as well as aqueous bark extract alone. These AgNPs, therefore, can be used safely for the control of dengue and filarial vectors that cause severe human health hazards.

In vitro elicitation, isolation, and characterization of conessine biomolecule from Holarrhena antidysenterica (L.) Wall. callus and its larvicidal activity against malaria vector, Anopheles stephensi Liston.

In vitro elicitation of an important compound conessine has been done in the bark-derived callus culture of Holarrhena antidysenterica (L.) Wall. employing different elicitors. For induction of callus, green bark explants excised from field-grown plants were cultured on MS medium augmented with different concentrations (0, 1, 2.5, 5, and 10 µM) of various growth regulators such as BA, IBA, NAA, and 2,4-D either alone or in combinations. The maximum amount of conessine (458.18 ± 0.89d µg/g dry wt.) was achieved in callus developed on MS medium supplemented with 5 µM BA and 5 µM 2,4-D through HPLC analysis. Elicitation in conessine content in the above callus was achieved employing a variety of organic (phenylalanine, tyrosine, chitosan, tryptophan, casein hydrolysate, proline, sucrose, and yeast extract) as well as inorganic elicitors (Pb(NO3)2, As2O3, CuSO4, NaCl, and CdCl2) in different concentrations. The optimum enhancement in conessine content (3518.58 ± 0.28g µg/g dry wt.) was seen at the highest concentration (200 mg/L) of phenylalanine. The enhancement was elicitor specific and dose dependent. The overall increment of the conessine content was seen in the order of phenylalanine > tryptophan > Pb(NO3)2 > sucrose > NaCl > As2O3 > casein hydrolysate > CdCl2 > chitosan > proline > yeast extract > CuSO4 > tyrosine. The isolation and purification of conessine was done using methanol as a solvent system through column chromatography (CC) and TLC. The isolated compound was characterized by FT-IR, 1H-NMR, and HRMS which confirmed with the structure of conessine. The bioassays conducted with the isolated compound revealed a strong larvicidal activity against Anopheles stephensi Liston with LC50 and LC90 values being 1.93 and 5.67 ppm, respectively, without harming the nontarget organism, Mesocyclops thermocyclopoides Harada, after 48 h of treatment. This is our first report for the isolation and elicitation of conessine in the callus culture of H. antidysenterica.

Compounds with inhibitory activity on peristalsis from the seeds of Holarrhena antidysenterica.

Eight compounds were isolated from the seeds of Holarrhena antidysenterica Wall.ex A.DC. On the basis of physico-chemical properties and spectroscopic data, holarrhenanan (1) was identified as a new compound, compounds 2-3 were isolated from H. antidysenterica for the first time, and five known compounds were also obtained. Inhibitory effects of some compounds and extracts to the intestinal peristalsis were evaluated. Results showed that the extracts and compounds 4, 6 exhibited remarkable inhibitory effects with tension inhibition rate of 32.77, 32.77% and amplitude inhibition rate of 59.51, 55.98%, respectively on the vitro rabbit intestinal peristalsis.

Conessine as a novel inhibitor of multidrug efflux pump systems in Pseudomonas aeruginosa.

BACKGROUND: Holarrhena antidysenterica has been employed as an ethnobotanical plant for the treatment of dysentery, diarrhoea, fever, and bacterial infections. Biological activities of the principle compound, conessine including anti-diarrhoea and anti-plasmodial effects were documented. Our previous study reported potency of Holarrhena antidysenterica extract and conessine as resistance modifying agents against extensively drug-resistant Acinetobacter baumannii. This study aimed to investigate (i) whether conessine, a steroidal alkaloid compound, could act as a resistance modifying agent against multidrug-resistant Pseudomonas aeruginosa, and (ii) whether MexAB-OprM efflux pump involved in the mechanism. METHODS: Conessine combined with various antibiotics were determined for synergistic activity against P. aeruginosa PAO1 strain K767 (wild-type), K1455 (MexAB-OprM overexpressed), and K1523 (MexB deletion). H33342 accumulation assay was used to evaluate efflux pump inhibition while NPN uptake assay was assessed membrane permeabilization. RESULTS: Conessine significantly reduced MICs of all antibiotics by at least 8-fold in MexAB-OprM overexpressed strain. The levels were comparable to those obtained in wild-type strain for cefotaxime, levofloxacin, and tetracycline. With erythromycin, novobiocin, and rifampicin, MICs were 4- to 8-fold less than MICs of the wild-type strain. Loss of MexAB-OprM due to deletion of mexB affected susceptibility to almost all antibiotics, except novobiocin. Synergistic activities between other antibiotics (except novobiocin) and conessine observed in MexB deletion strain suggested that conessine might inhibit other efflux systems present in P. aeruginosa. Inhibition of H33342 efflux in the tested strains clearly demonstrated that conessine inhibited MexAB-OprM pump. In contrast, the mode of action as a membrane permeabilizer was not observed after treatment with conessine as evidenced by no accumulation of 1-N-phenylnaphthylamine. CONCLUSIONS: The results suggested that conessine could be applied as a novel efflux pump inhibitor to restore antibiotic activity by inhibiting efflux pump systems in P. aeruginosa. The findings speculated that conessine may also have a potential to be active against homologous resistance-nodulation-division (RND) family in other Gram-negative pathogens.

Antibacterial steroidal alkaloids from Holarrhena antidysenteriaca.

Two new steroidal alkaloids, isoconkuressine and N-formylconessimine, together with 6 known steroidal alkaloids including conkuressine, conessine, isoconessimine, conimine, conarrhimine, and funtudienine, were isolated from the seeds of Holarrhena antidysenteriaca Wall.ex A.DC. Their intrinsic antibacterial activities and synergistic effects with penicillin and vancomycin were analyzed in methicillin sensitive staphylococcus aureus (MSSA) and methicillin resistant staphylococcus aureus (MRSA). Two of the steroidal alkaloids including one new compound (N-formylconessimine) showed potential antibacterial activity and possessed synergistic effects with penicillin and vancomycin, respectively.

Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense.

In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, "sleeping sickness"), we have investigated extracts from the leaves and bark of the West African Holarrhenaafricana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM) against Tbr were recorded for 3ß-holaphyllamine (0.40 ± 0.28), 3α-holaphyllamine (0.37 ± 0.16), 3ß-dihydroholaphyllamine (0.67 ± 0.03), N-methylholaphyllamine (0.08 ± 0.01), conessimine (0.17 ± 0.08), conessine (0.42 ± 0.09), isoconessimine (0.17 ± 0.11) and holarrhesine (0.12 ± 0.08) with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs) could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆5,6 unsaturation slightly increased the activity while hydrolysis of C-12ß ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

THERAPEUTIC POTENTIALS OF ETHANOLIC EXTRACT OF LEAVES OF HOLARRHENA FLORIBUNDA (G. DON) DUR. AND SCHINZ (APOCYNACEAE).

BACKGROUND: Holarrhena floribunda is a plant of wide usage in the Togolese folk medicine. A previous ethnobotanical survey on the latex plants of the Maritime region of the country revealed that this plant was included in several recipes curing malaria and microbial infections. Therefore, this study aimed to seek for the effectiveness of the ethanolic extract of the plant in the treatment of these diseases. METHODS: The antimicrobial test was performed using the agar well-diffusion and the NCCLS broth microdilution methods, while the in vivo antimalarial activity was evaluated following the four-day suppressive test of Peters. The acute toxic effects of the extract were monitored after a single oral dose (5,000 mg/kg body weight) administration in NMRI mice. RESULTS: The results indicated that the ethanolic extract of leaves of H. floribunda was active on Staphylococcus aureus ATCC 29213 and clinical strains of Staphylococcus aureus, Salmonella typhi and Klebsiella pneumoniae with MICs ranging from 0.62 to 1.25 mg/mL. The extract also showed significant parasitaemia suppression in a dose-dependent manner. In the acute toxicity assay, the oral administration of the extract to the mice did not affect the relative weight of vital organs, and there were no signs of toxicity or death during the study period. The LD50 of the tested extract was found to be greater than 5,000 mg/kg, indicating its safety. CONCLUSION: This study demonstrates the antibacterial and antimalarial activities of leaves of H. floribunda and then, supports its medicinal use in the treatment of microbial infections.

Antibacterial steroidal alkaloids from Holarrhena antidysenteriaca / 中国天然药物

Two new steroidal alkaloids, isoconkuressine and N-formylconessimine, together with 6 known steroidal alkaloids including conkuressine, conessine, isoconessimine, conimine, conarrhimine, and funtudienine, were isolated from the seeds of Holarrhena antidysenteriaca Wall.ex A.DC. Their intrinsic antibacterial activities and synergistic effects with penicillin and vancomycin were analyzed in methicillin sensitive staphylococcus aureus (MSSA) and methicillin resistant staphylococcus aureus (MRSA). Two of the steroidal alkaloids including one new compound (N-formylconessimine) showed potential antibacterial activity and possessed synergistic effects with penicillin and vancomycin, respectively.