Anti-mycobacterial diynes from the Canadian medicinal plant Aralia nudicaulis

Ethnopharmacological relevance

Aralia nudicaulis, or wild sarsaparilla, is used as a traditional medicinal plant for the treatment of various illnesses by many of the Canadian First Nations. Iroquois and Algonquin First Nations of Eastern Canada use a tea prepared from dried Aralia nudicaulis rhizome as a cough medicine and for the treatment of tuberculosis. Previous investigations of aqueous extracts of Aralia nudicaulis rhizomes have shown it to possess antimycobacterial activity.

Aim of the study

To isolate and identify antimycobacterial constituents from Aralia nudicaulis rhizomes.

Materials and methods

Methanolic extracts of Aralia nudicaulis rhizomes were subjected to bioassay guided fractionation using the microplate resazurin assay (MRA) to assess inhibitory activity against Mycobacterium tuberculosis strain H37Ra. The antimycobacterial constituents were identified by NMR, MS and polarimetry.

Results

Two C17 polyacetylenes with significant antimycobacterial activity were isolated from the Aralia nudicaulis rhizome extract. The polyacetylenes were identified as (3R)-falcarinol and (3R, 9R, 10S)-panaxydol. Falcarinol and panaxydol displayed MICs of 25.6 μM and 36.0 μM and IC₅₀s of 15.3 μM and 23.5 μM against Mycobacterium tuberculosis H37Ra.

Conclusions

Falcarinol and panaxydol were identified as the principal constituents responsible for the antimycobacterial activity of Aralia nudicaulis rhizomes validating an ethnopharmacological use of this plant by the Canadian First Nations.     

Pharmacological mechanisms underlying the vascular activities of Loranthus ferrugineus Roxb. in rat thoracic aorta

Aim of the study

The present study was aimed to investigate the pharmacological basis for the use of Loranthus ferrugineus in hypertension.

Materials and methods

Loranthus ferrugineus methanol extract (LFME) was obtained using Soxhelt extractor and then successively fractionated using chloroform, ethyl acetate and n-butanol. The n-butanol fraction of LFME (NBF-LFME) was studied using isolated rat thoracic aorta.

Results

NBF-LFME (1.0 × 10⁻⁵ to 3.0 mg/ml) was found to be the most potent to concentration-dependently relax the endothelium-intact phenyephrine (PE, 1 μM)- and high K⁺ (80 mM)-precontracted rat aortic rings. Removal of the endothelium completely abolished the vascular relaxing properties of NBF-LFME. Pretreatment with atropine (1 μM), l-NAME (10 μM), indomethacin (10 μM) and methylene blue (10 μM) significantly blocked NBF-LFME-mediated relaxation. Endothelium-dependent and -independent relaxations induced by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, were significantly enhanced in aortic rings pretreated with NBF-LFME when compared to those observed in control aortic rings. On the contrary, glibenclamide (10 μM), propranolol (1 μM) and prazosin (0.01 μM) did not alter NBF-LFME-induced relaxation.

Conclusions

The results suggest that NBF-LFME induced vascular relaxation by stimulating muscarinic receptors, activating the endothelium-derived nitric oxide-cGMP-relaxant pathway, promoting prostacyclin release and/or possibly through its ability to lengthen the released nitric oxide half-life. The present data further supports previous in vivo findings and explain the traditional use of Loranthus ferrugineus as an anti-hypertensive agent.     

Anti-mycobacterial natural products from the Canadian medicinal plant Juniperus communis

Ethnopharmacological relevance

Common juniper, Juniperus communis, is amongst the plants most frequently used by the indigenous peoples of North America for medicinal purposes. The First Nations of the Canadian Maritimes use infusions of juniper primarily as a tonic and for the treatment of tuberculosis. Previous investigations of extracts derived from the aerial parts of J. communis have shown it to possess anti-mycobacterial activity. The aim of the study is to isolate and identify anti-mycobacterial constituents from the aerial parts of J. communis.

Materials and Methods

Methanolic extracts of J. communis needles and branches were subjected to bioassay guided fractionation using the microplate resazurin assay (MRA) to assess inhibitory activity against Mycobacterium tuberculosis strain H37Ra. The anti-mycobacterial constituents were identified by NMR, MS and polarimetry.

Results

The diterpenes isocupressic acid and communic acid and the aryltetralin lignan deoxypodophyllotoxin were isolated from the J. communis extract. Isocupressic acid and communic acid (isolated as an inseparable 3:2 mixture of cis and trans isomers) displayed MICs of 78 μM and 31 μM and IC₅₀s of 46 μM and 15 μM against M. tuberculosis H37Ra respectively. Deoxypodophyllotoxin was less active, with a MIC of 1004 μM and an IC₅₀ of 287 μM.

Conclusions

Isocupressic acid, communic acid and deoxypodophyllotoxin were identified as the principal constituents responsible for the anti-mycobacterial activity of the aerial parts of J. communis. Although further research will be required to evaluate the relative activities of the two communic acid isomers, this work validates an ethnopharmacological use of this plant by Canadian First Nations and Native American communities.     

Antimycobacterial terpenoids from Juniperus communis L. (Cuppressaceae)

Ethnopharmacological relevance

Juniperus communis is a plant which has been reported as a traditional cure for tuberculosis (TB) and other respiratory diseases.

Aim of the study

The aim of this study was to isolate and identify the constituents responsible for the activity of the n-hexane extract of Juniperus communis roots against Mycobacterium tuberculosis H₃₇Rv and Juniperus communis aerial parts against Mycobacterium aurum. Subsequently, it was to evaluate the activity of the pure isolated compounds against (i) drug-resistant Mycobacterium tuberculosis variants, (ii) non-replicating Mycobacterium tuberculosis and (iii) a range of non-tuberculous mycobacteria (NTM).

Materials and methods

The antimycobacterial activity of Juniperus communis extracts, fractions and constituents was determined against Mycobacterium tuberculosis H₃₇Rv, and against rifampicin-, isoniazid-, streptomycin- and moxifloxacin-resistant variants, using the microplate broth Alamar Blue assay (MABA) method. Isolated constituents were tested against non-replicating Mycobacterium tuberculosis H₃₇Rv, using the low oxygen recovery assay (LORA), and against NTM (Mycobacterium aurum, Mycobacterium phlei, Mycobacterium fortuitum and Mycobacterium smegmatis), using a broth microdilution method. Cytotoxicty studies were performed using mammalian Vero cells.

Results

The antimycobacterial activity of Juniperus communis was attributed to a sesquiterpene identified as longifolene (1) and two diterpenes, characterised as totarol (2) and trans-communic acid (3). All compounds were identified following analysis of their spectroscopic data (1D- and 2D-NMR, MS) and by comparison with the literature and commercial authentic standards when available. Revised assignments for 3 are reported. Totarol showed the best activity against Mycobacterium tuberculosis H₃₇Rv (MIC of 73.7 μM). It was also most active against the isoniazid-, streptomycin-, and moxifloxacin-resistant variants (MIC of 38.4, 83.4 and 60 μM, respectively). Longifolene and totarol were most active against the rifampicin-resistant variant (MICs of 24 and 20.2 μM, respectively). Totarol showed the best activity in the LORA assay (MIC of 81.3 μM) and against all NTM species (MICs in the range of 7–14 μM). Trans-communic acid showed good activity against Mycobacterium aurum (MIC of 13.2 μM). The low selectivity indices (SI) obtained following cytotoxicity studies indicated that the isolated terpenoids were relatively toxic towards mammalian cells. This is the first report of the isolation of (1) and (2) from Juniperus communis roots, and of (3) from the aerial parts. The antimycobacterial activity of (1) and (3), and the activity of (2) against Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium phlei, is reported for the first time. The effect of totarol on drug-resistant variants and non-replicating Mycobacterium tuberculosis has never been published.

Conclusions

The presence of antimycobacterial terpenoids in Juniperus communis aerial parts and roots justifies, to some extent, the ethnomedicinal use of this species as a traditional anti-TB remedy.     

Antiarrhythmic effects of dehydroevodiamine in isolated human myocardium and cardiomyocytes

Ethnopharmacological relevance

Dehydroevodiamine alkaloid (DeHE), a bioactive component of the Chinese herbal medicine Wu-Chu-Yu (Evodiae frutus), exerted antiarrhythmic effect in guinea-pig ventricular myocytes. We further characterize the electromechanical effects of DeHE in the human atrial and ventricular tissues obtained from hearts of patients undergoing corrective cardiac surgery or heart transplantation.

Materials and methods

The transmembrane potentials of human myocardia were recorded with a traditional microelectrode technique while sarcolemmal Na⁺ and Ca²⁺ currents in single human cardiomyocytes were measured by a whole-cell patch-clamp technique. The intracellular pH (pH_i) and Na⁺–H⁺ exchanger (NHE) activity were determined using BCECF-fluorescence in human atria.

Results

In human atria, DeHE (0.1–0.3 μM) depressed upstroke velocity, amplitude of action potential, and contractile force, both in slow and fast response action potential. Moreover, the similar depressant effects of DeHE were found in human ventricular myocardium. Both in isolated human atrial and ventricular myocytes, DeHE (0.1–1 μM) reversibly, concentration-dependently decreased the Na⁺ and Ca²⁺currents. Moreover, DeHE (0.1 and 0.3 μM) suppressed delayed afterdepolarizations and aftercontractions, induced by epinephrine and high [Ca²⁺]_o in atria. In human ventricular myocardium, the strophanthidin-induced triggered activities were attenuated by pretreating DeHE (0.3 μM). The resting pH_i and NHE activity were also significantly increased by DeHE (0.1–0.3 μM).

Conclusions

We concluded for the first time that, in the human hearts, DeHE could antagonize triggered arrhythmias induced by cardiotonic agents through a general reduction of the Na⁺ and Ca²⁺ inward currents, while increase of resting pH_i and NHE activity.     

Cytotoxicity and modes of action of four Cameroonian dietary spices ethno-medically used to treat Cancers: Echinops giganteus, Xylopia aethiopica, Imperata cylindrica and Piper capense

Ethnopharmacological relevance.

Echinops giganteus, Imperata cylindrica, Piper capense and Xylopia aethiopica are four medicinal spices used in Cameroon to treat cancers.

Aim of the study

The above plants previously displayed cytotoxicty against leukemia CCRF-CEM and CEM/ADR5000 cell lines as well as human pancreatic MiaPaCa-2 cells. The present study aims at emphasizing the study of the cytotoxicity and the modes of action of the above plants on a panel of ten cancer cell lines including various sensitive and drug-resistant phenotypes. The study has been extended to the isolation of the bioactive constituents from Echinops giganteus.

Materials and methods

The cytotoxicity of the extracts was determined using a resazurin reduction assay, whereas the caspase-Glo assay was used to detect the activation of caspases 3/7, caspase 8 and caspase 9 in cells treated with the four extracts. Flow cytometry was used for cell cycle analysis and detection of apoptotic cells, analysis of mitochondrial membrane potential (MMP) as well as measurement of reactive oxygen species (ROS).

Results

The four tested extracts inhibited the proliferation of all tested cancer cell lines including sensitive and drug-resistant phenotypes. Collateral sensitivity of cancer cells to the extract of Echinops giganteus was generally better than to doxorubicin. The recorded IC₅₀ ranges were 3.29 µg/mL [against human knockout clones HCT116 (p53^−/−) colon cancer cells] to 14.32 µg/mL (against human liver hepatocellular carcinoma HepG2 cells) for the crude extract from Echinops giganteus, 4.17 µg/mL (against breast cancer cells transduced with control vector MDA-MB231 cells) to 19.45 µg/mL (against MDA-MB-231 BCRP cells) for that of Piper capense, 4.11 µg/mL (against leukemia CCRF-CEM cells) to 30.60 µg/mL (against leukemia HL60AR cells) for Xylopia aethiopica, 3.28 µg/mL [against HCT116 (p53^−/−) cells] to 33.43 µg/mL (against HepG2 cells) for Imperata cylindica and 0.11 µg/mL (against CCRF-CEM cells) to 132.47 µg/mL (against HL60AR cells) for doxorubicin. The four tested extracts induced apoptosis in CCRF-CEM cells via the alteration loss of MMP whilst that of Piper capense also enhanced the production of ROS.

Conclusion

The studied plants are potential cytotoxic drugs that deserve more detailed exploration in the future, to develop novel anticancer drugs against sensitive and otherwise drug-resistant phenotypes.     

In vitro biological activities of niloticane,a new bioactive cassane diterpene from the bark of Acacia nilotica subsp. kraussiana

Ethnopharmacological relevance

Acacia nilotica subsp. kraussiana was reported in African traditional medicine for the treatment of various ailments. Isolation of an active compound in this study from the bark extract may lead to the validation of its efficiency as a traditional crude drug.

Aims of the study

This study aimed to isolate active compound(s) from an ethyl acetate bark extract of Acacia nilotica subsp. kraussiana and to investigate some of its biological activity.

Materials and methods

The isolation process was carried out using bioassay-guided fractionation. The isolated compound was tested for antibacterial activity using the micro-dilution assay; anti-inflammatory activity using the COX-1 and COX-2 assays and investigated for inhibitory effect against acetylcholinesterase using the microplate assay.

Results

A new bioactive compound was isolated and identified as a cassane diterpene, niloticane. Niloticane showed antibacterial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus with MIC values of 4 and 8 μg/mL, respectively. With Gram-negative bacteria, niloticane showed weak activity. MIC values obtained were 16 and 33 μg/mL against Klebsiella pneumonia and Escherichia coli, respectively. In the cyclooxygenase test, niloticane possessed activity with IC₅₀ values of 28 and 210 μM against COX-1 and COX-2, respectively. IC₅₀ values observed with indomethacin (positive control) were 3.6 μM for COX-1 and 189 μM for COX-2. In the acetylcholinesterase test, niloticane showed anti-cholinesterase activity with an IC₅₀ value of 4 μM. IC₅₀ values obtained by the galanthamine (positive control) was 2.0 μM.

Conclusion

The results obtained support the traditional uses of the bark of Acacia nilotica subsp. kraussiana in African traditional medicine for the treatment of some ailments that relate to microbial diseases, inflammation and central nervous system disorders.     

Pharmacokinetic study of salvianolic acid A in beagle dog after oral administration by a liquid chromatography–mass spectrometry method: A study on bioavailability and dose proportionality

Ethnopharmacological relevance

Salvianolic acid A (SAA) is one of the main water-soluble components isolated from Salvia miltiorrhiza Bunge. Pharmacological researches revealed that it had various curative activities after oral and intravenous administration, including beneficial effects on diabetes and its complications, cardioprotective effect, anti-platelet aggregation, and so on. However, there is no report regarding the pharmacokinetics of SAA in beagle dogs after oral administration up to now.

Aim of the study

To study the pharmacokinetics of different doses of SAA in beagle dogs and figure out the absolute bioavailability and dose proportionality of SAA after oral administration.

Materials and methods

Male and female beagle dogs were orally administered SAA 5, 10 and 20 mg/kg randomly. The plasma drug concentration was detected by a rapid, sensitive and reproducible liquid chromatography–mass spectrometry (LC–MS) method. The pharmacokinetic parameters were calculated from plasma concentration–time data using the DAS pharmacokinetic software Data Analysis System Version 3.0 program.

Results

After single-dose oral administration of SAA, the mean peak plasma concentration (C_max) values for groups treated with 5, 10 and 20 mg/kg doses ranged from 14.38 to 38.18 µg/L, and the mean area under the concentration–time curve (AUC_(0–t)) values ranged from 38.77 to 130.33 (µg/L⋅ h). SAA showed lack of dose proportionality over the dose range 5–20 mg/kg, based on the power model. However, the increase in systemic exposure with dose appeared linear. The absolute bioavailability was calculated to range from 1.47% to 1.84%.

Conclusion

The pharmacokinetic properties of SAA in beagle dogs after oral administration were characterized as rapid oral absorption, quick clearance, and poor absolute bioavailability. Systemic exposure exhibited lack of dose proportionality over the dose range 5–20 mg/kg. Furthermore, a readily preparative LC–MS method was demonstrated in this study for the research of traditional Chinese medicine.     

Cochlospermum vitifolium induces vasorelaxant and antihypertensive effects mainly by activation of NO/cGMP signaling pathway

Aim of the study

Cochlospermum vitifolium is a medicinal plant used for the treatment of diabetes, hepatobilary and cardiovascular illnesses. The aim of current study was to determine the in vivo antihypertensive and in vitro functional vasorelaxant mechanism of methanol extract of Cochlospermum vitifolium (MECv) and naringenin (NG).

Materials and methods

Test material was assayed on rat isolated aorta rings test with- and without-endothelium to determine their vasorelaxant mechanism. Also, the in vivo antihypertensive effect was evaluated on spontaneously hypertensive rat (SHR) model. In addition, presence of NG into the extract was confirmed by reverse phase high performance liquid chromatography (RP-HPLC) analysis.

Results

MECv (120 mg/kg) and NG (50 and 160 mg/kg) showed acute antihypertensive effects on SHR when systolic and diastolic pressure were decreased at 1 h and 24 h after administration, respectively. Vasorelaxant effect of MECv and NG was shifted to the right when endothelium-intact aortic rings were pre-incubated with L-NAME (10 μM) and ODQ (1 μM). Also, NG relaxant curves were displaced to the right in the presence of tetraethylammonium (TEA, 1 mM) and 2-aminopyridine (2-AP, 100 μM) on endothelium-denuded aortic rings.

Conclusion

Experiments described above showed that MECv play an important role in hypertension regulation through NO synthesis and may be PGI₂ production and potassium channel activation on excessive endothelial dysfunction conditions. Unfortunately, presence of NG into the extract is not significant on bioactivity of the extract; however, this compound could be tested and evaluated as structural scaffold for future drug design for development of antihypertensive agents.     

Effect of an alkaloidal fraction of Tabernaemontana elegans (Stapf.) on selected micro-organisms

Ethnopharmacological relevance

Bacterial infections remain a significant threat to human health. Due to the emergence of widespread antibiotic resistance, development of novel antibiotics is required in order to ensure that effective treatment remains available. There are several reports on the ethnomedical use of Tabernaemontana elegans pertaining to antibacterial activity.

Aim of the study

The aim of this study was to isolate and identify the fraction responsible for the antimicrobial activity in Tabernaemontana elegans (Stapf.) root extracts.

Materials and methods

The active fraction was characterized by thin layer chromatography (TLC) and gas chromatography–mass spectrometry (GC–MS). Antibacterial activity was determined using the broth micro-dilution assay and antimycobacterial activity using the BACTEC radiometric assay. Cytotoxicity of the crude extract and fractions was assessed against primary cell cultures; lymphocytes and fibroblasts; as well as a hepatocarcinoma (HepG2) and macrophage (THP-1) cell line using the Neutral Red uptake and MTT assays.

Results

The crude root extracts were found to contain a high concentration of alkaloids (1.2%, w/w). GC–MS analysis identified the indole alkaloids, voacangine and dregamine, as major components. Antibacterial activity was limited to the Gram-positive bacteria and Mycobacterium species, with MIC values in the range of 64–256 μg/ml. When combined with antibiotics, additive antibacterial effects were observed. Marked cytotoxicity to all cell lines tested was evident in the MTT and Neutral Red uptake assays, with IC₅₀ values <9.81 μg/ml.

Conclusions

This study confirms the antibacterial activity of Tabernaemontana elegans and supports its potential for being investigated further for the development of a novel antibacterial compound.     

Comparative antioxidant and anti-inflammatory effects of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol

Ethnopharmacological relevance

Zingiber officinale Rosc. (Zingiberaceae) has been traditionally used in Ayurvedic, Chinese and Tibb-Unani herbal medicines for the treatment of various illnesses that involve inflammation and which are caused by oxidative stress. Although gingerols and shogaols are the major bioactive compounds present in Zingiber officinale, their molecular mechanisms of actions and the relationship between their structural features and the activity have not been well studied.

Aim of the study

The aim of the present study was to examine and compare the antioxidant and anti-inflammatory activities of gingerols and their natural analogues to determine their structure–activity relationship and molecular mechanisms.

Materials and methods

The in vitro activities of the compounds [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol were evaluated for scavenging of 1,1-diphenyl-2-picyrlhydrazyl (DPPH), superoxide and hydroxyl radicals, inhibition of N-formyl-methionyl-leucyl-phenylalanine (f-MLP) induced reactive oxygen species (ROS) production in human polymorphonuclear neutrophils (PMN), inhibition of lipopolysaccharide induced nitrite and prostaglandin E₂ production in RAW 264.7 cells.

Results

In the antioxidant activity assay, [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol exhibited substantial scavenging activities with IC₅₀ values of 26.3, 19.47, 10.47 and 8.05 μM against DPPH radical, IC₅₀ values of 4.05, 2.5, 1.68 and 0.85 μM against superoxide radical and IC₅₀ values of 4.62, 1.97, 1.35 and 0.72 μM against hydroxyl radical, respectively. The free radical scavenging activity of these compounds also enhanced with increasing concentration (P < 0.05). On the other hand, all the compounds at a concentration of 6 μM have significantly inhibited (P < 0.05) f-MLP-stimulated oxidative burst in PMN. In addition, production of inflammatory mediators (NO and PGE₂) has been inhibited significantly (P < 0.05) and dose-dependently.

Conclusions

6-Shogaol has exhibited the most potent antioxidant and anti-inflammatory properties which can be attributed to the presence of α,β-unsaturated ketone moiety. The carbon chain length has also played a significant role in making 10-gingerol as the most potent among all the gingerols. This study justifies the use of dry ginger in traditional systems of medicine.     

The antimicrobial,antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea

Ethnopharmacological importance

Many Bauhinia species, including those indigenous to South Africa, are used in traditional medicine across the world for treating ailments such as gastrointestinal tract (GIT) disorders, diabetes, infectious diseases and inflammation.

Aims

Several relevant aspects of different fractions of leaf extracts of Bauhinia bowkeri (BAB), Bauhinia galpinii (BAG), Bauhinia petersiana (BAP), and Bauhinia variegata (BAV) used in South African traditional medicine to alleviate diarrhoea related symptoms were evaluated.

Materials and Methods

The antioxidative activities of the extracts were determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging and ferric reducing antioxidant power (FRAP) methods. In vitro antimicrobial activities of the extracts were determined against bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) and clinical isolates of the opportunistic fungal strains (Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans) using a serial dilution microplate method. The polyphenolic contents were quantified using standard methods, and anti-inflammatory activities of the crude extracts were determined using the cyclooxygenase and soybean 15-lipoxygenase enzyme inhibitory assays. The safety of the extracts was evaluated by determining the cytotoxicity against Vero cell lines.

Results

The acidified 70% acetone crude extract and their fractions had good antiradical potency against the DPPH and ABTS radicals. The methanol soluble portions of the butanol fractions were more potent (EC₅₀ ranges from 0.64±0.05 to 1.51±0.07 and 0.88±0.18 to 1.49±0.09 μg/ml against DPPH and ABTS radical respectively) compared to the standard, trolox and ascorbic acid (EC₅₀ ranges from 1.47±0.24 to 1.70±0.27 μg/ml) for both DPPH and ABTS. The crude extracts contained variable quantities of phenolic content. The crude extracts and their fractions had weak to good antimicrobial activities, inhibiting the growth of the organisms at concentrations ranging from 39 to 2500 μg/ml. The BAG crude extract and its fractions were the most active against the fungi (MICs ranging from 39 to 625 μg/ml) while the BAB extract and its fractions were the least active with the MICs ranging between 39 and 2500 μg/ml. Aspergillus fumigatus was the least susceptible fungus while Cryptococcus neoformans was the most susceptible.The phenolic-rich crude extracts of BAB, BAG, and BAP had moderate to good dose-dependent cyclooxygenase-1 enzyme inhibitory activity with inhibitions between 22.8% and 71.4%. The extracts were however, inactive against cyclooxygenase-2. The extracts had some level of cytotoxicity towards Vero cell lines, reducing cell viability to less than 10% at concentrations more than 50 μg/ml.

Conclusion

The biological activities observed in Bauhinia species provide a scientific basis for the use of the plants in traditional medicines to treat diseases with multi-factorial pathogenesis such as diarrhoea, with each aspect of activity contributing to the ultimate therapeutic benefit of the plants. However, the use of the phenolic-rich extracts of these plants to treat diarrhoea or any other ailments in traditional medicine needs to be monitored closely because of potential toxic effects and selective inhibition of COX-1 with the associated GIT injury.     

Antimycobacterial activity of Citrullus colocynthis (L.) Schrad. against drug sensitive and drug resistant Mycobacterium tuberculosis and MOTT clinical isolates

Ethnopharmacological relevance

Citrullus colocynthis (Cucurbitaceae), a folk herbal medicine and traditionally used natural remedy for tuberculosis in India has been studied to validate its antitubercular activity against drug sensitive and drug resistant (including multidrug resistant) Mycobacterium tuberculosis and Mycobacterium other than tuberculosis (MOTT) bacilli.

Materials and methods

Inhibitory and bactericidal activities of crude extracts, fractions and compounds of Citrullus colocynthis plant, consisting of aerial parts and ripe deseeded fruits were performed against the drug sensitive standard strain of Mycobacterium tuberculosis H37Rv (ATCC 27294), 16 drug resistant strains of Mycobacterium tuberculosis and two MOTT strains, using radiometric BACTEC 460TB system.

Results

Methanolic extract of ripe deseeded fruit of Citrullus colocynthis has shown good activity (MIC≤62.5 µg/ml), whereas among the bioactive fractions, FC IX demonstrated the best activity (MIC 31.2 µg/ml) against Mycobacterium tuberculosis H37Rv. Bioactive FC III, IX and X also inhibited 16 clinical isolates of Mycobacterium tuberculosis consisting of seven non-multidrug resistants, eight multidrug resistants, one extensively drug resistant and two of MOTTs with MICs in the range of 50–125, 31.2–125 and 62.5–125 µg/ml, respectively. Ursolic acid and cucurbitacin E 2-0-β-d-glucopyranoside were identified as the main biomarkers active against Mycobacterium tuberculosis H37Rv (MICs 50 and 25 µg/ml respectively), as well as against the 18 clinical isolates. FC III and FC IX showed better inhibition of drug resistant and MOTT clinical isolates. Minimal bactericidal concentrations of extracts, fractions and compound C-2 were ≥two-fold MIC values.

Conclusions

The study provides a scientific rationale for the traditional use of Citrullus colocynthis fruit in the treatment of tuberculosis. In addition, the study elucidates a broad spectrum antimycobacterial action of Citrullus colocynthis fruit, which can contribute to the development of improved preparation of an antitubercular natural drug for the treatment of drug resistant tuberculosis and MOTT infection as well.     

In vitro relaxant and spasmolytic effects of constituents from Viburnum prunifolium and HPLC quantification of the bioactive isolated iridoids

Ethnopharmacological relevance

Viburnum prunifolium is a North America shrub used in ethnomedicine because of its spasmolytic, sedative, and anti-asthmatic properties.

Aim of the study

Contrasting results were reported in past literature about the active principles of this plant. Our aim was to clarify this matter by evaluating the relaxant and spasmolytic activities of the main constituents obtained from the drug.

Materials and methods

The pharmacological assays were carried out on rabbit jejunum spontaneous contractions and on guinea-pig carbachol-precontracted trachea.

Results

Cumulative concentration (1–100 μg/ml) of Viburnum prunifolium methanolic extract (MeOH extract), its purified fractions soluble in ethylacetate (EtOAc fraction) and in n-butanol (BuOH fraction), and the iridoid glucosides (2 × 10⁻⁵ to 4 × 10⁻⁴ M): 2′-O-acetyldihydropenstemide (1), 2′-O-trans-p-coumaroyldihydropenstemide (2), 2′-O-acetylpatrinoside (3), and patrinoside (4), isolated from EtOAc fraction (1 and 2) and BuOH fraction (3 and 4), induced both relaxant effect of rabbit jejunum spontaneous contractions and spasmolytic effect on guinea-pig carbachol (5.5 × 10⁻⁷ M)-precontracted trachea. Propranolol (10⁻⁶ M) antagonised all Viburnum prunifolium tested components relaxant and spasmolytic effects. At non-relaxing concentrations (0.5 μg/ml), MeOH extract and its fractions induced a potentiating effect of isoprenaline cumulative concentrations also in both isolated tissues.

Conclusion

In both tissues, the order of potency was EtOAc fraction > BuOH fraction > MeOH extract and 1 > 2 > 3 > 4 suggesting that the major iridoids of EtOAc fraction may be considered among the most active compounds.HPLC analysis of the bioactive iridoids indicates that 1 and 2 are present for 7.38% and 14.90% in EtOAc fraction, and 3 and 4 for 18.47% and 8.86% in BuOH fraction. By comparing the values of EC₅₀ of the fractions and compounds isolated from them, we may assume that the iridoids play a significant role in the biological activity of the corresponding fractions.     

The in vitro antiviral property of Azadirachta indica polysaccharides for poliovirus

Ethnopharmacological relevance

Azadirachta indica A. Juss, popularly known as neem, has been extensively used in Ayurvedic medicine by Indian population for over 2000 years. It is used traditionally for the healing of various diseases. Natural products and their derivatives provide an excellent source for new anti-viral drugs.

Aim of the study

The present study aims at evaluating the activity of two polysaccharides (P1 and P2) isolated from the leaves of Azadirachta indica and their chemical sulfated derivatives (P1S and P2S) against poliovirus type 1 (PV-1).

Materials and methods

The cytotoxicity of the compounds was analyzed by MTT and the antiviral effect was determined by plaque reduction assay in different protocols.

Results

The polysaccharides did not show any cytotoxic effects on HEp-2 cells at the highest tested concentration (200 μg/ml) and exhibited significant antiviral activity with inhibitory concentrations (IC₅₀) of 80 μg/ml, 37.5 μg/ml, 77.5 μg/ml, and 12.1 μg/ml for P1, P1S, P2 and P2S, respectively, and the selectivity indexes (SI) ranged from 18 to 131.9. The compounds demonstrated better inhibitory effect when added concomitantly with the virus infection with a dose-dependent curve inhibition. Lesser effect was observed when the compounds were added after viral infection and the least effect at pre-treatment.

Conclusions

We suggested that the polysaccharides obtained from Azadirachta indica act against PV-1 by inhibiting the initial stage of viral replication. Importantly, original polysaccharides showed better virucidal effect than their sulfated derivatives at all tested concentrations. This study provides a scientific basis for the past and present ethnomedical uses of this plant.     

Anti-inflammatory and analgesic properties of Heracleum persicum essential oil and hydroalcoholic extract in animal models

Ethnopharmacological relevance

Fruits of Heracleum persicum (Apiaceae) are used as pain killer in Iranian folkloric medicine.

Aims of study

To evaluate the anti-inflammatory and analgesic effects of the hydroalcoholic extract and essential oil of the plant fruits and analyzing the essential oil.

Materials and methods

Essential oil and hydroalcoholic extracts of the fruits were prepared according to standard methods and the components of essential oil were identified using GC–MS method. The acetic acid-induced writhing response and formalin test were used in male mice to assess analgesic activity. For evaluation of anti-inflammatory effect, carrageenan-induced rat paw edema was used.

Results

Hexyl butyrate (56.5%), octyl acetate (16.5%), hexyl 2-methylbutanoate (5.2%) and hexyl isobutyrate (3.4%) were identified as the major constituents of the oil. Oral or intraperitoneal administration of Heracleum persicum essential oil (HPEO) at doses of 50–200 mg/kg and Heracleum persicum hydroalcoholic extract (HPHE) at doses of 250 and 500 mg/kg significantly reduced acetic acid-induced abdominal constrictions. HPEO and HPHE also significantly attenuated the pain response of the second phase of formalin test.In carrageenan test HPEO at doses of 100 and 200 mg/kg and HPHE at a dose of 400 mg/kg induced a significant reduction of paw edema.

Conclusions

These results clearly show the analgesic and anti-inflammatory effects of the plant essential oil and hydroalcoholic extract. Further studies are needed to clarify the mechanism of action and the components responsible for these pharmacological effects.     

Protein tyrosine phosphatase 1B inhibitory by dammaranes from Vietnamese Giao-Co-Lam tea

Ethnopharmacological relevance

Gynostemma pentaphyllum (Thunb.) tea was used in Vietnamese folk medicine as anti-diabetic agent.

Aim of the study

This study was aimed to investigate the inhibitory activities of fractions and constituents isolated from Gynostemma pentaphyllum on protein tyrosine phosphatase 1B (PTP1B) since it has been proposed as a treatment therapy for type 2 diabetes and obesity.

Materials and methods

The 70% EtOH extract, CHCl₃ fraction, EtOAc fraction, BuOH fraction, and seven isolated dammarane triterpenes were evaluated for their inhibitory activity in protein phosphatase enzymes (PTP1B and VHR).

Results

CHCl₃-soluble fraction showed a dose-dependent inhibitory activity of the PTP1B enzyme with the IC₅₀ value of 30.5 μg/mL. Among seven tested compounds, compounds 6 showed the most potent PTP1B inhibitory activity with IC₅₀ value of 5.3 ± 0.4 μM compared to a range 15.7–28.5 μM for the other six compounds. The inhibition mode of 6 was competitive toward p-NPP with a K_i value of 2.8 μM.

Conclusion

These study results suggested that the PTP1B inhibitory activity of these dammaranes may enable this plant to play an important role in the treatment of diabetes.     

Zanthoxylum capense constituents with antimycobacterial activity against Mycobacterium tuberculosis in vitro and ex vivo within human macrophages

Ethnopharmacological relevance

Zanthoxylum capense Thunb. (Rutaceae) is a medicinal plant traditionally used in Mozambique to treat tuberculosis.

Aims of the study

The main aim of the study was to find antimycobacterial lead compounds from Zanthoxylum capense. Another goal was to provide scientific validation for the use of this plant in traditional medicine.

Methods and materials

By bioassay-guided fractionation, 16 compounds were isolated and screened for their in vitro antimycobacterial activity against two different strains of Mycobacterium tuberculosis. Their in vitro cytotoxicity to human THP-1 macrophages was also assessed. The compounds with favourable selectivity index values (SI>10) were further investigated for their ability to inhibit the growth of Mycobacterium tuberculosis H37Rv in an intracellular macrophage model of infection.

Results

The best results were obtained for a benzophenanthridine alkaloid, decarine (1), and an N-isobutylamide, N-isobutyl-(2E,4E)-2,4-tetradecadienamide (15), which showed high activity against Mycobacterium tuberculosis H37Rv (MIC of 1.6 μg/ml), and a low macrophage cytotoxicity (IC₅₀>60 μg/ml), indicating considerable selective activity. The benzophenanthridine alkaloid 6-acetonyldihydronitidine (6) revealed cytotoxicity (IC₅₀ 1.7 μg/ml), despite the determined MIC of 6.2–12.5 μg/ml. In infected macrophages, decarine (1) was able to reduce bacterial survival by almost two log units at a concentration of 6.2 μg/ml 5 days post-drug exposure. Compound 15 exhibited an intermediate activity at drug concentrations ranging from 6.2 to 25 μg/ml.

Conclusions

The high antimycobacterial activity of decarine found, both in vitro and ex vivo against mycobacteria, and the low cytotoxicity towards human macrophages indicate that it may be valuable as a lead scaffold for the development of anti-TB drugs.     

Pharmacokinetics and tissue distribution of resveratrol,emodin and their metabolites after intake of Polygonum cuspidatum in rats

Ethnopharmacological relevance

The rhizome of Polygonum cuspidatum SIEB. et ZUCC. (Polygonaceae, PC), a widely used Chinese medicine, is commonly prescribed for the treatments of amenorrhea, arthralgia, jaundice, abscess, scald and bruises.

Aim of the study

PC contains various polyphenols including stilbenes, anthraquinones and flavonoids. This study investigated the pharmacokinetics and tissue distribution of emodin and resveratrol in PC.

Material and methods

Male Sprague-Dawley rats were orally administered PC (2 and 4 g/kg) and blood samples were withdrawn at the designed time points via cardiopuncture. Moreover, after 7-dose administrations of PC (4 g/kg), brain, liver, lung, kidney and heart were collected. The concentrations of resveratrol and emodin in the plasma and tissues were assayed by HPLC before and after hydrolysis with β-glucuronidase and sulfatase.

Results

The glucuronides/sulfates of emodin and resveratrol were exclusively present in the plasma. In liver, kidney, lung and heart, the glucuronides/sulfates of resveratrol were the major forms. For emodin, its glucuronides/sulfates were the major forms in kidney and lung, whereas considerable concentration of emodin free form was found in liver. Neither free forms nor conjugated metabolites of resveratrol and emodin were detected in brain.

Conclusion

The sulfates/glucuronides of resveratrol and emodin were the major forms in circulation and most assayed organs after oral intake of PC. However, the free form of emodin was predominant in liver.