Hypoglycemic properties of some preparations and compounds from Artemisia ludoviciana Nutt

Ethnopharmacological relevance

Artemisia ludoviciana, commonly known as “estafiate”, plays an important role in contemporary Mexico for treating several diseases including diabetes. To establish the preclinical efficacy of Artemisia ludoviciana as hypoglycemic and/or antihyperglycemic agent using well-known animal models.

Materials and methods

Acute hypoglycemic as well as oral glucose (OGTT) and sucrose (OSTT) tolerance tests were used to demonstrate the value of the plant for treating diabetes. An essential oil (EO), an organic extract (OE) and an infusion (AE) were assayed in normal and NA-STZ-treated mice. The acute toxicity of the three preparations was analyzed by the Lorke method. The infusion was subjected to conventional phytochemical study using chromatographic conventional procedures. Some of the isolates were evaluated using the same pharmacological assays as well as an enzymatic test. The latter was employed to assess their potential inhibitory effect on yeast α-glucosidase.

Results

Oral administration of OE to normal mice significantly decreased blood glucose level only at the dose of 100 mg/kg; in NA-STZ-mice the hypoglycemic effect was observed at the three doses tested (31.6, 100, and 316 mg/kg). The infusion reduced significantly, blood sugar levels only in diabetic mice; the best effect was observed at the dose of 316 mg/kg. The EO was inactive when evaluated in normal mice. Regarding to the antihyperglycemic effect, the best effect was observed with the OE, during the OGTT and OSTT in diabetic mice. The infusion (AE) showed better effects during the OGTT in both normal and diabetic animals at the dose of 100 mg/kg. Finally, the EO was inactive during an OGTT at the three doses tested (31.6, 100, and 316 mg/kg) in diabetic mice. In addition, the results of AE on the enzymatic test using yeast α-glucosidase revealed an inhibition of 45%; this finding was consistent with the results obtained with the same preparation in vivo during an OSTT. Conventional phytochemical analysis of the active AE led to the isolation and characterization of eupatilin (1), jaceosidin (2), arglanin (3), salvinine (4), and 3,5-dicaffeoylquinic acid (5). Biological testing of 1 and 3 revealed their hypoglycemic effect. The hypoglycemic effect of arglanin (3) was attenuated in the presence of nicorandil, which suggested that the lactone behaved as an ATP-K⁺-channel blocker as glibenclamide. Salvinine (4) turned out to be a mixed α-glucosidase inhibitor, while 3 was inactive.

Conclusions

Artemisia ludoviciana preparations showed hypoglycemic and antihyperglycemic effects, which could explain its effectiveness for treating diabetes in contemporary Mexico. Some of the active principles of the plant included compounds 1–5. These compounds seem to be acting synergistically on different molecular targets which involved glucose absorption and insulin liberation.     

Lignans from the stems of Clematis armandii (“Chuan-Mu-Tong”) and their anti-neuroinflammatory activities

Ethnopharmacological relevance

The dried stems of Clematis armandii (Caulis clematidis armandii), named “Chuan-Mu-Tong” in Chinese Pharmacopoeia, have been traditionally used as an herbal remedy mainly for inflammation-associated diseases. The Aim of the study is to identify the potential anti-neuroinflammatory components from Clematis armandii.

Materials and methods

The ethanol extract of “Chuan-Mu-Tong” was suspended in H₂O and exhaustively extracted with CH₂Cl₂. The CH₂Cl₂ fraction was successively subjected to column chromatography (CC) over silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures of the isolated compounds were identified by spectroscopic methods and by comparison with those reported in the literature. Their anti-neuroinflammatory activities were evaluated by inhibitory effects on pro-inflammatory mediators [e.g. nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α)] in lipopolysaccharide (LPS)-activated BV-2 cells.

Results

One new and sixteen known lignans were isolated and characterized. The absolute configuration of the new lignan, (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol (1), was elucidated by a combination of 1D/2D NMR techniques and the Electronic Circular Dichroism (ECD) spectroscopy based on the empirical helicity rules. The anti-neuroinflammatory bioassay showed that compounds 1, (7R,8S)-dehydrodiconiferyl alcohol (2), erythro-guaiacylglycerol-β-coniferyl ether (5), and threo-guaiacylglycerol-β-coniferyl ether (6) displayed significant inhibitory effects on NO production. Among them, neolignans 1 and 2 exhibited more potent activities than the positive control (N^G-monomethyl-l-arginine, l-NMMA), with an IC₅₀ value of 9.3 and 3.9 μM, respectively. Moreover, both 1 and 2 were also found to concentration-dependently suppress the TNF-α release in LPS-stimulated BV-2 cells.

Conclusion

The results revealed that lignans are the major components of “Chuan-Mu-Tong”, and their anti-neuroinflammatory activities strongly support the traditional application of this herb medicine on inflammation. Moreover, the dihydrobenzo[b]furan neolignans 1 and 2 as well as Caulis clematidis armandii could be further exploited as new therapeutic agents to treat inflammation-mediated neurodegenerative and aging-associated diseases.     

Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents

Ethnopharmacological relevance

Arctium lappa and Tussilago farfara (Asteraceae) are two plant species used traditionally as antitubercular remedies. The aim of this study was (i) to screen Arctium lappa and Tussilago farfara extracts for activity against Mycobacterium tuberculosis and (ii) to isolate and identify the compound(s) responsible for this reputed anti-TB effect.

Materials and methods

The activity of extracts and isolated compounds was determined against Mycobacterium tuberculosis H₃₇R_v using a high throughput spot culture growth inhibition (HT-SPOTi) assay.

Results

The n-hexane extracts of both plants, the ethyl acetate extract of Tussilago farfara and the dichloromethane phase derived from the methanol extract of Arctium lappa displayed antitubercular activity (MIC 62.5 μg/mL). Further chemical investigation of Arctium lappa led to the isolation of n-nonacosane (1), taraxasterol acetate (2), taraxasterol (3), a (1:1) mixture of β sitosterol/stigmasterol (4), isololiolide (5), melitensin (6), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10). Compounds isolated from Tussilago farfara were identified as a (1:1) mixture of β sitosterol/stigmasterol (4), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10), loliolide (11), a (4:1) mixture of p-coumaric acid/4-hydroxybenzoic acid (12), p-coumaric acid (13). All compounds were identified following analyses of their physicochemical and spectroscopic data (MS, ¹H and ¹³C-NMR) and by comparison with published data. This is the first report of the isolation of n-nonacosane (1), isololiolide (5), melitensin (6) and kaempferol-3-O-glucoside (10) from Arctium lappa, and of loliolide (11) from Tussilago farfara. Amongst the isolated compounds, the best activity was observed for p-coumaric acid (13) (MIC 31.3 μg/mL or 190.9 μM) alone and in mixture with 4-hydroxybenzoic acid (12) (MIC 62.5 μg/mL).

Conclusions

The above results provide for the first time some scientific evidence to support, to some extent, the ethno-medicinal use of Arctium lappa and Tussilago farfara as traditional antitubercular remedies.     

Antimicrobial activity of the crude extract,fractions and compounds from stem bark of Ficus ovata (Moraceae)

Aim of the study

This study was designed to investigate the antimicrobial activities of the methanol extracts from the stem bark of Ficus ovata (FOB), fractions (FOB1–6) and compounds isolated following bio-guided fractionation [3-friedelanone (1), taraxeryl acetate (2), betulinic acid (3), oleanoïc acid (4), 2-hydroxyisoprunetin (5), 6,7-(2-isopropenyl furo)-5,2,4-trihydroxyisoflavone (6), Cajanin (7) and protocatechuic acid (8)].

Materials and Methods

The micro-dilution method was used for the determination of the minimal inhibition concentration (MIC) and the minimal microbicidal concentration (MMC) against fungi (two species), Gram-positive (three species) and Gram-negative bacteria (five species).

Results

The results of the MIC determinations indicated that the crude extract (FOB), fractions FOB2 and FOB4 as well as compound 5 were active on the entire studied organisms. Other samples showed selective activity, fractions FOB1, FOB3 and FOB5 being active against 50% of the tested microbial species while FOB6 was active on 40%. Compounds 8, 6, 2 and 7 prevented the growth of 80%, 70%, 50% and 20% of the organisms respectively. The lowest MIC value (156g/ml) observed with the crude extract was recorded on Streptococcus faecalis, Candida albicans and Microsporum audouinii. The corresponding value for fractions (39 μg/ml) was noted with FOB4 against Staphylococcus aureus, while that of the tested compounds (10 μg/ml) was observed with compound 8 on Microsporum audouinii. The results of the MMC determination suggested that the cidal effect of most of the tested samples on the studied microorganisms could be expected.

Conclusions

The overall results provided evidence that the studied plant extract, as well as some of the isolated compounds might be potential sources of new antimicrobial drug.     

Bioactive styryllactones and alkaloid from flowers of Goniothalamus laoticus

Ethnopharmacological relevance

Goniothalamus laoticus (Annonaceae) is being used traditionally as a tonic and a febrifuge by the local people in the northeastern part of Thailand.

Aim of study

To investigate the Thai medicinal plant, Goniothalamus laoticus, for antiplasmodial, antimycobacterial and cytotoxicity activities.

Materials and methods

The flowers extracts of Goniothalamus laoticus were isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods. The antiplasmodial, antimycobacterial and cytotoxicity evaluation of styryllactone derivatives and alkaloid were also performed.

Results

Ten compounds, cinnamic acid (1); dihydrochrysine (2); β-sitosterol (3); six styryllactones, (+)-3-acetylaltholactone (4), goniotriol (5), (+)-altholactone (6), (+)-goniofufurone (7), 9-deoxygoniopypyrone (8), howiinin A (9); and an aporphine alkaloid; (−)-nordicentrine (10) were isolated from flowers of Goniothalamus laoticus. Among these, compounds 1, 3–5, 8–10 are first isolated from the Goniothalamus laoticus. Besides, compound 10 is the first report from the Goniothalamus genus. The isolated compounds were evaluated in antiplasmodial, antimycobacterial and anticancer cell lines tests. Compounds 4–6 and 10 exhibited antiplasmodial activity against Plasmodium falciparum (IC₅₀ 2.6, 7.9, 2.6 and 0.3 μg/mL, respectively), while 5, 6, 9 and 10 showed antimycobacterial activity against Mycobacterium tuberculosis (MIC 100, 6.25, 6.25 and 12.5 μg/mL, respectively). In addition, compounds 4–10 showed cytotoxicity against cancer cells, KB, BC1, NCI-H187, and MCF-7 with IC₅₀ ranging from 0.4 to 22.7 μg/mL.

Conclusion

This finding showed that the styryllactone derivatives and alkaloid isolated from the flowers of Goniothalamus laoticus exhibited antiplasmodial activity against Plasmodium falciparum, antimycobacterail against Mycobacterium tuberculosis and cytotoxicity against four cancer cell lines.     

Antimicrobial activity of the methanolic extract and compounds from Morus mesozygia stem bark

Aim of the study

This study was aimed at investigating the antimicrobial activity of the methanolic extract (MMB) and compounds isolated from the stem bark of Morus mesozygia, namely 3β-acetoxyurs-12-en-11-one (1), moracin Q (2), moracin T (3), artocarpesin (4), cycloartocarpesin (5), moracin R (6), moracin U (8), moracin C (9), and moracin M (10).

Materials and Methods

The liquid microdilution assay was used in the determination of the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC), against nine bacterial and two fungal species.

Results

The results of the MIC determination showed that the compounds 3, 4, 8 and 9 were able to prevent the growth of all tested microbial species. All other samples showed selective activities. Their inhibitory effects were noted on 90.9% studied organisms for the crude extract, 81.8% for compound 6, 72.7% for compound 10, 63.6% for compound 1, 54.5% for compound 5, and 45.5% for compound 2. The lowest MIC value of 39 μg/ml was obtained with the crude extract against Escherichia coli. The corresponding value for compounds (5 μg/ml) was registered with compound 9 on Shigella dysenteriae and compound 3 on E. coli, S. dysenteriae, Pseudomonas aeruginosa, Salmonella typhi and Bacillus cereus. The lowest MIC value (39 μg/ml) observed with the crude extract (on E. coli) was only eightfold greater than that of gentamycin used as reference antibiotic (RA) while the corresponding value (5 μg/ml) recorded with compounds 3 and 9 was equal to that of RA on the corresponding microorganisms.

Conclusions

The obtained results highlighted the interesting antimicrobial potency of M. mesozygia as well as that of the studied compounds, and provided scientific basis for the traditional use of this species.     

Antiparasitic compounds from Cornus florida L. with activities against Plasmodium falciparum and Leishmania tarentolae

Aim of the study

The objective of this study was to identify the antiplasmodial constituents from the bark of Cornus florida L., a plant traditionally used in North America for the treatment of malaria.

Methods and materials

Dried and powdered bark was extracted with 95% ethanol. The resultant extract was subjected to in vitro antiplasmodial-guided fractionation against Plasmodium falciparum (D10 strain). Antiplasmodial IC₅₀ values were calculated for pure compounds. Compounds were also assayed against Leishmania tarentolae, and rat skeletal myoblast L6 cells to assess antileishmanial activity and cytotoxicity, respectively.

Results

Antiplasmodial-guided fractionation afforded 8 compounds: betulinic acid (1), ursolic acid (2), β-sitosterol (3), ergosta-4,6,8,22-tetraene-3-one (4), 3β-O-acetyl betulinic acid (5), 3-epideoxyflindissol (6), 3β-O-cis-coumaroyl betulinic acid (7), 3β-O-trans-coumaroyl betulinic acid (8), of which, (6) is for the first time here isolated from a natural product and (4), (7) and (8) are reported for the first time from this genus. In vitro IC₅₀ values against P. falciparum for (4) (61.0 μM) (6) (128.0 μM), (7) (10.4 μM), (8) (15.3 μM) are reported for the first time. Antileishmanial IC₅₀ values are reported here for the first time for (4) (11.5 μM), (6) (1.8 μM), (7) (8.3 μM) and (8) (2.2 μM). Cytotoxicity against L6 cells is reported for all compounds.

Conclusions

The compounds isolated in this study, while displaying moderate in vitro antiplasmodial activity, do not fully support the historical importance of C. florida as an antimalarial remedy in North America. The traditional remedy may exert its well documented effects by mechanisms unrelated to direct antiplasmodial action. While not traditionally used to treat Leishmania, this work shows that several constituents of C. florida possess promising in vitro antileishmanial activity.     

Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita

Aim of the study

Artemisia vestita is a common traditional Tibetan medicinal plant which has been used widely in China for treating various inflammatory diseases. Since little is known about its active components, the purpose of this study was to isolate and identify the immunosuppressive compounds from Artemisia vestita.

Materials and methods

A bioassay-guided isolation was performed with picryl chloride-induced contact hypersensitivity in mice. MTT assay and Flow cytometric analysis were used for determining Con A-induced lymphocyte proliferation and CD25 expression in T cells, respectively.

Results

The ethanol extract of the Artemisia vestita was found to possess significant inhibitory activity against the picryl chloride-induced contact hypersensitivity in mice. Then 4 fractions were isolated by macroporous adsorption resin and one of these fractions (AV3), which showed the highest activity in in vivo test, was further subjected to column chromatography. Nine known flavones were isolated and identified as pectolinarigenin (1), jaceosidin (2), cirsilineol (3), cirsimaritin (4), hispidulin (5), quercetin (6), 6-methoxytricin (7), acacetin (8), and apigenin (9). The structures of the 9 flavones were elucidated by spectral techniques. All the compounds were evaluated for their inhibitory activity on the proliferation and activation of T cells in vitro. Among the 9 flavones, cirsilineol (3), 6-methoxytricin (7) and apigenin (9) significantly inhibited T cell proliferation and activation in the bioassays.

Conclusion

The result suggests that cirsilineol, 6-methoxytricin and apigenin are the major active components in Artemisia vestita.     

Antimicrobial activities of methanol extract and compounds from stem bark of Vismia rubescens

Ethnopharmacological relevance

The plant, Vismia rubescens (Guttiferae) is popularly used in Cameroon and in several parts of Africa as febrifugal and for the treatment of various microbial infections (skin diseases, diarrhoea and venereal diseases).

Aim of the study

This study was mapped out to evaluate the antimicrobial activities of the methanol extract and compounds from the stem bark of Vismia rubescens.

Materials and methods

Structures of the compounds obtained after column chromatography of the methanol-soluble fraction were determined by spectroscopy and in comparison with published data. The broth micro-dilution method was used to evaluate the antimicrobial activities against three bacteria species (Salmonella typhi, Stahylococcus aureus and Pseudomonas aeruginosa) and four yeast species (Candida albicans, Candida tropicalis, Candida parapsilosis and Cryptococcus neoformans).

Results

Chemical analysis of the methanol extract from the stem bark of Vismia rubescens yielded five known compounds 1,4,8-trihydroxyxanthone (1), 1,7-dihydroxyxanthone (2), physcion (3), friedelin (4) and friedelanol (5). The crude extract and compounds 1, 2 and 3 exhibited both antibacterial and antifungal activities that varied between the microbial species (MIC = 3.12–1000 μg/ml). Compounds 2 and 3 were the most active (MIC = 3.12–100 μg/ml) while Stahylococcus aureus and Pseudomonas aeruginosa were sensitive to all the tested compounds. The antimicrobial activity of this plant as well as that of compounds 1 and 2 is being reported here for the first time.

Conclusion

These results provide promising baseline information for the potential use of this plant as well as some of the isolated compounds in the treatment of skin diseases and diarrhoea.     

Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson's disease

Aim of the study

Parkinson's disease is a neurological disorder mostly effecting the elder population of the world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the composition and constituents of the aqueous extract of Banisteriopsis caapi for monoamine oxidases (MAO) inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological disorders, including Parkinsonism.

Materials and methods

The aqueous extract of Banisteriopsis caapi stems was standardized and then fractionated using reversed-phase (RP) chromatography. Pure compounds were isolated either by reversed-phase (RP) chromatography or centrifugal preparative TLC, using a Chromatotron^®. Structure elucidation was carried out by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and chemical derivatization. Chemical profiling of the extract was carried out with RP-HPLC. The inhibitory activity of MAO-A, MAO-B, acetylcholinesterase, butyrylcholinesterase and catechol-O-methyl transferase enzymes, as well as antioxidant and cytotoxic activities of both Banisteriopsis caapi extract and isolated compounds was evaluated.

Results

An examination of the aqueous extracts of Banisteriopsis caapi cultivar Da Vine yielded two new alkaloidal glycosides, named banistenoside A (1) and banistenoside B (2), containing “azepino[1,2-a]tetrahydro-β-carboline” unique carbon framework. One additional new natural tetrahydronorharmine (4), four known β-carbolines harmol (3), tetrahydroharmine (5), harmaline (6) and harmine (7), two known proanthocyanidines (−)-epicatechin (8) and (−)-procyanidin B2 (9), and a new disaccharide β-d-fructofuranosyl-(2 → 5)-fructopyranose (14) together with known sacharose (15) and β-d-glucose (16) were also isolated. In addition, the acetates of 1, 2, 8, 9, 14 and 15 (compounds 10–13, 17, 18) were also prepared. Harmaline (6) and harmine (7) showed potent in vitro inhibitory activity against recombinant human brain monoamine oxidase (MAO)-A and -B enzymes (IC₅₀ 2.5 and 2.0 nM, and 25 and 20 μM, respectively), and (−)-epicatechin (8) and (−)-procyanidin B2 (9) showed potent antioxidant and moderate MAO-B inhibitory activities (IC₅₀ < 0.13 and 0.57 μg/mL, and 65 and 35 μM). HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7–9) were present in high concentrations in dried bark of large branch. Analysis of regular/commercial Banisteriopsis caapi dried stems showed a similar qualitative HPLC pattern, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency.

Conclusion

Collectively, these results give additional basis to the existing claim of Banisteriopsis caapi stem extract for the treatment of Parkinsonism, including other neurodegenerative disorders.     

Anti-diabetic activity of Swertia corymbosa (Griseb.) Wight ex C.B. Clarke aerial parts extract in streptozotocin induced diabetic rats

Ethnopharmacologicals relevance

Swertia corymbosa locally called as Shirattakuchi have a long history of use in Ayurveda herbal preparations in Indian traditional system of medicine. It has been used in folklore medicine for the treatment of diabetes.

Aim of the study

The present study aimed to investigate the effect of the methanolic extract of Swertia corymbosa (SC) in diabetic and to analyze its chemical composition by HPLC–ESI/MS that may correlate with their pharmacological activities.

Materials and methods

The in vitro anti-diabetic activity of the extracts was measured by using α-glucosidase and α-amylase enzyme inhibitory activity. The methanolic extract of Swertia corymbosa were administered orally (125, 250 and 500 mg/kg, for 28 days) to streptozotocin-induced diabetic rats. Hypoglycemic effects, oral glucose tolerance test, change in body weight and lipid profile, biochemical analysis and histopathological examination were assessed. High-performance liquid chromatography–electrospray ionization/mass spectrometry (HPLC–ESI/MS) method was also developed to analyze the chemical composition.

Results

In vitro anti-dabetic study, the methanol extract of SC is found to be a potent inhibitor of α-glucosidase and α-amylase activity. Oral administration of SC and standard drug for 28 days caused a significant decrease in the concentrations of blood glucose level, total cholesterol (TC), serum triglycerides (TGs), low-density lipoprotein-cholesterol (LDL-C), malondialdehyde (MDA) and significant increase in the concentrations of high density lipoprotein-cholesterol (HDL-C), serum insulin and body weight. Furthermore, activities of antioxidative enzymes, including SOD, GPx, GSH and CAT were enhanced dosed dependently with SC. Histopathological studies of the pancreas showed the regeneration of the β-cells by extract which were earlier necrosed by streptozotocin. Ten major compounds such as loganic acid (1), swertiamarin (2), sweroside (3), gentiopicroside (4), isovitexin (5), amoroswertin (6), amarogentin (7), gentiacaulein (8), decussatin (9) and swertianin (10) were analyzed by HPLC–ESI/MS system.

Conclusions

These results demonstrate that SC aerial parts of methanolic extract is an effective anti-diabetic and antioxidant activities which provides the scientific proof for the folklore medicine.     

UPLC/MS/MS method for quantification and cytotoxic activity of sesquiterpene lactones isolated from Saussurea lappa

Ethnopharmacological relevance

Saussurea lappa (Asteraceae) roots have been reputed for the usage in traditional medicinal systems of India, China and Japan for the treatment of various kinds of disorders such as anti-ulcer, anti-convulsant, anti-cancer, hepatoprotective, anti-arthritic and anti-viral activities.

Materials and methods

Compounds were isolated using a column chromatographic technique. The root extract, fractions and isolated compounds were tested for cytotoxicity against A549 (human lung carcinoma) and C-6 (rat glioma) cells using the Sulphorhodamine B assay. Chromatographic separations of active sesquiterpene lactones were accomplished on BEH-HSS-T3 column at 25 °C.

Results

Phytochemical investigation of Saussurea lappa root extract resulted in the isolation of isoalantolactone (1), β-cyclocostunolide (2) α-cyclocostunolide (3), 4-hydroxy-3,5-dimethoxycinnamyl-9-O-β-d-glucopyranoside (4), sucrose (5), and alantolactone (6). Their structures were determined by spectroscopic means. Ethanolic extract, chloroform fraction, compounds 1, 2, 3 and 6 possessed significant activity against both tested cells. The quantification was performed using the transitions of m/z 233/105 for isoalantolactone and m/z 233/105 for alantolactone respectively. Costunolide and dehydrocostus lactone were also characterised by comparison of MS/MS fragmentation pattern.

Conclusions

This is the first study on simultaneous quantification of isoalantolactone and alantolactone by the UPLC/MS/MS method in Saussurea lappa. Our study against A549 and C-6 cells showed higher cytotoxicity. It is suggested that roots of Saussurea lappa might be a potential source of anticancer compounds.     

Isolation and identification of antibacterial and cytotoxic compounds from the leaves of Muntingia calabura L.

Ethnopharmacological relevance

Muntingia calabura (Elaeocarpaceae) is one of the most common roadside trees in Malaysia. Its leaves, barks, flowers and roots have been used as a folk remedy for the treatment of fever, incipient cold, liver disease, as well as an antiseptic agent in Southeast Asia. The aim of this study is to isolate and identify the antibacterial and cytotoxic compounds from the leaves of Muntingia calabura L.

Materials and methods

Antibacterial and cytotoxic activities were determined by micro-broth dilution and MTT assays, respectively. Seven fractions (F1–F7), three flavones and a chalcone were isolated from the active EtOAc extract using bioassay-guided screening. The structures of four compounds were elucidated by spectroscopic methods and compared with published data. The compounds were further tested for their antibacterial and cytotoxic activities.

Results

Three flavones and a chalcone [5,7-dihydroxy-3,8-dimethoxyflavone (1), 2′,4′-dihydroxychalcone (2), 5-hydroxy-3,7-dimethoxyflavone (3) and 3,5,7-trihydroxy-8-methoxyflavone (4)] were isolated from the active fraction F5 of EtOAc extract. Compounds 1 and 3 were isolated for the first time from Muntingia calabura L. Antibacterial activity indicates that compound 2 exhibited the most significant activity with MIC value of 50 μg/mL and 100 μg/mL against MSSA and MRSA, respectively. Cytotoxic activity indicates that compounds 2 and 3 exhibited very strong activity against HL60 with IC₅₀ values of 3.43 μg/mL and 3.34 μg/mL, respectively.

Conclusion

The antibacterial activity of the leaves of Muntingia calabura L. is ascribable to the active compound 2 while the cytotoxic activity is ascribable to the active compounds 2 and 3.     

Non-toxic melanin production inhibitors from Garcinia livingstonei (Clusiaceae)

Ethnopharmacological relevance

The stem bark of Garcinia livingstonei is used traditionally as a skin lightening agent.

Aim of the study

To isolate and identify compounds responsible for the observed skin lightening activity of Garcinia livingstonei and to evaluate their cytotoxicity.

Materials and methods

Constituents of the stem bark and fruits of Garcinia livingstonei were isolated using chromatographic techniques and structures were determined using 1D and 2D NMR and MS analysis. MeWo cells were used to evaluate the cytotoxicity and impact on melanin levels of extracts and compounds isolated, in vitro.

Results

Twelve known compounds, morelloflavone (1), morelloflavone-7″-sulphate (2), guttiferone A (3), sargaol (4), isojacareubin (5), 6-deoxyisojacareubin (6) and in addition to the common triterpenoids, betulin, betulin aldehyde, lupeol, lupenone, euphol and stigmasterol were isolated in this investigation. Morelloflavone, morelloflavone-7″-sulphate and sargaol, were found to be considerably less cytotoxic and more effective as skin lightening agents than hydroquinone.

Conclusions

A range of compounds was isolated from the stem bark and fruit of Garcinia livingstonei. Although the bark extract contained the cytotoxic guttiferone A, it was found to be less toxic than hydroquinone, and morelloflavone, the 7″-sulphate derivative and sargaol show potential for development as depigmentation/skin lightening agents.     

Monoterpene bisindole alkaloids,from the African medicinal plant Tabernaemontana elegans, induce apoptosis in HCT116 human colon carcinoma cells

Ethnopharmacological relevance

Tabernaemontana elegans is a medicinal plant used in African traditional medicine to treat several ailments including cancer. The aims of the present study were to identify anti-cancer compounds, namely apoptosis inducers, from Tabernaemontana elegans, and hence to validate its usage in traditional medicine.

Methods and materials

Six alkaloids, including four monomeric indole (1–3, and 6) and two bisindole (4 and 5) alkaloids, were isolated from the methanolic extract of Tabernaemontana elegans roots. The structures of these compounds were characterized by 1D and 2D NMR spectroscopic and mass spectrometric data. Compounds 1−6 along with compound 7, previously isolated from the leaves of the same species, were evaluated for in vitro cytotoxicity against HCT116 human colon carcinoma cells by the MTS metabolism assay. The cytotoxicity of the most promising compounds was corroborated by Guava-ViaCount flow cytometry assays. Selected compounds were next studied for apoptosis induction activity in HCT116 cells, by evaluation of nuclear morphology following Hoechst staining, and by caspase-3 like activity assays.

Results

Among the tested compounds (1−7), the bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were found to be cytotoxic to HCT116 cells at 20 µM, with compound 5 being more cytotoxic than the positive control 5-Fluorouracil (5-FU), at a similar dose. In fact, even at 0.5 µM, compound 5 was more potent than 5-FU. Compounds 4 and 5 induced characteristic patterns of apoptosis in HCT116 cancer cells including, cell shrinkage, condensation, fragmentation of the nucleus, blebbing of the plasma membrane and chromatin condensation. Further, general caspase-3-like activity was increased in cells exposed to compounds 4 and 5, corroborating the nuclear morphology evaluation assays.

Conclusions

Bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were characterized as potent apoptosis inducers in HCT116 human colon carcinoma cells and as possible lead/scaffolds for the development of anti-cancer drugs. This study substantiates the usage of Tabernaemontana elegans in traditional medicine to treat cancer.     

Evaluation of antimicrobial activity of ethanol extract and compounds isolated from Trichodesma indicum (Linn.) R. Br. root

Ethnopharmacological relevance

The roots are reportedly used to treat diarrhoea, dysentery, leprosy, skin diseases and fever.

Aim of study

The aim of present study was to investigate the in vitro antimicrobial potential of ethanol extract of Trichdesma indicum root, and its purified compounds and to validate scientifically its use in traditional medicine.

Material and methods

The root of Trichdesma indicum was extracted with ethanol and subjected to chromatographic separation for isolation of phytochemical compounds. Structures of isolated compounds were elucidated by spectroscopic methods. The antimicrobial activities of the ethanol extract of T. indicum and isolated compounds were primarily evaluated by a disc diffusion test. The anti-microbial efficacy of the ethanol extract or isolated compounds was then assessed in vitro by determining minimal inhibition concentration (MIC) and minimal bactericidal or fungicidal concentration (MBC/MFC).

Results

n-Decanyl laurate (1), n-tetradecanyl laurate (2), n-nonacosanyl palmitate (3), stigmast-5-en-3β-ol-21(24)-olide (4), n-pentacos-9-one (5), n-dotriacont-9-one-13-ene (6), stigmast-5-en-3β-ol-23-one (7) and lanast-5-en-3β-D-glucopyranosyl-21 (24)-olide (8) were isolated from ethanol extract of T.indicum. The ethanol extract and isolated compounds (1–8) showed varying degrees of antimicrobial activities. The ethanol extract exhibited potent growth inhibitory activity against S. aureus, B. subtilis and C. albicans with an MIC value of 19.2 μg/ml. Among all the isolated compounds, lanast-5-en-3β-D-glucopyranosyl-21 (24)-olide (8) displayed strongest antibacterial activity against S. aureus with MIC value of 2.4 μg/ml.

Conclusions

The results of present study provide ground basis for the potential use of the ethanol extract Trichodesma indicum root as well as the some of the isolated compounds in the treatment of infections associated with the studied microorganisms.     

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.     

Quassinoid constituents of Quassia amara L. leaf herbal tea. Impact on its antimalarial activity and cytotoxicity

Aim of the study

Our objective was to assess whether it could be contemplated to recommend Quassia amara young leaf tea for treatment against malaria, and if yes, set up a standard protocol for preparing the herbal tea.

Materials and methods

The leaf tea was extracted with methylene chloride and the organic extract was fractionated with HPLC. Pure compounds were characterized and their in vitro cytotoxicity and antiplasmodial activity was determined.

Results and discussion

We discovered that antimalarial Quassia amara young leaf tea contains several quassinoids: simalikalactone D (SkD, 1), picrasin B (2), picrasin H (3), neoquassin (4), quassin (5), picrasin I (6) and picrasin J (7). These last two compounds are new. In addition, our experiments demonstrate that both biological activity and cytotoxicity of the remedy may be attributed solely to the presence of SkD.

Conclusion

In conclusion, this preparation should not be recommended for treatment of malaria until a clinical study in humans is performed with SkD.     

Topical analgesic,anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: Phytochemical characterization of active fractions

Ethnopharmacological relevance

Oxybaphus nyctagineus (Michx.) Sweet has traditionally been used by several Native American tribes predominantly as a topical anti-inflammatory and analgesic agent.

Aim of the study

To evaluate the antioxidant, analgesic and anti-inflammatory activity of the extracts prepared from the aerial parts of Oxybaphus nyctagineus and to characterize the major chemical constituents of the bioactive extracts.

Materials and methods

Crude polar and apolar extracts (PCE and ACE) of the herb of Oxybaphus nyctagineus were prepared and tested in the models of the CFA-induced hyperalgesia in rat knee and carrageenan-induced paw edema in rat. To identify the active compounds, subfractions were prepared by column chromatography and subjected in vitro assays, such as antioxidant assays (DPPH, peroxynitrite (ONOO⁻) scavenging), and the LPS-induced IL-1β release test in human monocytes. Preparative HPLC was employed for the isolation of active substances, while phytochemical analysis was performed by mean of LC–MS/MS and NMR.

Results

The topically administered PCE and ACE of Oxybaphus nyctagineus demonstrated a significant analgesic and anti-inflammatory effect in the inflammation animal models. The subfraction A4 of ACE and the subfraction P5 of PCE considerably inhibited the LPS-induced IL-1β release in human monocytes, while the strongest activity was localized in the subfraction P5 in the antioxidant assays. The HPLC–MS/MS and NMR analysis revealed that 6-methoxyflavonol diglycosides, namely patuletin-3-O-robinobioside (1), 6-methoxykaempferol-3-O-robinobioside (2), spinacetin-3-O-robinobioside (3), and hydroxy-polyenoic fatty acids, namely corchorifatty acid B (4), 9-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOT acid) (5), and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HOD acid) (6) were present in PCE, and in ACE as major compounds.

Conclusion

The results of this study established a pharmacological evidence for the traditional use of Oxybaphus nyctagineus as an anti-inflammatory agent used topically, and provided data on its phytochemical composition for the first time.     

New antiplasmodial alkaloids from Stephania rotunda

Ethnopharmacological relevance

Stephania rotunda Lour. (Menispermaceae) is a creeper growing in many countries of Asia and commonly found in the mountainous areas of Cambodia. As a folk medicine, it has been mainly used for the treatment of fever and malaria. The pharmacological activity is mostly due to alkaloids. Thus the aim of this study is to isolate new bioactive alkaloids from Stephania rotunda and to evaluate their in vitro antiplasmodial activity.

Materials and methods

Alkaloids were isolated and identified from dichloromethane and aqueous extracts using a combination of flash chromatography, high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The purified compounds were tested for in vitro antiplasmodial activity on chloroquine-resistant W2 strain of Plasmodium falciparum.

Results

A new aporphine alkaloid named vireakine (2) along with two known alkaloids stephanine (1) and pseudopalmatine (8), described for the first time in Stephania rotunda, and together five known alkaloids tetrahydropalmatine (3), xylopinine (4), roemerine (5), cepharanthine (6) and palmatine (7) were isolated and identified. The structure of the new alkaloid was established on the basis of 1D and 2D NMR experiments and mass spectrometry. The compounds were evaluated for their in vitro antiplasmodial and cytotoxic activities. All tested compounds showed significant antiplasmodial activities with IC₅₀ ranged from 1.2 μM to 52.3 μM with a good selectivity index for pseudopalmatine with IC₅₀ of 2.8 μM against W2 strain of Plasmodium falciparum and IC₅₀>25 μM on K562S cells.

Conclusions

This study provides evidence to support the use of Stephania rotunda for the treatment of malaria and/or fever by the healers. Alkaloids of the tuber exhibited antiplasmodial activity and particularly cepharanthine and pseudopalmatine.