Constituents isolated from Cordyceps militaris suppress enhanced inflammatory mediator's production and human cancer cell proliferation

Aim of the study

The purpose of this study is to isolate the pure compounds from the extracts of Cordyceps militaris obtained through solid-state cultivation process, and evaluate their anti-inflammatory and anticancer properties.

Materials and methods

Silica gel column chromatographic purification of Cordyceps militaris extracts resulted in the isolation of 10 pure compounds (1-10). The compounds 1-10 were examined for their growth inhibitory properties against nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-12 enhanced production from LPS/IFN-γ-stimulated macrophages. Additionally, the anti-proliferation effects of 1-10 on human cancer cell lines, colon (colon 205), prostate (PC-3), and hepatoma (HepG2) cells were also analyzed.

Results

Compound 8 displayed potent growth inhibition on NO, TNF-α and IL-12 production with an IC₅₀ value of 7.5, 6.3, and 7.6 μg/ml, respectively. A similar inhibitory trend on these inflammatory mediators was observed for 3, 7, 9 and 10 with an IC₅₀ values ranging from 10.8 to 17.2 μg/ml. On the other hand, compounds 3 and 8 were potent anti-proliferative agents with an IC₅₀ value of 35.6 and 32.6 μg/ml toward PC-3 and colon 205 cell lines, respectively. The compounds 1 and 2 showed potent anti-proliferation in PC-3 and colon 205 cells, while only 3 displayed such effect in HepG2 cells.

Conclusion

The present study provides scientific supporting information for the ethnopharmacological use of Cordyceps militaris as an anti-inflammatory and anticancer agent.     

Hypoglycemic evaluation of a new triterpene and other compounds isolated from Euclea undulata Thunb. var. myrtina (Ebenaceae) root bark

Aim of the study

Investigate the hypoglycaemic activity of the four isolated compounds from a crude acetone extract of the root bark of Euclea undulata var. myrtina, which is used by traditional healers in the Venda area, Limpopo Province in the treatment of diabetes.

Material and methods

The hypoglycaemic activity of the four compounds isolated from Euclea undulata was determined by in vitro screening of glucose utilization by C2C12 myocytes at a concentration of 25 μg/ml or 50 μg/ml. The inhibition of α-glucosidase was also tested at concentrations ranging from 0.02 to 200.00 μg/ml.

Results

Assay-guided isolation of the crude acetone extract of the root bark of Euclea undulata var. myrtina afforded a new triterpene, α-amyrin-3O-β-(5-hydroxy) ferulic acid (1), in addition to three known compounds; betulin (2), lupeol (3) and epicatechin (4). The in vitro results on C2C12 myocytes suggest that compound 4 may have some effect to lowers blood glucose levels, whereas compound 1 has the ability to inhibit α-glucosidase at a concentration of 200.0 μg/ml with an IC₅₀ value of 4.79 that correlates with that of the positive control acarbose IC₅₀ value 4.75.

Conclusion

The results suggest that 4 may have some ability to lower blood glucose levels, whereas 1 has the ability to inhibit α-glucosidase.

Ethnopharmacological relevance

These findings corroborate the ethnomedicinal use of Euclea undulata by traditional healers for the treatment of diabetes as two substances was isolated from the acetone plant extract that exhibit hypoglycaemic activity.     

Lipoxygenase inhibitory activity of crude bark extracts and isolated compounds from Commiphora berryi

Ethnopharmacological relevance

Commiphora berryi is traditionally used for the treatment of cold and fever as well as for wound healing in the southern parts of India.

Aim of study

The present study was designed to investigate in vitro soybean lipoxygenase inhibitory activity of crude extracts and compounds isolated from Commiphora berryi.

Materials and methods

The bark of Commiphora berryi was extracted with different organic solvents and subjected to chromatographic separation for isolation of bioactive compounds. Structures of isolated compounds were elucidated by spectroscopic methods. The anti-inflammatory activity of bark extracts and bioactive compounds were assessed by in vitro soybean lipoxygenase (SBL) assay.

Results

3β-Hydroxyglutin-5-ene (1), friedelin (2), cycloeucaneol (3) nimbiol (4), sugiol (5), surianol (6), daucosterol (7) and ursolic acid (8) were isolated from crude bark extracts of the Commiphora berryi. The structure of nimbiol (4) was also confirmed by single crystal X-ray analysis. The petroleum ether, methanol, chloroform and ethyl acetate extracts of bark of Commiphora berryi showed SBL inhibitory activity with the IC₅₀ values of 15.3, 54.2, 71.5 and 87.8 μg/ml respectively. Among all the isolates, friedelin (2) showed significant SBL inhibitory activity with IC₅₀ 35.8 μM.

Conclusion

The overall results provide evidence that the studied plant might be a potential source of anti-inflammatory agents.     

Bioactive polyphenols in Ximenia americana and the traditional use among Malian healers

Ethnopharmacological relevance

Ximenia americana is a medicinal bushy, spiny shrub or small tree used in Mali in West Africa for treatment of various diseases, most common are infectious and inflammatory ailments.

Aims of the study

(1) To perform an ethnopharmacological survey on the traditional use of Ximenia americana among healers in Mali. (2) To isolate and identify chemical constituents from the ethanol extract of Ximenia americana leaves and to study their radical scavenging and enzyme inhibitory effects.

Materials and methods

In five different districts in Mali, 38 healers were interviewed about their medicinal use of Ximenia americana. An aqueous ethanol extract of the leaves of this tree was prepared and further fractionated with liquid-liquid extraction, VersaFlash and Sephadex LH-20 column chromatography, and preparative HPLC. Isolated compounds were identified by 1D and 2D NMR spectroscopy. Extracts, subfractions and isolated compounds were investigated as DPPH radical scavengers and as inhibitors of xanthine oxidase and 15-lipoxygenase.

Results

Major areas of use by Malian healers were against throat infection, amenorrhea and as tonic. Fractionation of the ethanol extract led to the isolation and identification of the cyanogenic glycoside sambunigrin (1), which is previously known from the plant. Additionally, gallic acid (2) and the gallotannins β-glucogalline (3) and 1,6-digalloyl-β-glucopyranose (4) were found. The following flavonoids were isolated: quercetin (5), quercitrin (quercetin-3-O-α-rhamnopyranoside) (6), avicularin (quercetin-3-O-α-arabinofuranoside) (7), quercetin-3-O-β-xylopyranoside (8), quercetin-3-O-(6″-galloyl)-β-glucopyranoside (9) and kaempferol-3-O-(6″-galloyl)-β-glucopyranoside (10). The flavonoids were active both as enzyme inhibitors and DPPH radical scavengers.

Conclusion

Sambunigrin (1) was the main compound in the EtOAc soluble fraction of the alcoholic extract of Ximenia americana leaves. Gallic acid (2), gallotannins (3-4) and flavonoids (5-10) were identified for the first time in the genus Ximenia. The identified compounds may give a rationale for the traditional use of Ximenia americana in Mali. Healers interviewed reported the use against throat infections, amenorrhea, as tonic, for wound healing and against pain.     

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 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.     

Chemical constituents from the stem bark of Symplocos paniculata Thunb. with antimicrobial, analgesic and anti-inflammatory activities

Ethnopharmacological relevance

The stem bark of Symplocos paniculata Thunb. has been used to check abortion in folk medicine in India.

Aim of the study

The present study was undertaken to isolate the phytochemicals from the plant together with the evaluation of antimicrobial, analgesic and anti-inflammatory activities of the plant extract and isolated compounds.

Materials and methods

The plant extract was subjected to column chromatography for isolation of phytochemicals. The agar diffusion method was adopted for antimicrobial activity to determine MICs. Ethanolic extract and isolated compounds were selected for investigating their analgesic activity on acetic acid-induced writhing response in mice. The anti-inflammatory activity was performed on carrageenan-induced paw edema in rats.

Results

The stem bark of the plant afforded seven compounds, 4-(8-hydroxyethyl) cyclohexan-1-oic acid (1); androst-5(6)-ene 17-one 3β-O-(β-d-glucopyranoside) (2); 9β,25-cyclo 3β-O-(β-d glucopyranosyl)-echynocystic acid (3); 9β,19-cyclo 24-methylcholan-5,22-diene 3β-O-{β-d-glucopyranosyl (1 → 6) α-l-rhamnopyranoside} (4); 30-ethyl 2α,16α-dihydroxy 3β-O-(β-d-glucopyranosyl) hopan-24-oic acid (5); 32,33,34-trimethyl-bacteriohopan-16-ene 3-O-β-d-glucopyranoside (6) and flavone 3′,4′,5′,6-tetramethoxy 7-O-β-d-glucopyranosyl (1 → 3) β-d-glucopyranoside (7). The extract and isolated compounds exhibited antimicrobial, analgesic and anti-inflammatory activities.

Conclusion

The present study concludes that ethanolic extract of the plant and its constituents having significant antimicrobial, analgesic and anti-inflammatory activities.     

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.     

Pharmacological mechanisms underlying the anti-ulcer activity of methanol extract and canthin-6-one of Simaba ferruginea A. St-Hil. in animal models

Relevance

Simaba ferruginea A. St-Hil. (Simaroubaceae) is a subshrub typical of the Brazilian Cerrado, whose rhizomes are popularly used as infusion or decoction for the treatment of gastric ulcers, diarrhea and fever.

Aim of the study

To evaluate the pharmacological mechanism(s) of action of the antiulcer effects of the methanol extract of Simaba ferruginea and its alkaloid canthin-6-one.

Materials and methods

Rhizome of Simaba ferruginea was macerated with methanol to obtain the methanol extract (MESf) from which was obtained, the chloroform fraction. Canthin-6-one alkaloid (Cant) was purified and then isolated from the chloroform fraction (CFSf). The isolated Cant was identified by HPLC. Anti-ulcer assays were determined using ethanol and indomethacin-induced ulcer models in mice and rats respectively. In order to determine the probable mechanisms of actions of MESf and Cant animals were pretreated with l-NAME prior to anti-ulcer agent treatments and ulcer induction and nitric oxide (NO) level determined in order to assess NO involvement in the gastroprotective effects. Assays of malondialdehyde (MDA), myeloperoxidase (MPO), pro-inflammatory cytokines: interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and prostaglandin E₂ (PGE₂) were also carried out according to previously described methods.

Results

The results indicate that the antiulcerogenic effects of MESf and Cant in ethanol-induced ulcer is mediated in part through increase in the production of protective endogenous NO as the antiulcerogenic activity of MESf and Cant was reduced in animals pre-treated with l-NAME. In indomethacin-induced ulcer pre-treatment with MESf and Cant showed reduction in the levels of MPO and MDA in the gastric tissue, thus indicating the participation of the antioxidant mechanisms on the gastroprotective effects. The plasma levels of IL-8 in ulcerated rats with indomethacin were also reduced by Cant, but not by MESf, indicating that inhibition of this cytokine contributes to the gastroprotective effect of Cant. However MESf and Cant had no effect on the mucosal membrane levels of PGE₂, indicating that the gastroprotective effects of these agents is independent of PGE₂ modulation.

Conclusion

The results obtained in this study with MESf and Cant added insights into the pharmacological mechanisms involved in their mode of antiulcer action. The results indicate that Cant is one of the compounds responsible for these effects. Such findings are of extreme importance in the strive for future development of potent, safer and effective antiulcer agent. The efficacy of MESf and Cant in gastroprotection shows that Simaba ferruginea might be a promising antiulcer herbal medicine, in addition to confirming the popular use of this plant against gastric ulcer models utilised in this study.     

Hydrophobic constituents and their potential anticancer activities from Devil's Club (Oplopanax horridus Miq.)

Ethnopharmacological relevance

Devil's Club (Oplopanax horridus) is one of the most important spiritual and medicinal plants to many indigenous peoples of Alaska and the Pacific Northwest. It is widely used for external and internal infections as well as arthritis, respiratory ailments, digestive tract ailments, broken bones, fever, headaches, and cancer.

Aim of the study

To investigate hydrophobic constituents and their potential anticancer activity from Devil's Club, Oplopanax horridus.

Materials and methods

The root bark extract of Oplopanax horridus was isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods and comparison with published data. The anti-proliferation of isolated hydrophobic constituents in human breast cancer MCF-7 cells, human colon cancer SW-480 and HCT-116 cells were tested. The potential mechanism of anti-proliferation was also investigated using cell cycle and apoptosis assays.

Results and discussion

Six compounds were isolated and structurally identified as 9,17-octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate (1), oplopandiol acetate (2), falcarindiol (3), oplopandiol (4), trans-nerolidol (5) and t-cadinol (6). These compounds showed potential anticancer activities on human breast cancer and colon cancer cells, of which compound 3 possesses the strongest activity. Further cell cycle and apoptosis tests by flow cytometry showed the polyacetylenes 1-4 induced HCT-116 cell arresting in G2/M phase and inhibited proliferation by the induction of apoptosis at both earlier and later stages.

Conclusion

These results provide promising baseline information for the potential use of Oplopanax horridus, as well as some of the isolated compounds in the treatment of cancer.     

Bioassay-guided isolation of anti-inflammatory, antinociceptive and wound healer glycosides from the flowers of Verbascum mucronatum Lam.

Ethnopharmacological relevance

The leaves, flowers and whole aerial parts of Verbascum L. species have been used to treat respiratory problems, haemorrhoids and other types of inflammatory conditions in traditional Turkish medicine.

Aim of the study

In order to evaluate this traditional information, the anti-inflammatory, antinociceptive and wound healing activities of Verbascum mucronatum Lam. which is used as haemostatic in Turkish folk medicine were investigated.

Materials and methods

In vivo inhibitory effect of the extracts on the carrageenan-induced hind paw edema model in mice was studied for the assessment of anti-inflammatory activity. Moreover, the wound healing potential of the plant were evaluated by using in vivo wound healing experimental models, i.e. incision and excision models on mice and rats, were comparatively assessed with a reference ointment Madecassol^®. Skin samples were also evaluated histopathologically.

Results

The results of these experimental studies exhibited that Verbascum mucronatum displays anti-inflammatory, antinociceptive and wound healing activities. Through bioassay-guided fractionation and isolation procedures four iridoid glucosides, ajugol (1), aucubin (2), lasianthoside I (3), catalpol (4), two saponins, ilwensisaponin A (5) and C (6) and a phenylethanoid glycoside, verbascoside (7) were isolated and their structures were elucidated by spectral techniques. Verbascoside (7) was found to possess significant wound healing activity as well as antinociceptive and anti-inflammatory potentials, per os without inducing any apparent acute toxicity or gastric damage.

Conclusion

The experimental study revealed that Verbascum mucronatum displays remarkable antinociceptive, anti-inflammatory and wound healing activities.     

Encecalol angelate, an unstable chromene from Ageratum conyzoides L.: total synthesis and investigation of its antiprotozoal activity

Ethnopharmacological relevance

In agreement with ethnomedicinal reports, the dichloromethane extract of Ageratum conyzoides L. (Asteraceae) was recently shown to be of considerable activity against Trypanosoma brucei rhodesiense, the etiologic agent of East African Human Trypanosomiasis (East African Sleeping Sickness). Isolated compounds, namely, methoxylated flavonoids as well as the chromene derivative encecalol methyl ether, were less active than the crude extract. The activity of the extract was found to decrease considerably while stored in solution. An unstable compound was detected in the fresh extract by HPLC, which was converted rapidly into the encecalol methyl ether while stored in methanolic solution. This compound, deemed to represent a constituent with antitrypanosomal activity, could not be isolated from the extract in intact form.

Aim of the study

To elucidate the structure of this unstable compound and to investigate its potential role in the antitrypanosomal activity of the total extract.

Materials and Methods

UHPLC/ESI-qQTOF MSMS and NMR data of the degraded product indicated its chemical identity as encecalol angelate (1) which was therefore prepared by total synthesis via a linear six steps synthesis, starting from resorcinol and 2-methylbut-3-en-2-ol.

Results

Total synthesis, in an overall yield of 15%, led to pure 1, which was chromatographically and spectroscopically identical with the natural product. The compound degraded in methanol with a half-life of approximately 6 h to yield encecalol methyl ether (2). The antiprotozoal activity of synthetic encecalol angelate against T. brucei rhodesiense as well as T. cruzi, Leishmania donovani and Plasmodium falciparum was investigated and found to be quite low.

Conclusions

The synthetic approach applied here for the first time also provides access to the related bioactive chromenes encecalin (7) and encecalol (8) with improved yields compared with reported methods. Encecalol angelate, however, is most likely not responsible for the high antitrypanosomal activity of the freshly prepared dichloromethane extract of A. conyzoides.     

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana

Ethnopharmacological relevance

Tridax procumbens is an active herb against leishmaniasis.

Aim of the study

Leishmaniasis is a group of diseases caused by Leishmania protozoa. We investigated the antileishmanial activity of Tridax procumbens extracts and a pure compound against promastigotes of Leishmania mexicana, the causative agent of cutaneous leishmaniasis in the New World.

Materials and methods

Extracts and (3S)-16,17-didehydrofalcarinol (1) were obtained by chromatographic methods from Tridax procumbens, and the latter identified by spectroscopic analysis. The effect of these extracts and 1 on the growth inhibition of promastigotes of Leishmania mexicana was evaluated. In order to test the safety of extracts and 1, mammalian cells were treated with them, and cell viability was assessed using trypan blue and MTT.

Results

We demonstrated that extracts of Tridax procumbens and 1 showed a pronounced activity against Leishmania mexicana. The methanol extract inhibited promastigotes growth of Leishmania mexicana with a 50% inhibitory concentration (IC₅₀) of 3 μg/ml, while oxylipin 1 exhibited the highest inhibition at IC₅₀ = 0.478 μg/ml.

Conclusions

In this study we report the biological activity of extracts and (3S)-16,17-didehydrofalcarinol (1), obtained from Tridax procumbens, on the promastigote form of Leishmania mexicana, with no effect upon mammalian cells.     

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.     

Antinociceptive effect of extracts and compounds from Hofmeisteria schaffneri

Ethnopharmacological relevance

Hofmeisteria schaffneri (Asteraceae) is a medicinal plant widely commercialized in the most important Markets of Mexico City for the treatment of gastro-intestinal complaints and skin afflictions.

Aim of the study

The main goals of this study were to establish the potential acute toxicity and the antinociceptive activity in animal models of several preparations and compounds from Hofmeisteria schaffneri.

Materials and methods

The aqueous and organic extracts as well as the essential oil of Hofmeisteria schaffneri were prepared by infusion, maceration and hydrodistillation, respectively. Investigation of the acute toxicity was accomplished by the Lorke method. The antinociceptive effect was assessed using the writhing and the hot plate tests. Natural compounds were isolated by standard phytochemical procedures. In addition, a few thymol esters were prepared by chemical synthesis. The stability of natural and synthetic esters was qualitatively analyzed by measuring their susceptibility to hydrolysis by pig liver estearase and mouse plasma at 37 °C.

Results

The LD₅₀ for each preparation tested was higher than 5000 mg/kg revealing that they were not toxic to mice after exposure for short space of time. On the other hand, the extracts showed significant antinociceptive effect when tested in the hot plate model. The most active natural product as antinociceptive agent was hofmeisterin III (1) which also was the most stable in the stability study. Its pharmacological effect seems to be partially mediated by an opioid mechanism since naloxone inhibits its action. Using compound 1 as a lead molecule, several synthetic thymol esters were prepared and only compounds 13, 15 and 17 were antinoceptive at the dose of 1 mg/kg.

Conclusions

The present investigation provided evidence of the efficacy of several preparations of Hofmeisteria schaffneri as antinociceptive agents. The most active preparation was the essential oil which contained large amount of hofmeisterin III (1) and other thymol derivatives. Some novel synthetic analogs of hofmeisterin III with antinociceptive properties were discovered. The nature of the ester chain of these analogs did not have a clear impact on the antinociceptive activity. The phyto-preparations analyzed in this study were not toxic to mice according to the Lorke's test; therefore considering their long term use of the plant they might be secure for human consumption.     

Iridoids as chemical markers of false ipecac (Ronabea emetica), a previously confused medicinal plant

Ethnopharmacological relevance

Several roots or rhizomes of rubiaceous species are reportedly used as the emetic and antiamoebic drug ipecac. True ipecac (Carapichea ipecacuanha) is chemically well characterized, in contrast to striated or false ipecac derived from the rhizomes of Ronabea emetica (syn. Psychotria emetica). Besides its previous use as substitute of ipecac, the latter species is applied in traditional medicine of Panama and fruits of its relative Ronabea latifolia are reported as curare additives from Colombia.

Materials and methods

Compounds of Ronabea emetica were isolated using standard chromatographic techniques, and structurally characterized by NMR spectroscopy and mass spectrometry. Organ specific distribution in Ronabea emetica as well as in Ronabea latifolia was further assessed by comparative HPLC analysis.

Results

Four iridoid-glucosides, asperuloside (1), 6α-hydroxygeniposide (2), deacetylasperulosidic acid (3) and asperulosidic acid (4) were extracted from leaves of Ronabea emetica. Rhizomes, used in traditional medicine, were dominated by 3. HPLC profiles of Ronabea latifolia were largely corresponding. These results contrast to the general tendency of producing emetine-type and indole alkaloids in species of Psychotria and closely related genera and merit chemotaxonomic significance, characterizing the newly delimited genus Ronabea.

Conclusions

The aim of the work was to resolve the historic problem of adulteration of ipecac by establishing the chemical profile of Ronabea emetica, the false ipecac, as one of its less known sources. The paper demonstrates that different sources of ipecac can be distinguished by their phytochemistry, thus contributing to identifying adulterations of true ipecac.     

Identification of a new antinociceptive alkaloid isopropyl N-methylanthranilate from the essential oil of Choisya ternata Kunth

Ethnopharmacological relevance

Mexican people employed infusion of leaves of Choisya ternata Kunth for their antispasmodic and “simulative properties”.

Aim of the study

In the present study the detailed GC and GC-MS analyses of the essential oil of Choisya ternata Kunth (Rutaceae) were performed. The presence of a minor constituent isopropyl N-methylanthranilate (1) was revealed among other identified volatiles. A synthesis of 1 was undertaken in order to corroborate this find and obtain gram quantities that would allow the testing of its biological activity (peripheral and central antinociceptive activity).

Materials and methods

The oils were investigated by GC and GC-MS. Synthesized compounds were spectrally characterized (UV-Vis, IR, 1D and 2D NMR, MS). The obtained synthetic samples of compounds were assayed for peripheral and central antinociceptive activity in two models (effects on acetic acid induced writhing in mice and the hot plate test for nociception).

Results

Detailed GC and GC-MS analyses of the essential oil of Choisya ternata Kunth (Rutaceae) among 157 other identified volatiles revealed the presence of a minor constituent isopropyl N-methylanthranilate (1). Compound 1, named ternanthranin, is therefore detected as a natural product for the first time with a very restricted occurrence (samples of several citrus oils were screened for the presence of 1). The antinociceptive activities were assayed for ternanthranin, the two other synthetic analogs, methyl and propyl N-methylanthranilate, as well as the essential oil and the crude ethanol extract of the leaves. The results clearly demonstrate a very high (even significant at 0.3 mg/kg) dose dependent activity for the anthranilates (and the extracts). Isopropyl N-methylanthranilate showed the highest, while methyl N-methylanthranilate showed the lowest activity (with the methyl ester at 3 mg/kg still better than acetylsalicylic acid, at 200 mg/kg, in the first, or comparable with morphine, at 5 mg/kg, in the second test).

Conclusion

This study once again revealed that detailed investigations of plant species with ethnopharmacologically documented activity may yield new natural compounds—a new alkaloid (ternanthranin), a volatile simple anthranilate that can be considered responsible for the antinociceptive activity of the crude plant extracts.     

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.     

Detection of antifungal compounds in Polygonum ferrugineum Wedd. extracts by bioassay-guided fractionation. Some evidences of their mode of action

Ethnopharmacological relevance

Polygonum ferrugineum Wedd. (Polygonaceae) is used to heal infected wounds and as antiseptic, antibiotic or antifungal in the traditional Argentinean medicine. The present investigation was carried out to evaluate the antifungal properties of different extracts of aerial parts of Polygonum ferrugineum, in order to give support to its ethnopharmacological use and to isolate the compounds responsible for the antifungal properties. The most active compounds were tested for their capacity of producing hyphae malformations, similar to those previously observed for crude extracts.

Materials and methods

Agar Dilution Method (ADM) and Agar Overlay Bioautography (AOB) were used for bioassay-guided fractionation of the aerial part extracts against a panel of human opportunistic pathogenic fungi. The Neurospora crassa assay, followed by Optical Microscopy and Scanning Electron Microscopy observation, was used for studies of mechanisms of action.

Results

MeOH extract and DCM and Hex sub-extracts, but not Aq, EtOAc or BuOH ones possess antifungal activity. Of the seven isolated compounds, cardamonin 2 showed a selective inhibition of Epidermophyton floccosum with a very low MIC (=6.2 μg/mL) and pashanone 1 possessed moderate antifungal activity (MICs = 25-50 μg/mL) but a broader spectrum of action. Chalcone 2, but not 1, induced swelling and shortening of the Neurospora crassa hyphae, similar as those caused by the crude DCM extract.

Conclusions

The bioassay-guided fractionation of Polygonum ferrugineum DCM extract allowed the isolation of five active compounds. Among them, cardamonin 2 showed the highest antifungal activity and selectivity towards Epidermophyton floccosum; in addition, it induced Neurospora crassa malformations that are similar than those produced by the crude DCM extract. These results give additional support to the ethnopharmacological use of Polygonum ferrugineum as antifungal agent.