Isolation of active constituents from cherry laurel (Laurocerasus officinalis Roem.) leaves through bioassay-guided procedures

Ethnopharmacological relevance

The fresh leaves of Laurocerasus officinalis Roem. (Rosaceae) are externally used against pain and feverish symptoms in Turkish folk medicine.

Aim of the study

Effects of the extracts, fractions and isolated compounds from the leaves of L. officinalis were investigated using in vivo models of inflammation and pain in mice.

Methods

The crude ethanolic extract from the leaves of plant was sequentially fractionated into five subextracts; explicitly, n-hexane, chloroform, ethyl acetate (EtOAc), n-butanol, and remaining water extracts. Further studies were carried out on the most active EtOAc subextract was further subjected to fractionation through column chromatography. For the anti-inflammatory activity, carrageenan-induced hind paw edema and acetic acid-induced increase in capillary permeability models, and for the antinociceptive activity p-benzoquinone-induced writhing test in mice were employed.

Results

Ethanolic extract of the leaves was shown to possess significant inhibitory activity in the assay methods without inducing any gastric damage. Through bioassay-guided fractionation and isolation procedures three phenolic compounds, 2-O-β-d-glucopyranosyl-2-hydroxyphenyl-acetic acid (1), kaempferol-3-O-β-d-xylopyranosyl-(1→2)-O-β-d-glucopyranoside (2) and (+)-catechin (3) were isolated from the active fraction and their structures were elucidated by spectral techniques (1D and 2D NMR, ESIMS).

Conclusion

The experimental data verified that Laurocerasus officinalis leaves displayed remarkable anti-inflammatory and antinociceptive activity.     

Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells

Aim of the study

Pregnane glycosides are potent cytotoxic agents which may represent new leads in the development of anti-tumour drugs, particularly in the treatment of breast cancer, because of the structural similarity to estrogenic agonists. Caralluma species are natural sources of a wide variety of pregnane glycosides. The aim of the study was to isolate, using an activity-guided fractionation approach, novel pregnane glycosides for testing on breast cancer and other tumour lines.

Materials and methods

The effect of crude extracts, specific organic fractions and isolated compounds from Caralluma tuberculata was tested on the growth and viability of MCF-7 estrogen-dependent, and MDA-MB-468 estrogen-independent breast cancer cells, Caco-2 human colonic cells, HUVECs and U937 cells. Neutral red uptake and MTT assays were used. Apoptosis was detected by Western blot of poly-(ADP ribose) polymerase (PARP) as were other markers of nuclear fragmentation (DNA ladder assay, staining of cells with nuclear dye DAPI). The involvement of caspases was investigated using the pan-caspase inhibitor Z-VAD-FMK.

Results

The ethyl acetate fraction of Caralluma tuberculata was found to be the most potent anti-proliferative fraction against all three cancer cell lines. Two novel steroidal glycosides were isolated from the active fraction after a series of chromatographic experiments. The structure of the isolated compounds was elucidated solely based on 2D-NMR (HMBC, HETCOR, DQF-COSY) and MS spectral analysis as compound 1: 12-O-benzoyl-20-O-acetyl-3β,12β,14β,20β-tetrahydroxy-pregnan-3-ylO-β-D-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-3-methoxy-β-d-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3β,7β,12β,14β-tetrahydroxy-17β-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-β-d-glucopyranosyl-(1 → 4)-6-deoxy-β-d-allopyranosyl-(1 → 4)-β-d-cymaropyranosyl-(1 → 4)-β-d-cymapyranosyl-(1 → 4)-β-d-cymaropyranoside. Compound 1 (pregnane glycoside) and compound 2 (androstan glycoside) induced apoptosis at <25 μM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor.

Conclusions

Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design.     

An anti-ulcerogenic flavonol diglucoside from Equisetum palustre L.

Ethnopharmacological relevance

The aerial parts of Eqiusetum palustre L. is used to treat peptic ulcer in Karaman, Turkey.

Aim of the study

The present study was undertaken to evaluate the in vivo gastroprotective potential and structure elucidation of the active constituent(s) through bioassay-guided fractionation procedures by using ethanol (EtOH)-induced ulcerogenesis model in rats.

Material and methods

The 80% ethanol extract of the aerial parts was subjected to subsequent separation procedures such as solvent–solvent partition and column chromatography through bioassay-guided fractionation techniques to isolate the active anti-ulcer component(s) by using the EtOH-induced ulcer model in rats. The ulcer index was used to evaluate and to calculate the gastroprotection percentage.

Results

The subextracts, n-butanol and remaining H₂O, of Equisetum palustre showed potent in vivo gastroprotective activity. Bioassay-guided fractionation has revealed that a flavonol diglucoside, was isolated as an active constituent from the n-BuOH subextract of Equisetum palustre. Structure elucidation by standard spectroscopic techniques (¹H NMR, ¹³C NMR and DEPT, etc.) revealed that this compound as kaempferol-3-O-1″-β-d-glucopyranosyl-3-O-1″′-β-d-glucopyranoside, which provided remarkable protection at 250 and 500 mg/kg doses (69.5 and 98.5% ulcer inhibition, respectively) against ethanol-induced gastric lesions.

Conclusion

Experimental data confirmed the ethnobotanical usage of Equisetum palustre, with scientific evidence, in Turkey. Additionally, kaempferol-3-O-1″-β-d-glucopyranosyl-3-O-1″′-β-d-glucopyranoside, a known compound, was defined as an anti-ulcerogenic compound in the present work for the first time.     

Anti-inflammatory activity of the extracts and fractions from Erigeron multiradiatus through bioassay-guided procedures

Ethnopharmacological relevance

Erigeron multiradiatus (Lindl.) Benth., an herb that grows in the alpine and subalpine meadow of Qinghai-Tibet plateau, has been widely used as a folk remedy by the native people for treatment of various inflammatory ailments.

Aim of the study

In order to isolate and identify the active components of Erigeron multiradiatus for anti-inflammatory activity, a preliminary phytochemical study and a bioassay-guided fractionation and purification process was performed.

Material and Methods

The dry whole plant Erigeron multiradiatus was extracted with 50% ethanol and then separated into CHCl₃, n-BuOH, and aqueous fractions. The anti-inflammatory activities of each fraction were investigated using two in vivo inflammation models.

Results

These results exhibited varying degrees of anti-inflammatory activities and the n-BuOH fraction showed the strongest anti-inflammatory activities. The n-BuOH fraction was then subjected to separation and purification using macroporous resins column chromatography and Sephadex LH-20 leading to two flavonoids glucuronides identified as scutellarein-7-O-β-glucuronide and apigenin-7-O-β-glucuronide. Furthermore, LC–MS/MS identification and quantification of isolated compounds were also performed.

Conclusion

Scutellarein-7-O-β-glucuronide and apigenin-7-O-β-glucuronide were considered as major components and principally responsible for the anti-inflammatory activity of Erigeron multiradiatus. Thus the results of our study provide a scientific basis for the utilization of Erigeron multiradiatus in traditional Tibetan medicine.     

Valuation of anti-inflammatory and antinociceptive activities of Erica species native to Turkey

Erica L. species (Ericaceae) have been popularly used as antirheumatic, diuretic, astringent and treatment of urinary infections. In order to evaluate this information, anti-inflammatory and antinociceptive activities of different extracts prepared with methanol, chloroform, ethyl acetate, n-butanol and water from the aerial parts of Erica arborea L., Erica manipuliflora Salisb., Erica bocquetii (Pe?men) P.F. Stevens and Erica sicula Guss. subsp. libanotica (C.&;W. Barbey) P.F. Stevens (Ericaceae) of Turkish origin were investigated by using in vivo methods. For the anti-inflammatory activity, carrageenan-induced hind paw edema model, PGE₂-induced hind paw edema model, and 12-O-tetradecanoyl-13-acetate (TPA)-induced mouse ear edema model and for the antinociceptive activity p-benzoquinone-induced writhing test in mice were employed. The ethyl acetate extracts of Erica arborea (EAE), Erica bocquetii (EBE) and Erica manipuliflora (EME) exhibited notable inhibition against carrageenan-induced (24.1–32.3%, 23.8–36.1%, 29.2–35.1%, respectively) and PGE₂-induced (21.2–37.7%, 6.8–29.7%, and 6.2–34.1%, respectively) hind paw edema as well as TPA-induced mouse ear edema models in mice, while the ethyl acetate extract of Erica sicula subsp. libanotica (ESE) (10.7–29.7%) displayed potent anti-inflammatory activity only on the PGE₂-induced hind paw edema model. However, the remaining extracts were found to be inactive against inflammatory models. Same extracts, i.e., EAE, EBE and EME were also found to exhibit remarkable antinociceptive activity in p-benzoquinone-induced abdominal constriction test at a dose of 100 mg/kg (46.5%, 27.7% and 36.3%, respectively).     

Antinociceptive effectiveness of triterpenes from rosemary in visceral nociception

Ethnopharmacological relevance

Rosemary is a species used worldwide as a common spice, but also in folk medicine for their therapeutic properties against abdominal pain. The rationale of this study was to examine the involvement of triterpenes and to compare their effectiveness in the antinociceptive effect of an ethanol extract of Rosmarinus officinalis L. (Lamiaceae).

Material and methods

Fractionation and HPLC analyses allowed the identification of a mixture of micromeric (121 mg/g), oleanolic (64 mg/g) and ursolic (83 mg/g) acids as partial antinociceptive responsible in an ethyl acetate fraction of R. officinalis by using the acetic acid-induced abdominal constrictions model in mice.

Results

These triterpenes individually evaluated produced a significant and dose-dependent antinociceptive response with similar potency as follows: ED₅₀=1.1 mg/kg (0.9–1.3 mg/kg), 2.1 mg/kg (1.6–2.6 mg/kg) and 1.6 mg/kg (1.1–2.1 mg/kg), respectively, by using the intraperitoneal (i.p.) route of administration in mice. Their maximal antinociceptive efficacy resembled that produced by ketorolac (10 mg/kg, i.p.), a common clinic analgesic.

Conclusion

Our results provide evidence that these triterpenes participate in the antinociceptive activity of R. officinalis. In addition, each individual triterpene showed a similar potency to that observed with ketorolac, a non-steroidal anti-inflammatory drug, in this experimental model.     

Bioguided isolation of myricetin-3-O-β-galactopyranoside with antinociceptive activity from the aerial part of Davilla elliptica St.-Hil

Ethnopharmacological relevance

Davilla elliptica St.-Hil. (Dilleniaceae) is a medicinal plant traditionally used in Brazil to treat inflammatory processes, to relieve pain, as diuretic, gastro- and hepatoprotective agents.

Aim of the study

To undertake the fractionation of the ethanolic extract from Davilla elliptica leaves guided by an antinociceptive assay.

Materials and methods

The antinociceptive activity was evaluated through the formalin test in mice. Extract fractionation was performed by percolation through silica gel and partition between immiscible solvents, followed by successive column chromatography over Sephadex LH-20 and preparative RP-HPLC. Structure elucidation of the isolated compound was accomplished by spectroscopic data.

Results

The EtOAc and MeOH fractions derived from the crude extract reduced significantly the licking time in the late phase of the formalin test. The bioguided fractionation of the MeOH fraction resulted in the isolation of myricetin-3-O-β-galactopyranoside, which produced significant inhibition on nociception induced by formalin (ID₅₀=0.26 mg/kg; p.o.).

Conclusions

These results point out that myricetin-3-O-β-galactopyranoside contributes for the antinociceptive effect of Davilla elliptica extract, a constituent considerably more potent than diclofenac, employed as reference drug.     

Anti-inflammatory effect of extract and fractions from the leaves of Byrsonima intermedia A. Juss. in rats

Aim of the study

Byrsonima intermedia is commonly used for its antiseptic, antimicrobial, and anti-inflammatory properties in the treatment of diarrhea and dysentery in Brazilian folk medicine. The purpose of this study was to examine the anti-inflammatory activity of the aqueous extract and fractions of Byrsonima intermedia leaves.

Materials and methods

Rats with carrageenan-induced paw edema and fibrovascular tissue growth, which was induced by subcutaneous implantation of a cotton pellet, were used as acute and chronic animal models of inflammation to investigate the anti-inflammatory effects of the aqueous extract and the individual ethyl acetate (EtOAc) and aqueous fractions of Byrsonima intermedia and catechin. High-performance liquid chromatography (HPLC) was used to determine the fingerprint chromatogram of the aqueous extract and fractions of Byrsonima intermedia.

Results

The crude aqueous extract at test doses of 30-300 mg/kg p.o. clearly demonstrated anti-inflammatory effects by reducing carrageenan-induced paw edema, as did the ethyl acetate (100 mg/kg) and aqueous fractions (30-100 mg/kg). In the chronic inflammation rat animal model with fibrovascular tissue growth, the aqueous extract of Byrsonima intermedia (BiAE) at doses of 30-300 mg/kg and the individual EtOAc and aqueous fractions at doses of 30-100 mg/kg and catechin significantly reduced the formation of granulomatous tissue. The presence of catechin and phenolic compounds in the extract and fractions of Byrsonima intermedia was confirmed using HPLC.

Conclusion

BiAE and the individual EtOAc and aqueous fractions of Byrsonima intermedia exhibited chronic and acute anti-inflammatory efficacy in rats, which supports previous claims of its use in traditional medicine.     

Evaluation of the antiulcerogenic activity profile of a flavonol diglucoside from Equisetum palustre L.

Ethnopharmacological relevance

The aerial parts of Equisetum palustre L. are used to treat peptic ulcer disease in Turkey. In a previous study, a flavonol diglucoside i.e., kaempferol 3-O-1″-β-d-glucopyranosyl-3-O-1″′-β-d-glucopyranoside (KGG) was isolated as the major antiulcerogenic constituent from the plant.

Aim of the study

The present study was undertaken to evaluate the antiulcer activity profile of KGG using various in vivo experimental ulcer models as well as by assessing gastric biochemical parameters.

Material and methods

KGG was obtained from the ethanol extract of the aerial parts of the plant by successive chromatographical methods. The activity profile of the compound was investigated using several ulcerogenesis models such as indomethacin-, indomethacin plus HCl/EtOH-, cysteamine-, serotonin-, N^G-nitro-l-arginine methyl ester plus EtOH-, diethyldithiocarbamate-, N-ethylmaleimide plus EtOH-, water immersion and restraint stress-, pyloric ligation-induced ulcers. In addition, effects of KGG on the biochemical parameters of gastric juice; i.e., inhibition of titratable gastric acidity, acid output, gastric pH, gastric secretion volume and peptic activity were studied.

Results

KGG exerted statistically significant gastroprotective activity against indomethacin-, indomethacin plus HCl/EtOH- and N-ethylmaleimide plus EtOH-induced ulcerogenesis. Moreover, KGG demonstrated weak activity against N^G-nitro-l-arginine methyl ester plus EtOH, water immersion and immobilization-induced stress, pyloric ligation-induced and diethyldithiocarbamate-induced gastric ulcer models, and also it was ineffective in the prevention of ulcers induced by serotonin and cysteamine. On the other hand, among the gastric biochemical parameters studied, KGG was only found to increase the gastric acid pH from 2.03 to 3.35.

Conclusion

Results of this investigation have clearly demonstrated that KGG was found to improve the cytoprotective mechanisms of the gastric mucosa. On the other hand, a weak activity profile was observed on the parameters affecting the gastric acidity (water immersion and restraint-induced-, pyloric ligation-induced-ulcerogenesis and titratable acidity).     

Antinociceptive and anti-inflammatory kaempferol glycosides from Sedum dendroideum

Aim of the study

To identify the compounds responsible for the antinociceptive and anti-inflammatory effects previously described for Sedum dendroideum, through bioassay-guided fractionation procedures.

Materials and methods

Antinociceptive activity was evaluated through mouse acetic acid-induced writhing model. The anti-inflammatory activity was assessed through croton oil-induced mouse ear oedema and carrageenan-induced peritonitis.

Results

The Sedum dendroideum juice afforded seven known flavonoids identified with basis on NMR data. The oral administration of the major kaempferol glycosides kaempferitrin [1] (17.29 μmol/kg), kaempferol 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside [2] (16.82 μmol/kg), kaempferol 3-O-neohesperidoside-7-O-α-rhamnopyranoside [3] (13.50 μmol/kg) or α-rhamnoisorobin [5] (23.13 μmol/kg) inhibited by 47.3%, 25.7%, 60.2% and 58.0%, respectively, the acetic acid-induced nociception (indomethacin: 27.95 μmol/kg, p.o.; 68.9%). Flavonoids 1, 2, 3 or 5, at the same doses, reduced by 39.5%, 46.5%, 35.6% and 33.3%, respectively, the croton oil-induced oedema (dexamethasone: 5.09 μmol/kg, s.c.; 83.7%) and impaired leukocyte migration by 42.9%, 46.3%, 50.4% and 49.6%, respectively (dexamethasone: 5.09 μmol/kg, s.c.; 66.1%).

Conclusions

Our findings show that the major kaempferol glycosides may account for the renowned medicinal use of Sedum dendroideum against pain and inflammatory troubles.     

Estimation of anti-inflammatory,antinociceptive and antioxidant activities on Arctium minus (Hill) Bernh. ssp. minus

Ethnopharmacological relevance

Arctium minus (Hill) Bernh. ssp. minus (Asteraceae) leaves are used to alleviate rheumatic pain, against fever and sunstroke with externally application in Turkish folk medicine.

Aim of the study

To evaluate the anti-inflammatory, antinociceptive and antioxidant activities of aqueous and ethanol extracts prepared from the leaves of Arctium minus ssp. minus.

Materials and methods

The ethanolic and aqueous extracts from the leaves of Arctium minus ssp. minus were evaluated in mice for anti-inflammatory activity using carrageenan-induced hind paw edema model and for antinociceptive activity using p-benzoquinone-induced abdominal contractions test. Moreover, the antioxidant power of the extracts has been determined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and flow injection analysis-luminol chemiluminescence (FIA-CL). In addition, the total phenolic content in both extracts was determined with spectrophotometric method.

Results

Our results showed that only the ethanol extract exhibited a dose-dependent anti-inflammatory activity ranging between 11.1 and 23.6% at 200 mg/kg dose as well as displayed a significant antinociceptive activity without inducing any gastric damage. Although, both extracts were shown to possess significant DPPH radical-scavenging activity, that of aqueous extract was found to have more pronounced activity. In FIA-CL system, the ethanol extract was shown to possess a significant scavenger activity against H₂O₂ while the aqueous extract was much more potent antioxidant activity against HOCl-luminol CL than ethanol extract.

Conclusion

According to our results, it was concluded that Arctium minus ssp. minus contains potent natural antioxidants. In this study, in vivo experimental results have also supported the folk medicinal utilization of Arctium minus ssp. minus.     

Anti-inflammatory activities and mechanisms of action of the petroleum ether fraction of Rosa multiflora Thunb. hips

Ethnopharmacological relevance

The hip of Rosa multiflora Thunb. has been traditionally used as a dietary supplement and a herbal remedy for the treatment of various diseases including cold, flu, inflammation, osteoarthritis, rheumatoid arthritis and chronic pain in China.

Aims of the study

To explore the anti-inflammatory ingredient of the hip of R. multiflora Thunb. and its mechanism of action.

Materials and methods

The ethanol extract of the hip of R. multiflora Thunb. was fractioned with petroleum ether, ethyl acetate and water, and each fraction was screened for anti-inflammatory activity in xylene-induced mouse ear edema model. Three more models, acetic acid-induced mouse vascular permeation, cotton pellet-induced rat granuloma, and carrageenan-induced rat hind paw edema were also employed to verify the anti-inflammatory effect of the identified fraction. To explore the mechanism of action, the activity of inducible nitric oxide synthase (iNOS) and the level of nitric oxide (NO) in sera, as well as mRNA expression level of cyclo-oxygenase-2 (COX-2) in inflammatory tissues of rats with carrageenan-induced hind paw edema were measured. GC-MS technology was applied to identify the active components in the active fraction.

Results and conclusions

The petroleum ether fraction (PEF) was identified to be the active fraction in inflammation animal models (i.e., oral administration of PEF (168.48, 42.12 and 10.53 mg/kg) evoked a significantly (P < 0.001) dose-dependent inhibition of the xylene-induced mice ear edema). Down-regulating COX-2 expression (P < 0.001) and reducing NO production (P < 0.05) through inhibiting iNOS activity (P < 0.001) may be the partial mechanism of action of PEF. GC-MS analysis indicated that unsaturated fatty acids are enriched in PEF and may be responsible for the anti-inflammatory activity of PEF and this herb. The results of this study provide pharmacological and chemical basis for the application of the hip of R. multiflora Thunb. in inflammatory disorders.     

Investigation for anti-inflammatory and anti-thrombotic activities of methanol extract of Capparis ovata buds and fruits

Ethnopharmacological relevance

Capparis ovata Desf. has wide natural distribution in Turkey and it is consumed in pickled form. Flower buds, root bark, and fruits of the plant are used traditionally due to their analgesic, anti-inflammatory, wound healing, anti-rheumatismal, tonic, and diuretic effects.

Aim of the study

The aim of this study was to investigate the possible anti-inflammatory and anti-thrombotic effects of methanol extracts prepared from flower buds (CBE) and fruits (CFE) of C. ovata.

Materials and methods

Anti-inflammatory effects of CBE and CFE were assessed using carrageenan-induced and prostaglandin E₂-induced mouse paw edema models. For the anti-thrombotic effect evaluation, carrageenan-induced tail thrombosis model was performed in mice. The extracts were administered intraperitonally (i.p.) at the doses of 100, 200, and 300 mg/kg. The anti-inflammatory effect of Capparis extracts were tested in comparison to 10 mg/kg diclofenac and anti-thrombotic activity to 10 and 100 IU heparin.

Results

CBE at the doses of 200, and 300 mg/kg and CFE at the doses of 100, 200, and 300 mg/kg showed significant anti-inflammatory activity and CFE reached therapeutic concentration early than CBE in carrageenan inflammation model. In prostaglandin E₂ inflammation model, CBE and CFE exhibited significant inhibitory effects. The C. ovata extracts did not show remarkable anti-thrombotic effect.

Conclusions

Based on the results obtained, it can be concluded that fruits of C. ovata have more potent anti-inflammatory effect than flower buds. It has been suggested that inhibition of cyclooxygenase pathway is one of the mechanisms of the activity. C. ovata may be potentially used as therapeutic agents for inflammatory diseases.     

Antiparasitic activities of two sesquiterpenic lactones isolated from Acanthospermum hispidum D.C

Ethnopharmacological relevance

Aerial parts of Acanthospermum hispidum D.C. are often used by traditional healers in Benin for various diseases and especially for malaria.

Aim of the study

To identify active compounds from extracts of Acanthospermum hispidum D.CV. leaves previously shown to possess antimalarial properties and analyse in vivo activity and toxicity of crude extracts.

Materials and methods

Compounds were isolated from aerial part of Acanthospermum hispidum D.C. and structurally elucidated using extensive spectroscopic analysis. Antiplasmodial activity was evaluated in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity and in vivo against Plasmodium berghei berghei by the 4-day suppressive test. Selectivity of extract and purified compounds on Plasmodium parasites were evaluated by using MTT test on J774 macrophage like murine cells and WI38 human normal fibroblasts and also against two other parasites: Trypanosoma brucei brucei and Leishmania mexicana mexicana. Acute and sub-acute toxicities of a crude extract were evaluated on mice.

Results

Two known sesquiterpenic lactones were isolated: 1 (15-acetoxy-8β-[(2-methylbutyryloxy)]-14-oxo-4,5-cis-acanthospermolide) and 2 (9α-acetoxy-15-hydroxy-8β-(2-methylbutyryloxy)-14-oxo-4,5-trans-acanthospermolide). 1 and 2 showed in vitro antiplasmodial activity against the chloroquine-sensitive strain (3D7) with IC₅₀ of 2.9 ± 0.5 and 2.23 ± 0.09 μM respectively. Only 2 showed a high selectivity index (SI: 18.4) on Plasmodium compared to cytotoxicity against human fibroblasts cell line (WI38). 1 and 2 also showed interesting antiparasitic activities in vitro against Trypanosoma brucei brucei (IC₅₀ of 2.45 ± 0.49 and 6.36 ± 1.42 μM respectively) and Leishmania mexicana mexicana (IC₅₀ of 0.94 ± 0.05 and 2.54 ± 0.19 μM respectively). Furthermore, crude acidic water extract and fractions containing one of the two isolated compounds displayed a weak in vivo antimalarial activity against Plasmodium berghei berghei with a long half-life causing a delayed effect. In vivo acute (2000 mg/kg) and sub-acute (1000 mg/kg) toxicity tests on the crude acidic water extract did not show toxicity.

Conclusion

Crude acidic water extract, fractions and pure isolated compounds from Acanthospermum hispidum showed promising in vitro antiplasmodial activity. Despite our study did not show in vivo acute and subacute toxicities of the crude acidic water extract, its weak in vivo antimalarial activity and the in vitro cytotoxicity of pure compounds and enriched extracts containing 1 and 2 indicate that the aerial parts of Acanthospermum hispidum should be used with caution for malaria treatments.     

Anti-inflammatory and antinociceptive effects of Arrabidaea brachypoda (DC.) Bureau roots

Aim of the study

Arrabidaea brachypoda (DC.) Bureau has been used to relieve general pain, painful joints and kidney stones in Brazilian folk medicine. Nevertheless, scientific information regarding this species is scarce; there are no reports related to its possible analgesic and anti-inflammatory effects. This study was aimed at evaluating the traditional use of Arrabidaea brachypoda root using in vivo inflammatory and nociceptive models.

Materials and methods

Carrageenan-induced paw edema, peritonitis and fibrovascular tissue growth induced by s.c. cotton pellet implantation were used to investigate the anti-inflammatory activity of Arrabidaea brachypoda roots ethanolic extract (AbEE) in rats. Formalin and acetic acid-induced writhing tests were used to investigate the antinociceptive activity in mice. High-performance liquid chromatography (HPLC) was used to determine the fingerprint chromatogram of AbEE.

Results

The AbEE at test doses of 30-300 mg/kg p.o. demonstrated anti-inflammatory effects. AbEE reduced paw edema induced by carrageenan, inhibited leukocyte recruitment into the peritoneal cavity and, in the model of chronic inflammation using the cotton pellet-induced fibrovascular tissue growth in rats, significantly inhibited the formation of granulomatous tissue. The extracts at test doses of 30-300 mg/kg p.o. clearly demonstrated antinociceptive activity, except during the first phase of the formalin test. The presence of quercetin and phenolic compounds in the extract Arrabidaea brachypoda was confirmed using HPLC.

Conclusion

Arrabidaea brachypoda ethanol extract markedly demonstrated anti-inflammatory action in rats and antinociceptive activity in mice, which supports the previous claims of traditional use.     

Antiulcer principle from Zingiber montanum

Ethnopharmacological relevance

Rhizome of Zingiber montanum has been extensively used as a folk medicine to ameliorate peptic ulcer at northern part of Bangladesh.

Aim of the study

To identify the antiulcer principle of the MeOH extract of the rhizome of Zingiber montanum by an ex vivo bioassay guided chromatographic separation and purification, and structure elucidation of the purified compound by spectroscopic methods.

Materials and methods

Dried powder of Zingiber montanum rhizomes was extracted with MeOH. The antiulcer activity of the crude extract and its chromatographic fractions were evaluated by the inhibition of 1 N HCl induced gastric lesions in Swiss albino mice. The pure compound was purified from the active fraction by crystallization with hexanes. Structure of the pure compound was elucidated by spectroscopic methods. The antiulcer activity of the pure compound was evaluated by the inhibition of 1 N HCl, 95% ethanol and indomethacin induced gastric lesions in mice.

Results

The MeOH extract of Zingiber montanum showed 61.97% and 83.10% inhibition of the 1 N HCl induced gastric lesions at doses of 200 mg/kg and 400 mg/kg, respectively, in mice. Chromatographic separation on silica gel of the extract was yielded seven fractions and the fraction 2 was found to have most potent antiulcer activity in mice. This fraction showed 77.46% inhibition of the 1 N HCl induced gastric lesions at a dose of 40 mg/kg in mice. Crystallization of the fraction yielded 1 (zerumbone, 180 mg). It showed statistically 45.77% and 92.25% inhibition of 1 N HCl induced gastric lesions in mice at doses of 20 mg/kg and 40 mg/kg, respectively. It also showed 29.07% and 45.35% inhibition of 95% ethanol induced gastric mucosal damage, and 64.76% and 72.38% inhibition of indomethacin induced gastric lesions in mice at doses of 20 mg/kg and 40 mg/kg, respectively.

Conclusion

Zerumbone (1) showed potent cytoprotective effect against necrotizing agent (HCl) and non-steroidal anti-inflammatory drug (indomethacin) induced gastric ulceration. It also exhibited moderate cytoprotective effect against noxious agent (EtOH) induced gastric lesions. It can be considered as a promising new antiulcer natural drug lead.     

Potentiality of standardized extract and isolated flavonoids from Zuccagnia punctata for the treatment of respiratory infections by Streptococcus pneumoniae: in vitro and in vivo studies

Ethnopharmacological relevance

Zuccagnia punctata Cav. (Fabaceae) is a monotypic species distributed in western Argentina and is traditionally used for the treatment of bacterial and fungal infections. The aim of this study was to demonstrated the antibacterial activity of the Zuccagnia punctata standardized extract and the structurally related non-methoxylated flavonoids with similar pattern of substitution and differences in ring C present in this plant species: 7-hydroxyflavanone (HF), 2′,4′-dihydroxychalcone (DHC) and 3,7-dihydroxyflavone (DHF), against Streptococcus pneumoniae clinical isolates using in vitro and in vivo models.

Materials and Methods

MIC values of natural products were determined by agar macrodilution method. In vivo activities were investigated in a Streptococcus pneumoniae infection model in mice. Lung and blood samples were obtained for bacterial cell counts. The serum was used by biochemical analysis (alanine transaminase, aspartate transaminase, urea and creatinine) in order to evaluate the toxicity of natural products.

Results

All samples showed antimicrobial activity in vitro with MIC values between 50 and 500 μg/ml. Zuccagnia punctata extract (1 mg/mice) and HF (1 mg/mice) significantly reduced the number of viable Streptococcus pneumoniae in lung (p < 0.01) while lower quantities has not effect. Therefore, the present study has shown that intake once or twice a day of 1 mg of Zuccagnia punctata extract or HF for seven days did not result in toxicity.

Conclusions

Our results showed that Zuccagnia punctata extract as well as one of its isolated flavonoids, 7-hydroxyflavanone, could be useful for the development of a novel respiratory infections treatment.     

Characterization of the antinociceptive and anti-inflammatory activities from Cocos nucifera L. (Palmae)

Aim of the study

Cocos nucifera cultivated in Brazil is known as “coco-da-Bahia” or “coqueiro-da-Índia”. The tea from the husk fiber is widely used to several inflammatory disorders. Crude extract and fractions obtained from Cocos nucifera “common variety” were evaluated to test the anti-inflammatory and antinociceptive activities.

Materials and methods

Crude extract (CE, 50, 100, and 150 mg/kg), fraction 1 (F1, molecular weight lesser than 1 kDa, 1, 10, and 50 mg/kg), fraction 2 (F2, molecular weight higher than 1 kDa, 1, 10, and 50 mg/kg), and the references drugs morphine (5 mg/kg), acetilsalicilic acid (200 mg/kg), prometazine (30 mg/kg), and metisergide (5 mg/kg) were evaluated on models of analgesia and inflammation.

Results

CE, F1, and F2 significantly develop peripheral and central antinociceptive activity but with less effect on supra-spinal regions of the brain. Administration of the opioid antagonist, naloxone (5 mg/kg) inhibited the antinociceptive effect indicating that Cocos nucifera crude extract and fractions may be acting in opioid receptors. CE and F1 also inhibited rat paw edema induced by histamine, and serotonin.

Conclusions

results demonstrated that Cocos nucifera and its fractions have antinociceptive and anti-inflammatory activities which confirm the popular use of this plant in several inflammatory disorders.     

Neuropharmacological study of Dracocephalum moldavica L. (Lamiaceae) in mice: sedative effect and chemical analysis of an aqueous extract

Ethnopharmacological relevance

Dracocephalum moldavica is used as a tranquilizer and as remedy for nervous conditions relief in the Mexican traditional medicine. Despite its intensive use no literature reported neuropharmacological studies on Dracocephalum moldavica as yet.

Aim of the study

The sedative, anxiolytic-like and antidepressant-like effects of the aqueous extract of aerial parts of Dracocephalum moldavica (Lamiaceae) (DM) were evaluated in behavioral models in mice. The general toxic effects of DM were evaluated as well as their chemical analysis was performed.

Materials and methods

DM effects were evaluated on pentobarbital-induced sleeping time (SPT), the hole-board (HBT), and the avoidance exploratory behavior (AEBT) tests and on the forced swimming test (FST). General activity and motor coordination were evaluated in the open field (OFT) and Rota-rod tests, respectively. The acute toxicity of DM was determinate by its LD₅₀ dose. The chemical analyses DM were performed by chromatographic and HPLC–ESI-MS techniques.

Results

DM prolonged the pentobarbital-induced sleeping time, induced sedation in the HBT, decreased spontaneous activity and produced motor coordination impairment in mice. However, DM did not show anxiolytic effects in the AEBT or HBT and it was not effective in FST. The DM-treatment produced mortalities with LD₅₀ = 470 mg/kg body weight.The HPLC–ESI-MS analysis of DM revealed that (acacetin, apigenin and luteolin)-7-O-β-d-(6″-O-malonyl)-glucoside derivates are the main compounds of DM.

Conclusions

DM induced sedative actions and a general inhibition of CNS activity observed by the decrease of animals’ general activity, motor coordination and exploration.     

Antiplasmodial activity of the andiroba (Carapa guianensis Aubl., Meliaceae) oil and its limonoid-rich fraction

Ethnopharmacological relevance

From seeds of Carapa guianensis the Amazon native people extracts the andiroba oil, which is traditionally used as febrifuge, anti-malarial, insecticidal and repellant. The non-saponifiable fraction separated from the oil is rich in limonoids, which assigns its pharmacological effects.

Materials and methods

The andiroba oil and its limonoid-rich fraction were submitted to in vitro antiplasmodial bioassay using W₂ and Dd₂ strains of Plasmodium falciparum. The acute toxicity of andiroba oil was evaluated. The limonoid-rich fraction was subjected to fractionation and identified its major constituents.

Results

Andiroba oil and its limonoid-rich fraction inhibited the growth of W₂ clone in 100%, between 24 and 72 h, at concentrations of 8.2 μg/mL and 3.1 μg/mL, respectively. Under the same conditions, the parasitaemia of Dd₂ clone provoked by the andiroba oil showed inhibition of 31% (IC₅₀ >82 μg/mL) with a time-dependent relationship of 24 h and inhibition of 88% (IC₅₀ 8.4 μg/mL) after 72 h, while for the limonoid-rich fraction the inhibition of Dd₂ clone was 56% (IC₅₀ 2.8 μg/mL) at 24 h and 82% (IC₅₀ 0.4 μg/mL) after 72 h. Andiroba oil in acute toxicity test with a fixed dose (LD₅₀ >2000 mg/kg) was not toxic The limonoids identified in the oil were gedunin, 6α-acetoxygedunin, 7-deacetoxy-7-oxogedunin, 7-deacetylgedunin, 1,2-dihydro-3β-hydroxy-7-deacetoxy-7-oxogedunin and andirobin. Gedunin and derivatives has been reputed as anti-malarials.

Conclusion

The results support the traditional use of andiroba oil as antiplasmodial, which additionally proved not to be toxic in bioassays conducted with mice.