Healing potential of Rosmarinus officinalis L. on full-thickness excision cutaneous wounds in alloxan-induced-diabetic BALB/c mice

Aim of the study

Rosmarinus officinalis (Rosemary) used in Jordanian folk medicine for wound management and treatment. Therefore, the present study was conducted to assess the healing efficacy of both aqueous extract and essential oil of the aerial parts on alloxan-induced diabetic BALB/c mice.

Materials and methods

Two full-thickness round wounds were created in the dorsal area of each mouse. Animals were divided into four groups of twenty mice each: untreated normal, untreated diabetic, aqueous extract- (intraperitoneal injection of 0.2 ml at a dose of 10% for 3 days) and essential oil-treated diabetic mice (topical application of 25 μl/excision wound, twice a day for 3 days). For 15 days, the wounds were visually observed; blood glucose level, body weight, regenerated granulation tissue weight and the percentage of wound contraction were measured. On days 6 and 15 after wounding, the animals were sacrificed and the histology of wound area was examined.

Results

Significant positive differences (p < 0.01) between treated and control groups were observed at different aspects of diabetic wound healing process. Reduced inflammation and enhanced wound contraction, re-epithelialization, regeneration of granulation tissue, angiogenesis and collagen deposition were detected in the treated wounds.

Conclusions

Results indicated that the essential oil of Rosmarinus officinalis was the most active in healing diabetic wounds and provide a scientific evidence for the traditional use of this herb in wound treatment. However, further scientific verification is required to confirm and assess the range of wound healing potential of essential oils of Rosemary chemotypes.     

Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera L.

Aim of the study

The objective of this study was to isolate and characterize the active constituents of the traditionally used antimalarial plant Liriodendron tulipifera by antiplasmodial-assay guided fractionation.

Materials and methods

Bark and leaves were extracted with solvents of increasing polarity. Fractions were generated using flash chromatography, counter current chromatography and preparative HPLC and subjected to in vitro antiplasmodial and cytotoxicity assays. Active fractions were subjected to further fractionation until pure compounds were isolated, for which the IC₅₀ values were calculated.

Results and discussion

Six known aporphine alkaloids, asimilobine (1), norushinsunine (2), norglaucine (3), liriodenine (4), anonaine (5) and oxoglaucine (6) were found to be responsible for the antiplasmodial activity of the bark. Leaves yielded two known sesquiterpene lactones, peroxyferolide (7) and lipiferolide (8) with antiplasmodial activity. The antiplasmodial activity of (2) (IC₅₀ = 29.6 μg/mL), (3) (IC₅₀ = 22.0 μg/mL), (6) (IC₅₀ = 9.1 μg/mL), (7) (IC₅₀ = 6.2 μg/mL) and (8) (IC₅₀ = 1.8 μg/mL) are reported for the first time.

Conclusion

This work supports the historical use of Liriodendron tulipifera as an antimalarial remedy of the United States and characterizes its antiplasmodial constituents.     

In vitro antiplasmodial activity of indole alkaloids from the stem bark of Geissospermum vellosii

Ethnopharmacological relevance

The stem bark of Geissospermum vellosii has been traditionally used by the native population of northern South America to treat malaria. Indole alkaloids have been previously isolated from this plant, but the antiplasmodial constituents have not yet been described. As part of our ongoing investigations of new bioactive compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity of isolated fractions and purified alkaloids from Geissospermum vellosii.

Materials and methods

Indole alkaloids were isolated and identified from a methanolic crude extract of Geissospermum vellosii bark using a combination of high performance counter current chromatography, mass spectrometry and nuclear magnetic resonance technologies. The methanolic extract, the crude alkaloid fractions and the purified compounds were tested for in vitro antiplasmodial activity against the chloroquine-sensitive strain of Plasmodium falciparum (D10).

Results

An indole alkaloid (4) along with four known indole alkaloids, geissolosimine (1), geissospermine (2), geissoschizoline (3), and vellosiminol (5) were isolated and structure elucidated. The antiplasmodial activity (IC₅₀) of the methanolic crude extract was 2.22 μg/mL, while for the isolated compounds it ranged from 0.96 μM to 13.96 μM except for (5) which showed a low activity (157 μM). Geissolosimine (1) showed the highest antiplasmodial activity (0.96 μM).

Conclusions

This study provides evidence to support the use of Geissospermum vellosii as an antimalarial agent, as used by the native populations. Geissolosimine (1) is a lead molecular structure for possible antimalarial drug development.     

Investigation of plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts

Aim of the study

To investigate the activities of the 217 plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts.

Materials and methods

Plant extracts were prepared by the method of maceration using solvents of different polarities. The growth inhibition of chloroquine-resistant Plasmodium falciparum strain (FcB1) was determined by measuring the radioactivity of the tritiated hypoxanthine incorporated. Activity against Leishmania (Leishmania) chagasi and Trypanosoma cruzi was measured by the MTT colorimetric assay. The antifungal tests were carried out by using the CLSI method. The active extracts were tested also by cytotoxicity assay using NIH-3T3 cells of mammalian fibroblasts.

Results

Two hundred and seventeen extracts of plants were tested against Plasmodium falciparum. The eleven active extracts, belonging to eight plant species were evaluated against L. (L.) chagasi, Trypanosoma cruzi, yeasts and in NIH-3T3 cells. The results found in these biological models are consistent with the ethnopharmacological data of these plants. The ethyl acetate extract of Diospyros hispida root showed IC₅₀ values of 1 μg/mL against Plasmodium falciparum. This extract demonstrated no toxicity against mammalian cells, resulting in a significant selectivity index (SI) of 435.8. The dichloromethane extract of Calophyllum brasiliense root wood was active against Cryptococcus gattii LMGO 01 with MIC of 1.95 μg/mL; and Candida albicans ATCC 10231 and Candida krusei LMGO 174, both with MIC of 7.81 μg/mL. The same extract was also active against Plasmodium falciparum and L. (L.) chagasi with IC₅₀ of 6.7 and 27.6 μg/mL respectively. The ethyl acetate extract of Spiranthera odoratissima leaves was active against Cryptococcus gattii LMGO 01 with MIC of 31.25 μg/mL, and against Plasmodium falciparum with IC₅₀ of 9.2 μg/mL and Trypanosoma cruzi with IC₅₀ of 56.3 μg/mL.

Conclusion

The active extracts for protozoans and human pathogenic yeasts are considered promising to continue the search for the identification and development of leading compounds.     

Effect of Emblica officinalis (fruit) against UVB-induced photo-aging in human skin fibroblasts

Ethnopharmacological relevance

Emblica officinalis fruit (EO), commonly known as Amla is a reputed traditional medicine and functional food used in Indian subcontinent. It has long been used in Indian folk medicine to treat liver diseases, stomach ulcers, inflammatory diseases, metabolic disorders, geriatric complaints, skin disorders and beauty care.

Aim of the study

Recently, it has been shown to promote pro-collagen content and inhibit matrix metalloproteinase levels in skin fibroblast. The aim of the present study was to investigate the efficacy of EO to inhibit UVB-induced photo-aging in human skin fibroblasts.

Materials and methods

Mitochondrial activity of human skin fibroblasts was measured by MTT-assay. Quantifications of pro-collagen 1 and matrix metalloproteinase 1 (MMP-1) release were performed by immunoassay techniques. Hyaluronidase inhibition assay was studied in vitro using bovine testicular hyaluronidase and human umbilical cord hyaluronic acid. Cell cycle analysis was performed by flowcytometry using propidium iodide.

Results

EO stimulated, the otherwise UVB inhibited cellular proliferation and protected pro-collagen 1 against UVB-induced depletion via inhibition of UVB-induced MMP-1 in skin fibroblasts (10-40 μg/mL, p > 0.001). EO exhibited inhibitory activity of hyaluronidase (10-40 μg/mL, p > 0.001). Treatment with EO also prevented UVB disturbed cell cycle to normal phase.

Conclusion

The results of the present study suggests that EO effectively inhibits UVB-induced photo-aging in human skin fibroblast via its strong ROS scavenging ability and its therapeutic and cosmetic applications remain to be explored.     

Permeability of rosmarinic acid in Prunella vulgaris and ursolic acid in Salvia officinalis extracts across Caco-2 cell monolayers

Ethnopharmacological relevance

Rosmarinic acid (RA), a caffeic acid-related compound found in high concentrations in Prunella vulgaris (self-heal), and ursolic acid (UA), a pentacyclic triterpene acid concentrated in Salvia officinalis (sage), have been traditionally used to treat inflammation in the mouth, and may also be beneficial for gastrointestinal health in general.

Aim of the study

To investigate the permeabilities of RA and UA as pure compounds and in Prunella vulgaris and Salvia officinalis ethanol extracts across human intestinal epithelial Caco-2 cell monolayers.

Materials and methods

The permeabilities and phase II biotransformation of RA and UA as pure compounds and in herbal extracts were compared using Caco-2 cells with HPLC detection.

Results

The apparent permeability coefficient (P_app) for RA and RA in Prunella vulgaris extracts was 0.2 ± 0.05 × 10⁻⁶ cm/s, significantly increased to 0.9 ± 0.2 × 10⁻⁶ cm/s after β-glucuronidase/sulfatase treatment. P_app for UA and UA in Salvia officinalis extract was 2.7 ± 0.3 × 10⁻⁶ cm/s and 2.3 ± 0.5 × 10⁻⁶ cm/s before and after β-glucuronidase/sulfatase treatment, respectively. Neither compound was affected in permeability by the herbal extract matrix.

Conclusion

RA and UA in herbal extracts had similar uptake as that found using the pure compounds, which may simplify the prediction of compound efficacy, but the apparent lack of intestinal glucuronidation/sulfation of UA is likely to further enhance the bioavailability of that compound compared with RA.     

An aqueous birch leaf extract of Betula pendula inhibits the growth and cell division of inflammatory lymphocytes

Ethnopharmacological relevance

Leaf extracts of Betula pendula have been traditionally used for the treatment of patients with rheumatoid arthritis (RA) or osteoarthritis.

Aim of the study

We investigated the anti-proliferative capacity of an aqueous leaf extract of Betula pendula (BPE) on human primary lymphocytes in vitro, because activated lymphocytes play a major role in the initiation and maintenance of RA.

Materials and methods

Lymphocyte proliferation and cell division was measured by the activity of mitochondrial dehydrogenases and by using the membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE), respectively. Apoptosis was analyzed by surface staining of phosphatidylserine and intracellular activation of effector caspases 3 and 7 in comparison to the drug methotrexate using flow cytometric and photometrical analysis. In addition, the impact of the extract on cell cycle distribution was investigated by propidium iodide staining of DNA. For the bioassays BPE concentrations of 10-160 μg/mL were investigated. A phytochemical analysis, using LC-MS and HPLC, was conducted to identify the polyphenolic constituents of the birch leaf extract.

Results

Leaf extracts of Betula pendula inhibited the growth and cell division (CD8⁺: 40 μg/mL: 45%; 80 μg/mL: 60%; 160 μg/mL: 87%) (CD4⁺: 40 μg/mL: 33%; 80 μg/mL: 54%; 160 μg/mL: 79%) of activated, but not of resting T lymphocytes in a significant dose-dependent manner. The inhibition of lymphocyte proliferation due to apoptosis induction (compared to untreated control: 40 μg/mL: 163%; 80 μg/mL: 240%; 160 μg/mL: 348%) and cell cycle arrest was comparable to that of methotrexate. LC-MS analyses showed that the extract contains different quercetin-glycosides.

Conclusion

Our results give a rational basis for the use of Betula pendula leaf extract for the treatment of immune disorders, like rheumatoid arthritis, by diminishing proliferating inflammatory lymphocytes.     

Xanthine oxidase inhibitory properties of Czech medicinal plants

Aim of the study

To investigate in vitro xanthine oxidase inhibitory properties of plants traditionally used in Czech Republic and Central-East Europe region for gout, arthritis or rheumatism treatment.

Materials and methods

Methylene chloride-methanolic and two ethanolic extracts of 27 plant species were screened for in vitro xanthine oxidase inhibitory activity using a spectrophotometric method.

Results

Around 50% of the species exhibited some degree of xanthine oxidase inhibitory properties at 200 μg/mL, showing a moderate correlation (r = 0.59) with total phenol content. The most active were methylene chloride-methanolic extracts of Populus nigra and Betula pendula, with IC₅₀ of 8.3 and 25.9 μg/mL, respectively, followed by 80% ethanolic extract of Caryophyllus aromaticus and Hypericum perforatum, both under 50 μg/mL.

Conclusions

Populus nigra and Betula pendula were identified as species with the highest xanthine oxidase inhibitory potential in our study. This correlates with the ethnobotanical data on their use in Central European folklore and provides the basis for further investigation on these plants.     

Identification and pharmacological characterization of the anti-inflammatory principal of the leaves of dwarf elder (Sambucus ebulus L.)

Aim of the study

The performed investigations aimed on the identification of the anti-inflammatory principal of extracts of leaves of Sambucus ebulus L. (dwarf elder) in order to rationalize the traditional use of this plant for the treatment of chronically inflammatory diseases.

Materials and methods

Dwarf elder leaf extract was subjected to activity guided fractionation using inhibition of TNFα induced expression of vascular cell adhesion molecule 1 (VCAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) as monitoring tool (positive control: parthenolide 10 μM, VCAM-1 expression (% of control): 5.35 ± 0.38%).

Results

Bio-guided isolation resulted in identification of ursolic acid as anti-inflammatory principal. Besides its inhibitory effects against TNFα induced expression of VCAM-1 (IC₅₀ 6.25 μM), ursolic acid inhibits also TNFα induced expression of ICAM-1 (IC₅₀ value between 3.13 and 6.25 μM) (positive control: parthenolide 10 μM, ICAM-1 expression (% of control): 38.89 ± 16.6%). Toxic effects of ursolic acid on HUVECs can be drastically reduced using an enriched extract instead of the pure compound.

Conclusions

Our findings suggest an additional mechanism of the anti-inflammatory activity of ursolic acid by demonstrating its ability to inhibit TNFα-stimulated expression of VCAM-1 and ICAM-1 and support the traditional use of extracts and preparations of Sambucus ebulus L., rich in ursolic acid, for the treatment of chronically inflammatory processes.     

Deciphering molecular mechanism underlying antileishmanial activity of Nyctanthes arbortristis, an Indian medicinal plant

Ethnopharmacological relevance

Nyctanthes arbortristis L. (Oleaceae) is widely used in the traditional medicine of India. The plant is shown to have antibacterial and antileishmanial activities.

Aim of the study

Evaluation of iridoid glucosides from the plant as inhibitor of trypanothione reductase (TryR), a validated drug target enzyme of the Leishmania parasite. The study contributes towards understanding mechanism of antileishmanial effect of the plant.

Materials and methods

TryR of Leishmania parasite is expressed and purified. Iridoid glucosides are isolated from the plant and tested as inhibitor of TryR enzyme of the parasite.

Results

Inhibitory constant (K_i) of various iridoid glucosides ranges from 3.24 ± 0.05 μM to 6.49 ± 0.05 μM. Thus, the molecular mechanism underlying antileishmanial activity of these compounds is mediated through inhibition of TryR.

Conclusion

The current study also points out towards potential application of iridoid glucosides as novel drugs against the disease.     

LC-MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract

Ethnopharmacological relevance

Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM.

Aim of the study

A selective and sensitive HPLC-MS/MS method was first developed and validated for the determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin in rat plasma.

Materials and methods

The LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFire™ C₁₈ column (150 mm × 4.6 mm, 5 μm), and the column temperature was maintained at 25 °C and the sample injection volume was 20 μL. The current LC-MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability.

Results

The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds in rat plasma after a single oral administration of Verbena officinalis L. extract with a dosage of 8.0 mL/kg. The time to reach the maximum plasma concentration (T_max1) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma concentration (T_max2) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (t_1/2) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively.

Conclusions

This paper described a simple, sensitive and validated LC-MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min.     

In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio-guided fractionation of the most active extract

Ethnopharmacological relevance

The aim of the study was to evaluate the in vitro antitrypanosomal and antileishmanial activity of crude extracts of 10 plant species traditionally used in Benin to treat parasitic infections.

Materials and methods

For each species, dichloromethane, methanol and aqueous extracts were tested. Their antitrypanosomal and antileishmanial activities were evaluated in vitro on Trypanosoma brucei brucei (strain 427) (Tbb) and on promastigotes of Leishmania mexicana mexicana (MHOM/BZ/84/BEL46) (Lmm).

Results

The best growth inhibition was observed with the dichloromethane extracts of aerial parts of Acanthospermum hispidum DC. (Asteraceae) (IC₅₀ = 14.5 μg/ml on Tbb and 11.1 μg/ml on Lmm), twigs of Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) (IC₅₀ = 5.8 μg/ml on Tbb), aerial parts of Byrsocarpus coccineus Schumach. & Thonn (syn. Rourea coccinea (Schumach. & Thonn.) Hook.f.) (IC₅₀ = 14.7 μg/ml on Tbb) and aerial parts of Carpolobia lutea G.Don. (IC₅₀ = 18.3 μg/ml on Tbb). All these extracts had a low cytotoxicity. It is not the case for the methanolic and water extracts of roots of Anchomanes difformis (Blume) Engl. (IC₅₀ = 14.7 and 13.8 μg/ml on Tbb) which were toxic at the same concentration range on WI38, human cells. A bio-guided fractionation of the most active extract of Keetia leucantha allowed to identify oleanolic acid and ursolic acid as responsible for the observed activities.

Conclusion

Our study gives some justification for antiparasitic activity of some investigated plants.     

Vasorelaxation by extracts of Casimiroa spp. in rat resistance vessels and pharmacological study of cellular mechanisms

Aim of the study

Casimiroa spp. are Mexican plants traditionally used for treatment of hypertension. To study their antihypertensive action, we determined the arterial dilatation induced by extracts from leaves and seeds of Casimiroa calderoniae F. Chiang & Medrano, Casimiroa edulis Llave et Lex, and Casimiroa pubescens Ramirez.

Materials and methods

The vascular effects of Casimiroa spp. extracts were investigated on rat caudal and aortic arteries. In addition, the extracts were characterized by HPLC using heraclenol, isopimpinellin, heraclenin and phellopterin as external standards. The methanolic extract of Casimiroa pubescens seeds (Cp12) was also studied by H-NMR and LC-MS (ESI-TOF) for the determination of casimiroin and zapotin.

Results

The hexanic and methanolic extracts of Casimiroa spp. (20 μg/ml) showed vasorelaxation in arterial tissues precontracted by phenylephrine (0.5 μM); the extracts from seeds always caused a greater relaxation in comparison to those from leaves. The most active were the methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12). To study the pharmacological mechanisms of vasodilatation we used various inhibitors selective to different receptor subtypes or intracellular enzymes. The vasorelaxant effect of Ce8 (20 μg/ml) remained unaffected by the pretreatment with pyrilamine (10 μM), an antagonist of histamine H₁ receptors, but was inhibited by atropine (0.1 μM), a muscarinic receptor antagonist. Therefore, to determine muscarinic receptor subtypes, we used pirenzepine (1 μM), a selective inhibitor of M₁ receptor, and 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP, 0.01 μM), a selective inhibitor of M₃ receptor. Only the latter reduced the vasodilatation by Ce8 and Cp12. To investigate the role of the nitric oxide synthase (NOS), we used N^G-nitro-l-arginine methyl ester (l-NAME, 10 μM), a selective NOS inhibitor, which decreased the dilatation induced by Ce8 and Cp12. Finally, we studied the action of (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (ODQ, 3 μM), a selective guanylyl cyclase inhibitor, which inhibited the dilatation by Casimiroa extracts.

Conclusion

The data show that methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) induce vasorelaxation by M₃ receptor through the activation of cGMP-dependent NO signaling. These results support the traditional use of Casimiroa decoctions for antihypertensive treatments in the Mexican ethnomedicine.     

Activation of M3 muscarinic receptor and Ca²⁺ influx by crude fraction from Crotonis Fructus in isolated rabbit jejunum

Ethnopharmacological relevance

Crotonis Fructus is the mature fruit of Croton tiglium L. (Euphorbiaceae), which has been used in traditional Chinese medicine for the treatment of gastrointestinal (GI) diseases, such as constipation, abdominal pain, peptic ulcer, and intestinal inflammation for thousands of years. The aim of this study was to investigate the pharmacological effect of extracts and fractions from Crotonis Fructus on GI tract.

Materials and methods

The activities of methanol extract and fractions from Crotonis Fructus on the smooth muscle contractions were evaluated using isolated rabbit jejunum model.

Results

The results suggest that the n-BuOH and H₂O fractions showed spasmolytic activity, while the MeOH extract, PE and EtOAc fractions exerted spasmogenic effect. Moreover, bioassay-guided fractionation verified that the EtOAc fraction was more potent than others, followed by PE fraction and methanol extract. Additionally, atropine (10 μM), 4-DAMP (10 μM) and verapamil (0.1 μM) produced a significant inhibition of contractions caused by EtOAc fraction, while either hexamethonium (10 μM) or methoctramine (10 μM) was inactive. Additionally, a HPLC fingerprint of EtOAc fraction was appraised to ensure its chemical consistency and the main component has been identified as phorbol 12-acetate-13-tiglate.

Conclusions

These data indicate that the regulatory effect of EtOAc fraction on GI motility are medicated via the activation of M3 muscarinic receptor and Ca²⁺ influx through L-type Ca²⁺ channel. These provide a scientific basis for the traditional use of Crotonis Fructus in GI disorders.     

Cytotoxic, apoptotic and anti-α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from the polyphenolic-rich extract of Elephantopus mollis Kunth

Ethnopharmacological relevance

The decoction of the whole plant of Elephantopus mollis Kunth. is traditionally consumed to treat various free radical-mediated diseases including cancer and diabetes.

Aim of the study

This study was initiated to determine whether the most effective antioxidant compound isolated from the whole plant of Elephantopus mollis can also contribute to its claimed traditional values as anticancer and antidiabetes agents.

Materials and methods

An active antiradical phenolic compound (3,4-di-O-caffeoyl quinic acid) was isolated from the methanol extract (with the highest in polyphenolic content) and their antioxidant activities were compared using four different assays, that are DPPH, FRAP, metal chelating, and β-carotene bleaching tests. The compound was also evaluated for its cytotoxic activity, apoptotic induction and anti-glucosidase efficacies using methylene blue, DeadEnd™ assay and α-glucosidase assays, respectively.

Results

The compound acted as a greater primary antioxidant than its methanol extract, by having higher ferric reducing activity (EC₅₀ 2.18 ± 0.05 μg/ml), β-carotene bleaching activity (EC₅₀ 23.85 ± 0.65 μg/ml) and DPPH scavenging activity (EC₅₀ 68.91 ± 5.44 μg/ml), whereas the methanol extract exhibited higher secondary antioxidant activity as a metal chelator with lower EC₅₀ value (49.39 ± 3.68 μg/ml) than the compound. Cytotoxicity screening of this compound exhibited a remarkable dose-dependent inhibitory effect on NCI-H23 (human lung adenocarcinoma) cell lines (EC₅₀ 3.26 ± 0.35 μg/ml) and was found to be apoptotic in nature based on a clear indication of DNA fragmentation. This compound also displayed a concentration-dependent α-glucosidase inhibition with EC₅₀ 241.80 ± 14.29 μg/ml.

Conclusions

The findings indicate the major role of 3,4-di-O-caffeoyl quinic acid to antioxidant capacities of Elephantopus mollis extracts. The compound also exerted apoptosis-mediated cytotoxicity and α-glucosidase inhibitory effects and is thus a promising non toxic agent in treating cancer and type 2 diabetes mellitus.     

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

Ethnopharmacological relevance

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

Aims of the study

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

Materials and methods

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

Results

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

Conclusion

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

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19)

Ethno pharmacological relevance

Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date.

Aim of the study

In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated.

Materials and methods

S-Mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects.

Results

Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K_i values of 67.1 and 16.4 μg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K_i = 39.6 and 41.5 μg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K_i = 7.1 μg/ml or 20.6 μM).

Conclusions

It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).