Cytotoxic Properties of the Stem Bark of Citrus reticulata Blanco (Rutaceae)

The bioassay‐guided fractionation of the n‐hexane extract of Citrus reticulata Blanco (Rutaceae) stem bark yielded scoparone (1), xanthyletin (2), lupeol (3), β‐amyrin (4), stigmasterol (5), β‐sitosterol (6) and palmitic acid. The structures of these compounds were determined by comprehensive spectroscopic analyses, i.e., 1D and 2D NMR and EI‐MS, and by comparison with the reported data. Extracts, fractions and isolated compounds 1–6 were assessed for cytotoxicity by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐dphenyltetrazolium bromide (MTT) assay against three human cancer cell lines, i.e., human lung adenocarcinoma cell line A549, human breast adenocarcinoma cell line MCF7 and human Caucasian prostate adenocarcinoma cell line PC3. Significant activity of the n‐hexane and the dichloromethane extracts was observed against the breast cancer cell line MCF7 with IC₅₀s of 45.6 and 54.7 μg/mL, respectively. Moreover, the 70% ethyl acetate in n‐hexane chromatographic fraction showed significant activity displaying IC₅₀ values of 53.0, 52.4 and 49.1 μg/mL against the cancer cell lines A549, MCF7 and PC3, respectively. Encouragingly, an IC₅₀ of 510.0 μg/mL against the human normal prostate cell line PNT2 indicated very low toxicity and hence favourable selectivity indices for the 70% ethyl acetate in n‐hexane fraction in the range of 9.6–10.4 towards cell lines A549, MCF7 and PC3. Because compounds isolated from the above fraction only delivered IC₅₀ values in the range of 18.2–96.3, 9.2–34.1 and 7.5–97.2 μg/mL against A549, MCF7 and PC3 cell lines, respectively, synergistic action between compounds is suggested. Bioassay results valorize the anticancer effectivity of the stem bark of this plant in Cameroonian pharmacopoeia. Copyright © 2017 John Wiley & Sons, Ltd.     

Modulation of Cox‐1, 5‐, 12‐ and 15‐Lox by Popular Herbal Remedies Used in Southern Italy Against Psoriasis and Other Skin Diseases

Acanthus mollis (Acanthaceae), Achillea ligustica, Artemisia arborescens and Inula viscosa (Asteraceae) are used in Southern Italy against psoriasis and other skin diseases that occur with an imbalanced production of eicosanoids. We here assessed their in vitro effects upon 5‐, 12‐, 15‐LOX and COX‐1 enzymes as well as NFκB activation in intact cells as their possible therapeutic targets. All methanol crude extracts inhibited both 5‐LOX and COX‐1 activities under 200 µg/mL, without significant effects on the 12‐LOX pathway or any relevant in vitro free radical scavenging activity. NFκB activation was prevented by all extracts but A. mollis. Interestingly, A. ligustica, A. arborescens and A. mollis increased the biosynthesis of 15(S)‐HETE, an anti‐inflammatory eicosanoid. A. ligustica (IC₅₀ = 49.5 µg/mL) was superior to Silybum marianum (IC₅₀ = 147.8 µg/mL), which we used as antipsoriatic herbal medicine of reference. Its n‐hexane, dichloromethane and ethyl acetate fractions had also inhibitory effects on the LTB₄ biosynthesis (IC₅₀s = 9.6, 20.3 and 68 µg/mL, respectively) evidencing that the apolar extracts of A. ligustica are promising active herbal ingredients for future phytotherapeutical products targeting psoriasis. © 2014 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.     

Angiotensin‐Converting Enzyme Inhibitory Activity and Antioxidant Properties of Nepeta crassifolia Boiss & Buhse and Nepeta binaludensis Jamzad

This article reports phytochemical and biological studies on Nepeta binaludensis and Nepeta crassifolia. Both species were investigated for their angiotensin‐converting enzyme (ACE) inhibitory activity and antioxidant properties through three in vitro models [2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) assay]. Aerial parts were extracted with methanol and partitioned between water and subsequently n‐hexane, ethyl acetate and n‐butanol. N. binaludensis methanol extract exerted significantly higher reducing power (1.9 μM Fe(II)/g) than did the positive control butylhydroxytoluene (63.2 μM Fe(II)/g) in FRAP assay. The highest DPPH radical scavenging activity was found for N. crassifolia, with IC₅₀ values of 9.6 and 12.1 µg/mL for ethyl acetate and n‐butanol fractions, respectively. n‐Butanol fraction of both species showed the highest ACE inhibitory activity, with IC₅₀ values of 59.3 and 81.7 µg/mL for N. binaludensis and N. crassifolia, respectively. Phytochemical investigations resulted in the isolation of ursolic acid, oleanolic acid, apigenin, luteolin and ixoroside. Apigenin‐7‐O‐glucoside, 8‐hydroxycirsimaritin and cirsimaritin were furthermore identified in N. crassifolia ethyl acetate‐soluble fraction. Nepetanudoside B was isolated from the n‐butanol fraction of N. binaludensis. Copyright © 2012 John Wiley & Sons, Ltd.     

In Vitro and In Silico Evaluation of NF‐κB Targeted Costunolide Action on Estrogen Receptor‐Negative Breast Cancer Cells—A Comparison with Normal Breast Cells

Costunolide, a sesquiterpene lactone is a plant‐derived secondary metabolite found to be present in most of the pharmacologically active herbs, being the cause for their medicinal values. The present study aims to evaluate the cytotoxic effect of costunolide isolated from Costus speciosus rhizome extract on MDA‐MB‐231 cells and explore its targeted action in comparison with its action on the normal breast cells (MCF 10A). The effect of costunolide on cell viability of the cells was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide viability assay. The targeted action of the compound was analyzed comparing the effectiveness of the compound to alter the protein expression levels of NF‐κB subunits in the normal and the cancer cells using western blotting analysis. In silico studies were performed to predict the targeted interaction of costunolide with the NF‐κB subunit proteins. Costunolide inhibited the cell viability of MDA‐MB‐231 cells in a dose‐dependent manner leaving no significant change in the viability of the normal breast cells. The over expressed NF‐κB subunits – p65, 52 and 100 in the cancer cells were found to be downregulated when treated with costunolide at an effective dose of 20 and 40 μM costunolide. In silico results provided stable interactions between costunolide and the target proteins, supporting the in vitro results in addition. Thus, costunolide derived from C. speciosus plant source elevates a fresh conviction for its use in breast cancer therapy for its cytotoxic efficacy and non‐toxic nature. Copyright © 2014 John Wiley & Sons, Ltd.     

Cytotoxicity Effects and Apoptosis Induction by Bisbenzylisoquinoline Alkaloids from Triclisia subcordata

Triclisia subcordata Oliv (Menispermeaceae) is a medicinal plant traditionally used for the treatment of various diseases in West Africa. The ethanol extract of T. subcordata and its fractions were screened for in vitro anti‐ovarian cancer activities using the Sulforhodamine B assay. The crude alkaloids showed the strongest activity in cell growth assays on Ovcar‐8 and A2780 cell lines (IC₅₀ < 2.4 µg/mL). A bisbenzylisoquinoline alkaloid‐cycleanine was isolated using HPLC and identified by mass spectrometry and nuclear magnetic resonance analyses. The IC₅₀ values of cycleanine and tetrandrine (an alkaloid previously reported from this plant) ranged from 7 to 14 μM on Ovcar‐8, A2780, Ovcar‐4, and Igrov‐1 ovarian cancer cell lines. The IC₅₀ of cycleanine on human normal ovarian surface epithelial cells was 35 ± 1 μM, hinting at modest selectivity toward cancer cells. Both cycleanine and tetrandrine caused apoptosis as shown by activation of caspases 3/7 and cleavage of poly(ADP‐ribose) polymerase to form poly(ADP‐ribose) polymerase‐1 by using western blot analysis. Flow cytometry analyses showed that the percentages of apoptotic cells and cells in subG₁ phase increased after exposure of cycleanine and tetrandrine to Ovcar‐8 cells for 48 h compared with control. Cycleanine, like its isomer tetrandrine isolated from T. subcordata, could be a potential new anti‐ovarian cancer agent acting through the apoptosis pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Antimalarial and safety evaluation of extracts from Toddalia asiatica (L) Lam. (Rutaceae)

Ethnopharmacological relevance

Toddalia asiatica (L) Lam. (Rutaceae) is a medicinal plant traditionally used in Kenya by many communities for the treatment of malaria and other ailments. All parts of the plant are claimed to have medicinal value, but the root bark in particular is believed to be more potent. Decoctions or infusions of the roots are taken orally to treat malaria, fever and stomach ache.

Aim of the study

To evaluate antimalarial activity of aqueous and organic extracts prepared from Toddalia asiatica and determine in vitro and in vivo safety of the extracts.

Materials and methods

Aqueous, ethyl acetate, hexane and methanol extracts were obtained from Toddalia asiatica root bark, fruits and leaves. In vitro antiplasmodial activity was done using chloroquine-sensitive (D6) and chloroquine-resistant (W2) Plasmodium falciparum strains and the concentration causing 50% inhibition of radioisotope incorporation (IC₅₀) was determined. In vivo assay was done by administering mice infected with Plasmodium berghei four consecutive daily doses of the extracts through oral route following Peters 4-Day suppressive test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Quinine hydrochloride was used as positive control while double distilled water or 20% Tween-80 was used as a negative control. In vivo acute toxicity was determined in mice using standard procedures. In vitro cytotoxicity assay was carried out using actively dividing sub-confluent Vero cells.

Results

Inhibitory concentrations of ethyl acetate extract of Toddalia asiatica fruits showed high activity against chloroquine resistant (W2) strains of Plasmodium falciparum (IC₅₀=1.87 μg/ml), followed by root bark aqueous extract (IC₅₀=2.43 μg/ml). Tested in vivo against Plasmodium berghei, the fruit ethyl acetate extract (500 mg/kg) and root bark aqueous extract (250 mg/kg) reduced malaria parasitaemia by 81.34% and 56.8% respectively. Higher doses were found to be less effective in vivo. Acute toxicity and cytotoxictiy of the tested extracts, with the exception of hexane extract from the roots, showed LD₅₀>1000 mg/kg and CC₅₀>100 μg/ml respectively.

Conclusions

The results obtained contribute to the validation of traditional use of Toddalia asiatica and provides in vivo and safety data of the plant extracts tested for the first time. Ethyl acetate extract of the fruits was active against chloroquine resistant Plasmodium falciparum as well as against Plasmodium berghei. These findings confirm the suitability of Toddalia asiatica as a good candidate for further tests to obtain a prototype for antimalarial medicine.     

Ethyl acetate fraction of the root of rubia cordifolia L. inhibits keratinocyte proliferation in vitro and promotes keratinocyte differentiation in vivo: potential application for psoriasis treatment

Psoriasis is a skin disease associated with hyperproliferation and aberrant differentiation of keratinocytes. Our previous studies have identified the root of Rubia cordifolia L. as a potent antiproliferative and apoptogenic agent in cultured HaCaT cells (IC₅₀ 1.4 μg/ml). In the present study, ethanolic extract of Radix Rubiae was fractioned sequentially with hexane, ethyl acetate (EA), n‐butanol and water. EA fraction was found to possess most potent antiproliferative action on HaCaT cells (IC₅₀ 0.9 μg/ml). Mechanistic study revealed that EA fraction induced apoptosis on HaCaT cells, as it was capable of inducing apoptotic morphological changes. Annexin V‐PI staining assay also demonstrated that EA fraction significantly augmented HaCaT apoptosis. In addition, EA fraction decreased mitochondrial membrane potential in a concentration‐ and time‐dependent manner. The standardized EA fraction was formulated into topical gel and its keratinocyte‐modulating action was tested on mouse tail model. EA fraction dose‐dependently increased the number and thickness of granular layer and epidermal thickness on mouse tail skin, indicative of the keratinocyte differentiation‐inducing activity. Taking the in vitro and in vivo findings together, the present preclinical study confirms that EA fraction is a promising antipsoriatic agent warranting further development for psoriasis treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

Down‐regulatory effect of usnic acid on nuclear factor‐κB‐dependent tumor necrosis factor‐α and inducible nitric oxide synthase expression in lipopolysaccharide‐stimulated macrophages RAW 264.7

The purpose of this study was to investigate the molecular mechanisms that are responsible for the antiinflammatory effect of usnic acid (UA). UA is one of the most common and abundant lichen metabolites. The present study examined the effects of UA on the tumor necrosis factor‐α (TNF‐α) and nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and the underlying molecular mechanisms. UA decreased the TNF‐α level in LPS‐stimulated RAW264.7 macrophages in dose‐dependent manner, the IC₅₀ value was 12.8 µM. RT‐PCR analysis indicated that it inhibited TNF‐α mRNA expression. Furthermore, it inhibited NO production in LPS‐activated RAW264.7 macrophages, the IC₅₀ value was 4.7 µM. Western blot analysis showed that UA attenuated LPS‐induced synthesis of iNOS protein and nuclear translocation of NF‐κB p65 in the macrophages, in parallel. UA also inhibited LPS‐mediated I‐κBα degradation. Taken together, this suggests that UA has an antiinflammatory effect by inhibiting TNF‐α and iNOS expression, possibly through suppression of nuclear translocation of NF‐κB p65 and I‐κBα degradation. Copyright © 2008 John Wiley & Sons, Ltd.