Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

Plant extracts for developing mosquito larvicides: From laboratory to the field, with insights on the modes of action.

In the last decades, major research efforts have been done to investigate the insecticidal activity of plant-based products against mosquitoes. This is a modern and timely challenge in parasitology, aimed to reduce the frequent overuse of synthetic pesticides boosting resistance development in mosquitoes and causing serious threats to human health and environment. This review covers the huge amount of literature available on plant extracts tested as mosquito larvicides, particularly aqueous and alcoholic ones, due to their easy formulation in water without using surfactants. We analysed results obtained on more than 400 plant species, outlining that 29 of them have outstanding larvicidal activity (i.e., LC50 values below 10 ppm) against major vectors belonging to the genera Anopheles, Aedes and Culex, among others. Furthermore, synergistic and antagonistic effects between plant extracts and conventional pesticides, as well as among selected plant extracts are discussed. The efficacy of pure compounds isolated from the most effective plant extracts and - when available - their mechanism of action, as well as the impact on non-target species, is also covered. These belong to the following class of secondary metabolites: alkaloids, alkamides, sesquiterpenes, triterpenes, sterols, flavonoids, coumarins, anthraquinones, xanthones, acetogenonins and aliphatics. Their mode of action on mosquito larvae ranges from neurotoxic effects to inhibition of detoxificant enzymes and larval development and/or midugut damages. In the final section, current drawbacks as well as key challenges for future research, including technologies to synergize efficacy and improve stability - thus field performances - of the selected plant extracts, are outlined. Unfortunately, despite the huge amount of laboratory evidences about their efficacy, only a limited number of studies was aimed to validate their efficacy in the field, nor the epidemiological impact potentially arising from these vector control operations has been assessed. This strongly limits the development of commercial mosquito larvicides of botanical origin, at variance with plant-borne products developed in the latest decades to kill or repel other key arthropod species of medical and veterinary importance (e.g., ticks and lice), as well as mosquito adults. Further research on these issues is urgently needed.

Isofuranodiene synergizes with temozolomide in inducing glioma cells death.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and deadly brain form of tumor. GBM exhibits high resistance to the standard treatment consisting of temozolomide (TMZ) combined with radiotherapy. Isofuranodiene (IFD) is a bioactive sesquiterpene occurring in the essential oils obtained from Alexanders (Smyrnium olusatrum L., Apiaceae). This compound has shown a broad spectrum of antitumoral activities in different human cancer cell lines both in vitro and in vivo. However, the mechanism of action of IFD on GBM and its potential effects in combination with chemotherapeutic drugs, have not been fully elucidated. PURPOSE: The aim of the present study was to evaluate the anticancer effects of IFD itself and in combination with TMZ in GBM. METHODS: Sulforhodamine B-based proliferation assay, cell cycle analysis and Annexin V/PI staining were carried out to determine the IFD effects on three human GBM cell lines, U87, T98, U251 and in normal human astrocyte. Modulation of protein expression levels was determined by western blot analysis. Reactive oxygen species (ROS) production was evaluated by cytofluorimetry. Moreover, the effects on cell viability of the IFD and TMZ co-administration was evaluated through the calculation of combination index (CI). RESULTS: IFD exerted cytotoxic effects against the GBM cell lines, but not in normal cells (normal human astrocytes). This compound induced a cell cycle blockage and a necrotic cell death depending on the increase of intracellular ROS levels. Furthermore, the synergism between IFD and TMZ was demonstrated in GBM cell lines. CONCLUSION: This study demonstrated the glioma selectivity of IFD and its cytotoxic properties suggesting a new strategy for the treatment of GBM in order to overcome the TMZ resistance and to reduce its side effects.

Thyme extract increases mucociliary-beating frequency in primary cell lines from chronic obstructive pulmonary disease patients.

Chronic obstructive pulmonary disease (COPD) is a respiratory disorder characterized by a progressive and irreversible airflow limitation. COPD is associated to a chronic inflammatory response with infiltration of inflammatory cells in the surface epithelium of large airways and abnormalities in structure and functions of cilia. Thyme (Thymus vulgaris L.) is a traditional medicinal plant of the Mediterranean area used to treat respiratory disorders. We previously evidenced that thyme extract reduce IL-1beta and IL-8, by downregulating the activated NF-κB levels, suggesting its potential therapeutically use in COPD. Cilia beating frequency (CBF) is dramatically impaired in COPD and different pharmacological agents can modulate cilia function. Herein we evaluated the effect of a commercial thyme extract in modulating CBF by measuring its activity in stimulating cAMP, Ca2+ levels and CBF in a MucilAir 3D human COPD airway epithelia reconstituted in vitro system using salmeterol, YM976, isoproterenol and GSK1016790 A as positive controls. Results showed that thyme extract increased cAMP levels starting from 12 h post-treatment, decreased extracellular Ca2+ levels and increased the CBF in airway epithelia from COPD donors. Overall, this work demonstrated that thyme extract is effective in stimulating CBF by inducing an increase of cAMP and Ca2+ levels, thus supporting its therapeutical use in the treatment of COPD.

Identification of highly effective antitrypanosomal compounds in essential oils from the Apiaceae family.

The Apiaceae family encompasses aromatic plants of economic importance employed in foodstuffs, beverages, perfumery, pharmaceuticals and cosmetics. Apiaceae are rich sources of essential oils because of the wealth of secretory structures (ducts and vittae) they are endowed with. The Apiaceae essential oils are available on an industrial level because of the wide cultivation and disposability of the bulky material from which they are extracted as well as their relatively cheap price. In the fight against protozoal infections, essential oils may represent new therapeutic options. In the present work, we focused on a panel of nine Apiaceae species (Siler montanum, Sison amomum, Echinophora spinosa, Kundmannia sicula, Crithmum maritimum, Helosciadium nodiflorum, Pimpinella anisum, Heracleum sphondylium and Trachyspermum ammi) and their essential oils as a model for the identification of trypanocidal compounds to be used as alternative/integrative therapies in the treatment of Human African trypanosomiasis (HAT) and as starting material for drug design. The evaluation of inhibitory effects of the Apiaceae essential oils against Trypanosoma brucei showed that some of them (E. spinosa, S. amomum, C. maritimum and H. nodiflorum) were active, with EC50 in the range 2.7-10.7 µg/mL. Most of these oils were selective against T. brucei, except the one from C. maritimum that was highly selective against the BALB/3T3 mammalian cells. Testing nine characteristic individual components (α-pinene, sabinene, α-phellandrene, p-cymene, limonene, ß-ocimene, γ-terpinene, terpinolene, and myristicin) of these oils, we showed that some of them had much higher selectivity than the oils themselves. Terpinolene was particularly active with an EC50 value of 0.035 µg/mL (0.26 µM) and a selectivity index (SI) of 180. Four other compounds with EC50 in the range 1.0-6.0 µg/mL (7.4-44 µM) had also good SI: α-pinene (>100), ß-ocimene (>91), limonene (>18) and sabinene (>17). In conclusion, these results highlight that the essential oils from the Apiaceae family are a reservoir of substances to be used as leading compounds for the development of natural drugs for the treatment of HAT.

The water extract of tutsan (Hypericum androsaemum L.) red berries exerts antidepressive-like effects and in vivo antioxidant activity in a mouse model of post-stroke depression.

Hypericum androsaemum L., commonly known as 'tutsan' or 'shrubby St. John's Wort', is a member of the Hypericum genus found growing spontaneously in the Mediterranean area and is cultivated extensively as an ornamental plant due to the showy color variation in its fresh berry-like capsules, which turn from red to shiny black as they ripen. Tutsan has also been used in Portuguese and Spanish folk medicine to treat depression. In this study, we assessed the beneficial role of the water extract of H. androsaemum red berries (WE) in an experimental animal model of post-stroke depression. WE was obtained by decoction of H. androsaemum red berries, and its content of ten bioactive compounds was determined through HPLC-DAD analysis. Behavioral tests were carried out using a mouse model of post stroke depression to examine the antidepressive-like activity of WE at two doses (15 and 30 mg/kg bw). In addition, the in vivo antioxidant activity in the mouse brain was evaluated by measuring CAT, GSH, and SOD activity and TBARS levels. WE contained significant amounts of shikimic acid (110.0 g/kg), chlorogenic acid (56.9 g/kg), catechin (5.8 g/kg) and hyperoside (2.7 g/kg). Overall, the highest dosage of WE was found to significantly reduce the symptoms of depression, restoring normal behaviour and reducing levels of oxidative stress by increasing endogenous antioxidant defenses. The protective effects of WE in post-stroke depression in a mouse model were demonstrated in vivo for the first time, and correlated with the antioxidant capacity of its bioactive constituents.

Chemical constituents, radical scavenging activity and enzyme inhibitory capacity of fruits from Cotoneaster pannosus Franch.

Cotoneaster pannosus (Rosaceae) is a semievergreen shrub, producing globose dark red pomes, native to China and widely used as an ornamental plant all over the world. Despite its extensive cultivation, little information is available on the chemical composition and biological activities of its fruits. In this work, the analysis of the chemical composition of C. pannosus fruits, in terms of phenolic components, carotenoids and ascorbic acid by HPLC/DAD, HPLC/ESI-MS and MS/MS as well as in terms of macro- and micro-nutrients was performed. The fruits proved to be a good source of shikimic acid and caffeoylquinic acids, whereas ß-carotene, pelargonidin-3-O glucoside and cyanidin-3,5-rutinoside gave an important contribution to the color of the fruit. Both the polar and apolar fruit extracts showed noteworthy radical scavenger activity and inhibitory effects against monoamine oxidase A (MAO-A), tyrosinase (TYR) and α-glucosidase, making C. pannosus red pomes a promising candidate ingredient in functional foods and dietary supplements.

An overlooked horticultural crop, Smyrnium olusatrum, as a potential source of compounds effective against African trypanosomiasis.

Among natural products, sesquiterpenes have shown promising inhibitory effects against bloodstream forms of Trypanosoma brucei, the protozoan parasite causing human African trypanosomiasis (HAT). Smyrnium olusatrum (Apiaceae), also known as Alexanders or wild celery, is a neglected horticultural crop characterized by oxygenated sesquiterpenes containing a furan ring. In the present work we explored the potential of its essential oils obtained from different organs and the main oxygenated sesquiterpenes, namely isofuranodiene, germacrone and ß-acetoxyfuranoeudesm-4(15)-ene, as inhibitors of Trypanosoma brucei. All essential oils effectively inhibited the growth of parasite showing IC50 values of 1.9-4.0µg/ml. Among the main essential oil constituents, isofuranodiene exhibited a significant and selective inhibitory activity against T. brucei (IC50 of 0.6µg/ml, SI=30), with ß-acetoxyfuranoeudesm-4(15)-ene giving a moderate potentiating effect. These results shed light on the possible application of isofuranodiene as an antiprotozoal agent to be included in combination treatments aimed not only at curing patients but also at preventing the diffusion of HAT.

Polar Constituents, Essential Oil and Antioxidant Activity of Marsh Woundwort (Stachys palustris L.).

Stachys palustris, well-known as marsh woundwort, is a perennial herb growing in wet environments of Europe. Its tubers, leaves and seeds are eaten raw or cooked. Alike other Stachys species, the plant is also used as a traditional remedy. Despite S. palustris has been consumed for centuries, little is known about its chemical constituents. In this work the main secondary metabolites of S. palustris from Hungary and France have been analysed. From the plant ethanolic extracts, ethanoid glucosides, isoscutellarein derivatives, caffeoyl-quinic acids and iridoids have been isolated and structurally characterized by NMR. The essential oils were analysed by GC/MS and showed (E)-phytol, fatty acids and carbonylic compounds as the most abundant compounds. The radical scavenging capacity of plant ethanolic extracts, as evaluated by the DPPH assay, was noteworthy, with IC50 values of 92.08 - 105.42 µg/ml.

Phenolic acids, antioxidant and antiproliferative activities of Naviglio® extracts from Schizogyne sericea (Asteraceae).

Schizogyne sericea, well-known as 'salado', is a halophytic shrub widespread on coastal rocks of Tenerife (Canary Islands). This plant is used traditionally as analgesic, astringent, anti-inflammatory and vulnerary agent. In the present work, we have analysed the aqueous and ethanolic extracts of S. sericea for the content of phenolic acids by HPLC-DAD. The dynamic solid-liquid Naviglio® extractor was used to extract the flowering aerial parts. Aqueous extracts showed higher levels of phenolics than ethanolic extracts. S. sericea extracts were rich in chlorogenic and isochlorogenic acids. The Naviglio® extracts obtained were assayed for in vitro biological activities, namely antioxidant, antimicrobial and cytotoxicity on tumour cells by DPPH, ABTS, FRAP, agar disc-diffusion and MTT methods, respectively. Results showed that aqueous extracts, being richer in phenolic acids, are endowed with relevant radical scavenging activity (TEAC values in the range 208-960 µmol TE/g) while ethanolic extracts exhibited noteworthy antiproliferative effects on tumour cells.

Polar extracts from the berry-like fruits of Hypericum androsaemum L. as a promising ingredient in skin care formulations.

ETHNOPHARMACOLOGICAL RELEVANCE: The top flowering aerial parts of the Hypericum species are traditionally used to prepare ointments to heal cuts and burns. Sometimes even the fruits are used for these purposes. Hypericum androsaemum L., commonly known as tutsan or shrubby St. John's Wort, is a Mediterranean medicinal plant which has been traditionally used to prepare an ointment for treating cuts and wounds. AIM OF THE STUDY: To evaluate the extracts obtained from H. androsaemum red berries as functional ingredients for skin care formulations. MATERIALS AND METHODS: The methanolic extract was obtained by Soxhlet extraction while the aqueous extract was prepared by decoction; their composition was determined by HPLC analysis. Their biological activities were measured in terms of proliferation and migration of human fibroblasts, inhibition of collagenase activity, and immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). In addition, we evaluated their photostability by UV spectroscopy and their protective effects against APPH-induced hemolysis in red blood cells (RBC). RESULTS: The polar extracts contained significant amounts of shikimic (108,143.7-115,901.3mg/kg) and chlorogenic acids (45,781.1-57,002.7mg/kg). The main components of these extracts made an important contribution to a significant increase in human fibroblast migration. Both extracts were also active as collagenase inhibitors, with the aqueous one showing a greater inhibitory capacity (IC50 value of 88.1µg/mL), similar to that of chlorogenic acid. The kinetic parameters determined for the enzymatic reaction revealed for both aqueous extract and chlorogenic acid an uncompetitive mechanism of inhibition. The methanolic extract showed important effects on PBMCs by modulating IL-6. Both extracts proved to be photostable in the UVA/B range and protected RBC against peroxidation at low concentrations. CONCLUSIONS: H. androsaemum red berries were proven to contain phytochemicals that improve skin regeneration, hence potentially employable in skin care formulations.

Bioactive Secondary Metabolites from Schizogyne sericea (Asteraceae) Endemic to Canary Islands.

Schizogyne sericea (Asteraceae) is a halophytic shrub endemic to the Canary Islands and traditionally employed as analgesic, astringent, anti-inflammatory, and vulnerary. A comprehensive phytochemical investigation was conducted on the flowering aerial parts by analyzing both essential oil constituents and polar compounds. The essential oil was dominated by p-cymene, with the noteworthy occurrence of ß-pinene and thymol esters. From the EtOH extract, eight compounds were isolated and structurally elucidated. Essential oil, polar fractions, and isolates (2), (4), and (5) were separately in vitro assayed for antiproliferative activity on human tumor cell lines (A375, MDA-MB 231, and HCT116) by MTT assay, for antioxidant potential by DPPH, ABTS, and FRAP assays, and for antimicrobial activity by the agar disk diffusion method. Results revealed that essential oil and compounds 1 and 2 exert a strong inhibition on tumor cells, and in some cases, higher than that of cisplatin. Fractions containing thymol derivatives (1 and 2) and caffeoylquinic acid derivatives 4 and 5 displayed antioxidant activity comparable to that of Trolox, making S. sericea extract an interesting natural product with potential applications as preservative or in the treatment of diseases in which oxidative stress plays an important role.

Blue honeysuckle fruit (Lonicera caerulea L.) from eastern Russia: phenolic composition, nutritional value and biological activities of its polar extracts.

In the present work we conducted a comprehensive chemical analysis of blue honeysuckle (Lonicera caerulea) spontaneously growing in eastern Russia. HPLC-DAD-ESI/MS analysis showed cyanidin-3-glucoside as the major constituent among phenolics, while nutritional analysis revealed fibre, protein, calcium and magnesium as the most important macro- and micronutrients, respectively. Fatty acid composition was dominated by polyunsaturated fatty acids, linoleic acid being the most abundant. Furthermore, we evaluated several in vitro biological activities such as antioxidant, antimicrobial, antiproliferative, wound healing and immunomodulatory effects of blue honeysuckle aqueous and ethanolic extracts that are often incorporated in food and nutraceutical preparations. While the fruit extracts were revealed to be potent radical scavengers with significant inhibition of ABTS radical, thus confirming the literature data, their inhibitory effects against microbial pathogens and tumor cell lines were negligible. The fruit aqueous extract did not show toxicity to human fibroblasts, but 24 h treatment with 150-200 µg per mL of extract slightly enhanced the cell migration when tested by scratched wound assay. Worth mentioning was the inhibitory effect displayed by the blue honeysuckle fruit aqueous extract on human lymphocytes.

Polar Constituents and Biological Activity of the Berry-Like Fruits from Hypericum androsaemum L.

Hypericum androsaemum, also known as Tutsan, is a small evergreen shrub common in the Mediterranean basin where it is traditionally used as diuretic and hepatoprotective herbal drug. This plant possesses the peculiarity to produce fleshy and berry-like fruits that ripen from red to shiny black. In the present work, the chemical constituents of methanolic extracts and infusions of red and black fruits were analyzed by HPLC, and correlated with their antioxidant properties which were evaluated by the DPPH, ß-Carotene/linoleic acid, and hypochlorous acid tests. In addition, the red pigment of the fruit was isolated by column chromatography and structurally elucidated by NMR. Results showed that H. androsaemum fruits contain high amounts of shikimic and chlorogenic acids, while their color was given by a tetraoxygenated-type xanthone, reported for the first time in Hypericum species. The red berries infusion gave the highest content of total phenolic compounds, DPPH, and hypochlorous acid scavenging activity, and ß-carotene bleaching. Cytotoxicity of the berries extracts on three human tumor cell lines (malignant melanoma, breast adenocarcinoma, and colon carcinoma) was evaluated by MTT assay, and relevant inhibition on colon carcinoma cells (IC50 value of 8.4 µg/mL) was found. Finally, the effects of red berries extract on the immune system were evaluated by peripheral blood mononuclear cell (PBMC) proliferation assay that revealed a strong stimulation on lymphocytes at low doses (0.4-6 µg/mL).

Antioxidant activity and cytotoxicity on tumour cells of the essential oil from Cedronella canariensis var. canariensis (L.) Webb & Berthel. (Lamiaceae).

Cedronella canariensis is a lemon-scented species of the family Lamiaceae endemic to the Canary Islands where it is used in the traditional medicine to prepare infusions or inhalations for anti-catarrhal, tonic, diuretic, hypoglycaemiant, hypotensive, anti-inflammatory and decongestant of the respiratory tract. In this work we investigated for the first time the antioxidant activity of the essential oil and its inhibitory effects on tumour cells (A375, MDA-MB-231, HCT 116) proliferation by DPPH, ABTS, FRAP and MTT assays, respectively. The oil, analysed by GC-ionisation flame detector and GC-MS, was characterised by pinocarvone (58.0%) and ß-pinene (10.8%) as the major constituents, being typical of the chemotype 'canariensis'. Noteworthy was the cytotoxic activity of the oil against the tumour cells examined, with IC50 values of 4.3, 7.3 and 11.4 µg/mL on A375, MDA-MB-231 and HCT 116 tumour cells, respectively, as well as the scavenging activity against the ABTS radical (IC50 of 10.5 µg/mL).