Phytochemical study and biological activities of diterpenes and derivatives from Plectranthus species

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2011This study focused on the research of new bioactive constituents from four species of the Plectranthus plants. Previous works on plants of the genus Plectranthus (Lamiaceæ) evidenced that some of their constituents possess interesting biological activities. The antimicrobial activity of the plant extracts and of the isolated metabolites was thoroughly searched. Antioxidant, anticholinesterase and anti-inflammatory properties of some compounds were also screened. The phytochemical study of the acetone extracts of Plectranthus ornatus Codd., P. ecklonii Benth., P. porcatus Winter & Van Jaarsv and P. saccatus Benth. rendered several terpenoid constituents mostly diterpenes. From P. ornatus three new forskolin-like labdane diterpenes (6-O-acetylforskolin, 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin), a new diterpene with the rare halimane skeleton (11R*-acetoxyhalima-5,13E-dien-15-oic acid), and two known labdane diterpenes were isolated; the rhinocerotinoic acid which was found in Plectranthus species for the first time, and plectrornatin C. Six known triterpenoids were also identified as mixtures. The study of P. ecklonii led to the isolation of two known abietanes, sugiol and parvifloron D. Sugiol was obtained from Plectranthus species for the first time. Four known triterpenoids were also identified as mixtures. P. porcatus, a plant not hitherto studied, yield a new spiro-abietane diterpene [(13S,15S)-6β,7α,12α,19-tetrahydroxy-13β,16-cyclo-8-abietene-11,14-dione]. A new beyerane diterpene (ent-7α-acetoxy-15-beyeren-18-oic acid) was isolated from P. saccatus. Attempting to find novel bioactive prototypes from the more potent antibacterial diterpenes, isolated in higher yields, some diterpene derivatives were prepared. Nine new derivatives were obtained from (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (P. ornatus). A new 2β-(4-hydroxy)benzoyloxy derivative of microstegiol was prepared from parvifloron D (P. ecklonii). From the 7α-acetoxy-6β-hydroxyroyleanone (isolated in the past from P. grandidentatus) thirteen ester derivatives were synthesized, whereof ten were new compounds. The unequivocal chemical structures of pure compounds (natural and derivatives) were deduced from their spectroscopic (IR, MS, 1D and 2D NMR experiments) and physico-chemical data, as well as from literature information. The preliminary antimicrobial activity screenings of all the isolated metabolites showed that several diterpenes inhibited the growth of the Gram positive bacteria tested. In addition, the minimum inhibitory concentration against standard and clinical isolates of sensitive and resistant Staphylococcus and Enterococcus strains was determined for the antibacterial metabolites and their synthesized derivatives. The (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid and its (11R*,13E)-halima-5,13-diene-11,15-diol derivative were the more active halimanes. Parvifloron D was less active than its microstegiol 2β-(4-hydroxy)benzoate derivative, but both showed more potent antibacterial activities than the halimane diterpenoids. The three 12-O-benzoyl esters derivatives of the 7α-acetoxy-6β-hydroxyroyleanone prototype revealed to be more potent growth inhibitors against Staphylococcus and Enterococcus strains than the prototype. The 6β-propionyloxy-12-O-propionyl derivative also showed to be more active against Enterococcus than the viii prototype. Generally, the 12-esters and the 6,12-diesters were more active against Enterococcus than Staphylococcus strains. The hydrophobic extra-interactions with the bacterial targets seem to play an important role on the activity of royleanones derivatives prepared. Taking into account the IC50 values which expressed the scavenging DPPH radical ability, the isolated metabolite parvifloron D as well as 7α-acetoxy-6β-hydroxyroyleanone showed in vitro antioxidant activity. The in vitro acetylcholinesterase assay did not detect any activity for all the newly isolated diterpenes and 7α-acetoxy-6β-hydroxyroyleanone. The COX inhibitor screening assay was tested on 6-O-acetylforskolin, rhinocerotinoic acid, plectrornatin C, (11R*,13E)-halima-5,13-diene-11,15- diol, 11R*-acetoxyhalima-5,13E-dien-15-oic acid and on its methyl ester, for their ability to inhibit COX-2. The preliminary results encourage further studies aiming to confirm and to examine its potential anti-inflammatory activity in a more robust approach.estudo teve como objectivo a pesquisa de novos constituintes bioactivos de quatro espécies de plantas do género Plectranthus. A actividade antimicrobiana dos extractos obtidos e dos metabolitos isolados foi realizada e foram testadas as propriedades anti-oxidante, anti-colinesterase e anti-inflamatória de alguns compostos. O estudo fitoquímico dos extractos de acetona de Plectranthus ornatus Codd., P. ecklonii Benth., P. porcatus Winter & Van Jaarsv. e P. saccatus Benth. originou diversos constituintes terpénicos, principalmente diterpenos. Três novos diterpenos do tipo forskolina (6-O-acetilforskolina; 1,6-di-O-acetilforskolina e 1,6-di-O-acetil-9-deoxiforskolina) foram isolados de P. ornatus. Foram também identificados um novo diterpeno com o raro esqueleto de halimano (ácido 11R*-acetoxihalima-5,13E-dien-15-óico), dois diterpenos labdânicos conhecidos; o ácido rinocerotinóico encontrado pela primeira vez em espécies do género Plectranthus, e a plectrornatina C. Seis triterpenos já conhecidos foram igualmente identificados na forma de misturas. O estudo de P. ecklonii originou o isolamento de dois abietanos conhecidos: o sugiol e a parviflorona D. O sugiol foi isolado pela primeira vez de espécies Plectranthus. Outros quatro triterpenos conhecidos foram identificados também como misturas. A planta P. porcatus, até à data não estudada, originou um novo diterpeno spiro-abietânico [(13S,15S)-6β,7α,12α,19-tetrahidroxi-13β,16-ciclo-8-abietene-11,14-diona]. Um novo diterpeno com esqueleto de beierano (ácido ent-7α-acetoxi-15-beieren-18-óico) foi isolado de P. saccatus. Na tentativa de obter novos protótipos bioactivos, vários derivados foram preparados, a partir dos diterpenos antibacterianos mais potentes e isolados em maior quantidade. Nove novos derivados foram obtidos do ácido (11R*,13E)-11-acetoxihalima-5,13-dien-15-óico (P. ornatus). Um novo derivado 2β-(4-hidroxi)benzoilado do microstegiol, foi preparado a partir da parviflorona D (P. ecklonii). Treze ésteres derivados da 7α-acetoxi-6β-hidroxiroyleanona (isolada anteriormente de P. grandidentatus) foram sintetizados, sendo de assinalar que dez dos derivados são compostos novos. A determinação estrutural dos compostos puros (naturais e derivados) foi deduzida por espectroscopia (IV, EM, RMN 1D e 2D), propriedades físico-químicas e com base na informação obtida da literatura. O estudo preliminar da actividade antimicrobiana de todos os metabolitos isolados, mostrou que diversos diterpenos inibem o crescimento de bactérias de Gram positivo. A concentração mínima inibitória (CMI) dos metabolitos e seus derivados foi determinada em estirpes de Staphylococcus e Enterococcus, tanto em bactérias padrão como em isolados clínicos resistentes e sensíveis a antibióticos. O ácido (11R*,13E)-11-acetoxihalima-5,13-dien-15-óico e o seu derivado (11R*,13E)-halima-5,13-diene-11,15-diol foram os halimanos mais activos. A parviflorona D foi menos activa do que o seu correspondente derivado 2β-(4-hidroxi)benzoilado, mas ambos apresentaram uma actividade antibacteriana mais potente do que os diterpenos com esqueleto de halimano. Os três 12-O-benzoil-ésteres derivados do protótipo 7α-acetoxi-6β-hidroxiroyleanona revelaram ser inibidores mais potentes do que a royleanona-protótipo, contra as estirpes testadas de Staphylococcus e Enterococcus. O derivado 6β-propioniloxi-12-O-propionilo mostrou ser o mais activo contra as estirpes testadas de Enterococcus do que o protótipo. De um modo geral, os derivados 12-ésteres e os 6,12-diésteres foram mais activos contra as estirpes de Enterococcus do que as estirpes de Staphylococcus testadas. As interacções hidrofóbicas com os alvos bacterianos parecem ter um papel importante na actividade antibacteriana dos derivados de royleanona preparados. Os metabolitos parviflorona D e a 7α-acetoxi-6β-hidroxiroyleanona demostraram possuir actividade antioxidante in vitro, tendo em conta os valores de IC50 que expressam a actividade anti-oxidante com base na captura do radical DPPH. Todos os novos diterpenos isolados e derivados obtidos neste trabalho foram testados e não revelaram possuir actividade inibitória da acetilcolinesterase in vitro. A actividade anti-inflamatória foi testada nos compostos 6-O-acetilforskolina, ácido rinocerotinóico, plectrornatina C, (11R*,13E)-halima-5,13-diene-11,15-diol, ácido 11R*-acetoxihalima-5,13E-dien-15-óico e no seu éster metílico, através da sua capacidade de inibir a COX-2. Os resultados preliminares obtidos apoiam a necessidade de estudos futuros de forma a confirmar, explorar e discutir uma potencial actividade anti-inflamatória.The research work was performed, mostly, in the Faculdade de Farmácia da Universidade de Lisboa at the Medicinal Chemistry Group (former Centro de Estudos de Ciências Farmacêuticas – CECF) of the Institute for Medicines and Pharmaceutical Sciences (iMed.UL). Funding to these research centres and the attribution of a Doctoral degree grant (SFRH/BD/19250/2004) were provided by the Fundação para a Ciência e a Tecnologia - Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES)

Characterization of lipid extracts from the Hermetia illucens larvae and their bioactivities for potential use as pharmaceutical and cosmetic ingredients

There is an increasingly growing demand for the use of natural and sustainable bioactives in the field of the pharmaceutical and cosmetic industries. The biomass from black soldier fly larvae (Hermetia illucens) can be viewed as an innovative source of compounds with high aggregate value and marketing potential due to the sustainable organic matter bioconversion process used as substrate for its development. This insect can be a source of lipid compounds with high added value, mainly due to its high content in fatty acids (FA) with potential applicability in the pharmaceutical and cosmetic industry. In this context, in this work different extraction methods were tested (decoction, microwaves, maceration and ultrasound), using water, acetone, n-hexane as extraction solvents, to evaluate yields of the BSF larvae lipid extracts, as well as their lipid profile, and a pre- liminary safety screening was conducted. Results show that despite using different extraction techniques and solvents, similar FA composition profiles were obtained. The lauric acid content (C12: 0) is elevated in all the extracts in relation to the other FA, ranging 37%–62%. The contents in palmitic (C16: 0) and oleic (C18: 1n-9) acids, were also high in all applied extraction methods. The omega-6 FA (ω-6 PUFAs), mainly linoleic acid (C18: 2n6c), were also identified in the lipid fraction of BSF larvae biomass, with a content variation between 4.5% and 17.7%, while the omega-3 group, namely α-Linolenic acid (C18: 3n3), presented values between 0.66% and 1.95%. None of the extracts presented toxicity in preliminary tests with the Artemia salina model. Through this study, it was possible to confirm that BSF larvae oil can be obtained by sustainable methods, containing a broad mixture of FA and being highly rich in lauric acid, with a promising skin care applicability.This work was supported by national funds through FCT - Foundation for Science and Technology, I.P., under the UIDB/04567/2020 and UIDP/04567/2020 projects attributed to CBIOS, and UIDB/00690/2020 to CIMO and by the PhD grant attributed to C.A. (UI/BD/151423/2021). National funding by FCT, P.I., through the institutional scientific employment program-contract for the contracts of A. Fernandes and L. Barros. Additionally, the authors would like to acknowledge funding of the projects ENTOVALOR (POCI-01-0247-FEDER294 017675/ 2016–2019) and NETA (POCI-01-0247-046959).info:eu-repo/semantics/publishedVersio

Amelioration effect of 18β-Glycyrrhetinic acid on methylation inhibitors in hepatocarcinogenesis -induced by diethylnitrosamine

Aimsuppression of methylation inhibitors (epigenetic genes) in hepatocarcinogenesis induced by diethylnitrosamine using glycyrrhetinic acid.MethodIn the current work, we investigated the effect of sole GA combined with different agents such as doxorubicin (DOX) or probiotic bacteria (Lactobacillus rhamanosus) against hepatocarcinogenesis induced by diethylnitrosamine to improve efficiency. The genomic DNA was isolated from rats’ liver tissues to evaluate either methylation-sensitive or methylation-dependent resection enzymes. The methylation activity of the targeting genes DLC-1, TET-1, NF-kB, and STAT-3 was examined using specific primers and cleaved DNA products. Furthermore, flow cytometry was used to determine the protein expression profiles of DLC-1 and TET-1 in treated rats’ liver tissue.ResultsOur results demonstrated the activity of GA to reduce the methylation activity in TET-1 and DLC-1 by 33.6% and 78%, respectively. As compared with the positive control. Furthermore, the association of GA with DOX avoided the methylation activity by 88% and 91% for TET-1 and DLC-1, respectively, as compared with the positive control. Similarly, the combined use of GA with probiotics suppressed the methylation activity in the TET-1 and DLC-1 genes by 75% and 81% for TET-1 and DLC-1, respectively. Also, GA and its combination with bacteria attenuated the adverse effect in hepatocarcinogenesis rats by altering potential methylomic genes such as NF-kb and STAT3 genes by 76% and 83%, respectively.ConclusionGA has an ameliorative effect against methylation inhibitors in hepatocellular carcinoma (HCC) by decreasing the methylation activity genes

Unveiling the mechanism of action of 7α-acetoxy-6β-hydroxyroyleanone on an mrsa/visa strain: Membrane and cell wall interactions

UIDB/00100/2020 PTDC/MED-QUI/29036/2017 CEECIND/03414/2018 UIDB/04378/2020 PTDC/BIA-MIC/31645/2017 UID/DTP/04138/2019 UID/DTP/04567/2019 CBIOS/PRUID/BI1/2017 UIDB/04567/2020 UID/AMB/50017 UIDP/50017/2020 UIDB/50017/2020The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7α-Acetoxy-6β-hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.publishersversionpublishe

Roots and rhizomes of wild Asparagus: Nutritional composition, bioactivity and nanoencapsulation of the most potent extract

The nutritional composition and bioactive properties of roots and rhizomes of Asparagus stipularis were evaluated. Antioxidant activity of extracts obtained by infusion was evaluated using free radicals scavenging and reducing power methods. Porcine liver primary cell was used to check the hepatotoxicity of infusions. Results revealed that Asparagus samples are likely a source of nutrients, such as dietary fibre and essential fatty acids. HPLC-DAD-ESI/MS characterization of infusions allowed the identification and quantitation of 7 phenolic compounds, all hydroxycinnamoyl derivatives, with caffeic acid as the most abundant. Roots infusion contained the highest amounts of these compounds. It also exhibited the highest antioxidant activity in all assays, with EC50 values of 0.44 ± 0.01, 0.98 ± 0.03 and 0.64 ± 0.01 mg/mL for DPPH, ABTS and FRAP assays, respectively, with no toxicity towards PLP2 primary cell cultures (GI50 > 400 μg/mL). PLGA nanoparticles loaded with root extract were prepared using solvent-evaporation double emulsion method. Nanoparticles size was about 260 nm and a polydispersity index around 0.1, with a zeta potential of about -36 mV, as well as a good encapsulation efficiency of approximately 83%. Their morphology was analysed by SEM and spherical polymeric nanoparticles with a smooth surface were observed. FTIR and DSC were also performed, which allowed corroborating the efficacy of the encapsulation and to confirm the production of a stable and robust system to load Asparagus extracts. The developed nanoparticles are expected to be used as delivery systems for bioactive compounds of A. stipularis and they could be used as an innovative dietary supplement.The UCM authors would like to thank to ALIMNOVA Research Group (UCM GR105/18) and Spanish Government through the project PID2019-109365RA-I00. Authors are also grateful to Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020), LAQV (UIDB/50006/2020), CCMar (UIDB/04326/2020), CBIOS (UIDB/ 04567/2020) and iBB-IST (UIDB/04565/2020). A. Fernandes contract was provided by National funding by FCT, P. I., through the institutional scientific employment program-contract. The authors are also grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. The GIP-USAL is financially supported by the Spanish Government through the project AGL2015- 64522-C2-2-R.info:eu-repo/semantics/publishedVersio

Antibacterial, Anti-Inflammatory, Antioxidant, and Antiproliferative Properties of Essential Oils from Hairy and Normal Roots of Leonurus sibiricus

Essential oils obtained from the NR (normal roots) and HR (hairy roots) of the medicinal plant Leonurus sibiricus root were used in this study. The essential oil compositions were detected by GC-MS. Eighty-five components were identified in total. Seventy components were identified for NR essential oil. The major constituents in NR essential oil were β-selinene (9.9%), selina-4,7-diene (9.7%), (E)-β-caryophyllene (7.3%),myli-4(15)-ene (6.4%), and guaia-1(10),11-diene (5.9%). Sixty-seven components were identified in HR essential oil, the main constituents being (E)-β-caryophyllene (22.6%), and germacrene D (19.8%). The essential oils were tested for cytotoxic effect, antimicrobial, anti-inflammatory, and antioxidant activities. Both essential oils showed activity against grade IV glioma cell lines (IC50 = 400 μg/mL), antimicrobial (MIC and MFC values of 2500 to 125 μg/mL), and anti-inflammatory (decreased level of IL-1β, IL-6, TNF-α, and IFN-γ in LPS-stimulated cells).The essential oils exhibited moderate antioxidant activity in ABTS (EC50 = 98 and 88 μg/mL) assay. This is the first study to examine composition of the essential oils and their antimicrobial, antioxidant, antiproliferative, and anti-inflammatory activities. The results indicate that essential oils form L. sibiricus root may be used in future as an alternative to synthetic antimicrobial agents with potential application in the food and pharmaceutical industries

a preliminary report

© European Association of Hospital Pharmacists 2021. No commercial re-use. See rights and permissions. Published by BMJ.OBJECTIVES: Since the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the pressure to minimise its impact on public health has led to the implementation of different therapeutic strategies, the efficacy of which for the treatment of coronavirus disease 2019 (COVID-19) was unknown at the time. Remdesivir (REM) was granted its first conditional marketing authorisation in the EU in June 2020. The European Medicines Agency (EMA) and local health authorities all across the EU have since strongly recommended the implementation of pharmacovigilance activities aimed at further evaluating the safety of this new drug. The objective of this study was to evaluate adverse drug reactions (ADRs) attributed to either REM or hydroxychloroquine (HCQ) in patients hospitalised for COVID-19 in Centro Hospitalar de Lisboa Ocidental, a Portuguese hospital centre based in Lisbon. We present the preliminary results reporting plausible adverse effects of either HCQ or REM. METHODS: An observational cohort study was carried out between 16 March and 15 August 2020. Participants were divided into two cohorts: those prescribed an HCQ regimen, and those prescribed REM. Suspected ADRs were identified using an active monitoring model and reported to the Portuguese Pharmacovigilance System through its online notification tool. The ADR cumulative incidence was compared between the two cohorts. RESULTS: The study included 149 patients, of whom 101 were treated with HCQ and the remaining 48 with REM. The baseline characteristics were similar between the two cohorts. A total of 102 ADRs were identified during the study period, with a greater incidence in the HCQ cohort compared with the REM cohort (47.5% vs 12.5%; p<0.001). Causality was assessed in 81 ADRs, all of which were considered possible. CONCLUSIONS: Real-world data are crucial to further establish the safety profile for REM. HCQ is no longer recommended for the treatment of COVID-19.publishersversionpublishe

Chemical composition and antimicrobial activity of essential oil of peruvian Dalea strobilacea Barneby

Dedicado en homenaje al Dr. Isidoro Manuel Sánchez Vega (Moche 1938 – Lima 2015).The composition of the essential oil obtained by hydrodistillation from Dalea strobilacea Barneby (Fabaceae) aerial parts was examined by GC and GC/MS. β-Phellandrene (44%) together with α-pinene (18%) were the main essential oil components. Antimicrobial activity of the essential oil was evaluated against eight bacterial strains. A moderate growth inhibition of Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis was shown by the essential oil.  

Increased antibacterial properties of indoline-derived phenolic Mannich bases

The search for antibacterial agents for the combat of nosocomial infections is a timely problem, as antibiotic-resistant bacteria continue to thrive. The effect of indoline substituents on the antibacterial properties of aminoalkylphenols was studied, leading to the development of a library of compounds with minimum inhibitory concentrations (MICs) as low as 1.18 μM. Two novel aminoalkylphenols were identified as particularly promising, after MIC and minimum bactericidal concentrations (MBC) determination against a panel of reference strain Gram-positive bacteria, and further confirmed against 40 clinical isolates (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, and Listeria monocytogenes). The same two aminoalkylphenols displayed low toxicity against two in vivo models (Artemia salina brine shrimp and Saccharomyces cerevisiae). The in vitro cytotoxicity evaluation (on human keratinocytes and human embryonic lung fibroblast cell lines) of the same compounds was also carried out. They demonstrated a particularly toxic effect on the fibroblast cell lines, with IC50 in the 1.7-5.1 μM range, thus narrowing their clinical use. The desired increase in the antibacterial properties of the aminoalkylphenols, particularly indoline-derived phenolic Mannich bases, was reached by introducing an additional nitro group in the indolinyl substituent or by the replacement of a methyl by a bioisosteric trifluoromethyl substituent in the benzyl group introduced through use of boronic acids in the Petasis borono-Mannich reaction. Notably, the introduction of an additional nitro moiety did not confer added toxicity to the aminoalkylphenols.acceptedVersionPeer reviewe