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Abstract

West Nile virus (WNV) is a mosquito-borne flavivirus responsible of neuroinvasive manifestations. Natural products are well-known for their biological activities and pharmaceutical application. In this study, the inhibitory effects of essential oils (EOs) of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. on WNV replication were investigated. WNV was incubated with EOs before adsorption on Vero cells, viral replication was carried out in the absence or presence of EO. Cells were exposed to EO before the adsorption of untreated-virus. GC-MS and GC-FID were used for chemical characterization of EOs. Cell protection from infection was observed for both EOs. P. aduncum EO was characterized by dillapiole as main compound (48.21%) and O. quixos EO by 1,8-cineole (39.15%). Further investigations, such as the study of molecular and cellular mechanisms of action and in vivo evaluation, should be performed on these essential oils to derive new potential drugs against WNV.

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... Pino et al. (2018) identified more than one hundred volatile compounds in EOs, of which 1,8-cineole (21.4%) and p-cymene (12.6%) stand out. Radice et al. (2019) also identified these constituents to be the most abundant in the oil, with concentration of 39.15% and 6.12% respectively. Other compounds present and likewise reported by the previous authors are limonene (1.84-9.2%), ...
... There are some studies regarding the biological properties of EO from O. quixos, including the following studies on the following activities: antiplatelet [18], antithrombotic [18,19], antifungal [20], antimicrobial [21,22], antioxidant [21,23], anti-inflammatory [24] and antiviral [25]. Nevertheless, this continues to be a novel field for scientific research, since reports are scarce. ...
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Termites are one of the most challenging pests that agriculture and urban environments are faced with. They bring substantial losses in annual and perennial crops and damage wood components at construction sites. The development of natural products with biological activity for termite control is an ecological alternative in the search to replace conventional products. Hence, the aim of this research was to predict the termiticidal and repellent effects of the essential oil from Ocotea quixos leaves on Nasutitermes corniger using a one-factor response surface methodology design. The variable analysed was the concentration of essential oil in ethanol at an interval of 0.3–0.05% for anti-termite activity and between 0.12 and 0.01% for repellent action. A 100% mortality rate was found at concentrations higher than 0.12% and at the minimum concentration analysed, the effect was 22.2%. As for the repellent action, the concentration of 0.12% was able to repel 100% of the termites and at 0.01% it repelled 48.9%. The analysis of the essential oil from Ocotea quixos leaves by GC-MS resulted in the presence of 42 compounds, 39 of them elucidated. The main compounds were (E)-cinnamyl acetate (36.44%), (E)-cinnamaldehyde (27.03%), (E)-β-caryophyllene (5.21%) and (E)-methyl isoeugenol (4.18%).
... The compounds of O. campechianum essential oil are reported in Table 1; distillation yields were of 0.68%. As previously published by us [34], the yields for O. quixos essential oil were 0.13%, for P. aduncum 0.12%. The essential oil of O. campechianum was found to be rich in eugenol (18.6%), b-caryophyllene (17.0%), 1,8-cineole (11.4%), bicyclogermacrene (9%), cis-ocimene (8%) and a-caryophyllene (4.5%), that of O. quixos was characterised by 1,8-cineole (39.2%), sabinene (6.5%), a-pinene (6.3%), b-caryophyllene (4.7%) and terpinen-4-ol (4.2%), and P. aduncum essential oil showed a high percentage of dillapiole (48.2%) followed by transocimene (7.5%) and b-caryophyllene (4.8%) [34]. ...
... As previously published by us [34], the yields for O. quixos essential oil were 0.13%, for P. aduncum 0.12%. The essential oil of O. campechianum was found to be rich in eugenol (18.6%), b-caryophyllene (17.0%), 1,8-cineole (11.4%), bicyclogermacrene (9%), cis-ocimene (8%) and a-caryophyllene (4.5%), that of O. quixos was characterised by 1,8-cineole (39.2%), sabinene (6.5%), a-pinene (6.3%), b-caryophyllene (4.7%) and terpinen-4-ol (4.2%), and P. aduncum essential oil showed a high percentage of dillapiole (48.2%) followed by transocimene (7.5%) and b-caryophyllene (4.8%) [34]. ...
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Aedes aegypti , the main arbovirus vector of the Yellow fever, Dengue, Chikungunya and Zika viruses, is widely distributed in tropical and subtropical areas throughout the world. Preventive control efforts have been implemented worldwide aimed at reducing its impact on human health. The recent reduction of chemicals available for vector control due to their negative impact on the environment and human health and the increase in mosquito resistance to insecticides have driven the research community to identify and evaluate sustainable alternatives to synthetic insecticides. In this study, the potential larvicidal effect of essential oils extracted from Ocimum campechianum , Ocotea quixos and Piper aduncum were tested in vitro . GC and GC–MS analyses showed that the main compounds were eugenol (18%), 1,8-cineole (39%) and dillapiole (48%), respectively. Susceptibility to essential oils was measured according to the WHO protocol. After 24 h, the mean percentage mortality ranged from 2.7 to 100% for P. aduncum , from 2.2 to 100% for O. campechianum , and from 2.9 to 100% for O. quixos . The highest potential was displayed by P. aduncum , followed by O. campechianum and O. quixos , with LC 50 values of 25.7, 69.3 and 75.5 ppm, respectively. The rapid and effective larvicidal activity of these three oils led us to consider these results to be promising, also considering the possibility of local cultivation of these plants in tropical and subtropical areas and the simple technology for their large-scale preparation and production. Further studies are needed to evaluate the individual components and their activity as larvicides.
... This study is part of a large research project characterized by the biodiversity mapping of Amazonian Ecuador flora belonging to ethnomedical traditional uses through chemical and biological characterization of EOs [1,2]. ...
... Since most of the plant species used as traditional health remedies are characterized by flavour and aromatic smell, the study of EOs is one of the best strategies to draw a research profile matching biodiversity and phytomedicine. For this reason, the chemical fingerprinting is focused on the characterization of EOs, while the biological activities are assayed through in vitro tests to confirm traditional properties or to find new health applications [1,2]. ...
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Piper carpunya Ruiz & Pav. (Piperaceae) is a perennial aromatic shrub of Amazonian area of Ecuador and Peru, belonging to the ethnomedicine of these countries. The traditional preparations of the crude drug (fresh leaves used topically as is, and dried leaves in infusions or decoctions) are known for anti-inflammatory, antiulcer, antidiarrheal, antiparasitic effects, and wound healing properties. In light of this traditional evidence, chemical composition (GC-MS) and biological activity, i.e., antioxidant, antifungal (yeast) capacities, and genotoxicity, of Amazonian P. carpunya leaf essential oil (EO) have been investigated in order to valorize some of the putative ethnomedical effects. The EO was obtained through steam distillation of fresh leaves (yield: 7.6 g/kg [0.76%]; refractive index at 20°C: 1.49; density: 0.928 g/mL). Chemical characterization performed through GC-MS evidenced the presence of 21 compounds (96.2% of the total). The most abundant constituents were piperitone (26.2%), limonene (9.5%), elemicin (7.2%), and β -phellandrene (5.6%). In vitro DPPH antioxidant assay showed a weak radical scavenging ability (IC 50 ) if compared to positive control. Low bioactivity of the EO was also demonstrated against yeast, but it showed an interesting synergistic activity (FIC index of EO+fluconazole) against Candida sp. strains. Ames test evidenced the safety of the EO concerning genotoxicity.
... In a recent review, Taher et al. (2020) summarized the traditional uses of P. aduncum and indicated that the plant contains 23 essential oil components, benzoic acid derivatives, phenylpropanoid, flavonoids, chromenes, chalcones, sesquiterpenes and monoterpenes. Radice et al. (2018) reported the antiviral effects of dillapiole, a significant component of P. aduncum's essential oil, on the West Nile virus. The anti-viral effects of P. aduncum on poliovirus have also been reported (Loh ezic-Le D ev ehat, Bakhtiar, B ezivin, Amoros, & Boustie, 2002). ...
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Purpose This review aims to provide synoptic documentation on acclaimed anecdotal plant-based remedies used by Latin America and the Caribbean (LAC) communities to manage COVID-19. The theoretical approaches that form the basis for using the anecdotally claimed phytotherapies were reviewed against current scientific evidence. Design/methodology/approach In this paper plant-based remedies for managing COVID-19 were searched on social and print media to identify testimonies of people from different communities in LAC countries. Information was extracted, evaluated and reviewed against current scientific evidence based on a literature search from databases such as Journal Storage (JSTOR), Excerpta Medica Database (EMBASE), SpringerLink, Scopus, ScienceDirect, PubMed, Google Scholar and Medline to explore the scientific basis for anecdotal claims. Findings A total of 23 medicinal plants belonging to 15 families were identified as phytotherapies used in managing COVID-19 in LAC communities. Originality/value The plant-based remedies contained valuable phytochemicals scientifically reported for their anti-inflammatory, antiviral, antioxidant and anticancer effects. Anecdotal information helps researchers investigate disease patterns, management and new drug discoveries. The identified acclaimed plant-based remedies are potential candidates for pharmacological evaluations for possible drug discovery for future pandemics.
... The EO from inflorescences from Brazilian samples consisted mainly of oxygenated monoterpenoids (43.9%), followed by sesquiterpene hydrocarbons (30.5%) and phenylpropanoids (10.0%) [32,42] (See Table 5). There is evidence of at least two chemotypes of leaf EOs from Equadorian specimens, one from the western Amazonian region characterized by dillapiole (31.5% to 97.3%) [43] and the second from the Atlantic Forest dominated by monoterpenoid compounds such as (E)-nerolidol and linalool [32] Da Silva et al. [32] describe EOs of aerial parts from two different chemotypes, one from Equador with dillapiole at 45.9%, (E)-β-ocimene at 19.0%, and piperitone at 8.4%; and one from Cuba with piperitone between 19.0% and 23.7%, camphor between 9.4% and 17.1%, and viridiflorol between 13.0% and 14.5%; the review of the literature reveals a general preference for dillapiole in the species from the Neotropical region, and a clear distinction for the specimen from China, dominated by eugenol. ...
Article
Piper species are aromatic plants used as spices in the kitchen, but their secondary metabolites have also shown biological effects on human health. These plants are rich in essential oils, which can be found in their fruits, seeds, leaves, branches, roots and stems. Some Piper species have simple chemical profiles, while others, such as Piper nigrum, Piper betle, and Piper auritum, contain very diverse suites of secondary metabolites. In traditional medicine, Piper species have been used worldwide to treat several diseases such as urological problems, skin, liver and stomach ailments, for wound healing, and as antipyretic and anti-inflammatory agents. In addition, Piper species could be used as natural antioxidants and antimicrobial agents in food preservation. The phytochemicals and essential oils of Piper species have shown strong antioxidant activity, in comparison with synthetic antioxidants, and demonstrated antibacterial and antifungal activities against human pathogens. Moreover, Piper species possess therapeutic and preventive potential against several chronic disorders. Among the functional properties of Piper plants/extracts/active components the antiproliferative, anti-inflammatory, and neuropharmacological activities of the extracts and extract-derived bioactive constituents are thought to be key effects for the protection against chronic conditions, based on preclinical in vitro and in vivo studies, besides clinical studies. Habitats and cultivation of Piper species are also covered in this review. In this current work, available literature of chemical constituents of the essential oils Piper plants, their use in traditional medicine, their applications as a food preservative, their antiparasitic activities and other important biological activities are reviewed.
... The occurrence of alkamides, piperamides, kavalactones, and benzoic acid derivatives has been reported for plants of the Piper genus(36,38). Likewise, several biological activities, including biocidal activity against bacteria like Mycobacterium tuberculosis, activities against various protozoa like Leishmania, Plasmodium, and Trypanosoma(39,40), and antiviral activity against West Nile Virus and Human immuno deficienty virus resistant strains(41,42). Among those compounds, alkamides and piperamides containing amides and aromatic groups similar to remdesivir, an approved drug for COVID-19, can exert a potential inhibition of SARS-CoV-2, given the interaction of these structures with Mpro, RdRp (RNA dependent RNA polymerase), and ACE2 (angiotensin converting enzyme)(43)(44)(45).The interaction of Piper compounds with SARS-CoV-2 viral proteins has been evaluated in silico(35). ...
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Background: Coronavirus infectious disease 2019 (COVID-19) caused by the infection with the new coronavirus SARS-CoV-2 has affected the life and health of more than 222 million people. In the absence of any specific pharmacological treatment, the need to find new therapeutic alternatives is clear. Medicinal plants are widely used worldwide to treat different conditions, including COVID-19; however, in most cases, there are no specific studies to evaluate the efficacy of these treatments. Objective: This article evaluates the antiviral effect of six plant extracts used by indigenous and afro Colombian people against SARS-CoV-2 in vitro Methods: The antiviral effect of six extracts prepared from plants used in Colombian traditional medicine was evaluated against SARS-CoV-2 through a pre-post treatment strategy on the Vero E6 cell line. Once cytotoxicity was established through an MTT assay, the antiviral effect of the extracts was calculated based on the reduction in the viral titer determined by plaque assay. Results: Gliricidia sepium inhibited SARS-CoV-2 in a 75.6%, 56.8%, 62.5% and 40.0% at 10 mg/mL, 8 mg/mL, 6 mg/mL, and 2 mg/mL, respectively, while Piper tuberculatum treatment reduced viral titer in 33.3% at 6 mg/mL after 48h. Conclusion: G. sepium and P. tuberculatum extracts exhibit antiviral activity against SARS-CoV-2 in vitro.
... The main compounds identified in other study with the leaves of O. quixos were caryophyllene (19.0%), humulene (14.3%) and eremophyllene (11.4%) [97]. In more recently studies with leaves EOs, the most prominent compounds reported in EO of O. quixos were 1,8-cineol (21.4%) and p-cymene (13) (12.6%) [98], 1,8-cineole (39%) [99,100] and the two related compounds: (E)-cinnamyl acetate (85) (37-27%) and (E)-cinnamaldehyde (94) (27%) [101,102]. ...
Article
This review describes the chemical composition and biological properties of essential oils from plants of the Ocotea genus, from different origin. This taxon belongs to the Laureaceae family, which in turn, is best known for medicinal use, often related to the essential oils. The text includes studies about Ocotea species distributed mainly on the South American continent, but also reporting species located in North America and Africa. Brazil, Colombia, Costa Rica and Ecuador are countries with highest number of species mentioned in the review. Also, the major components of each essential oil, its chemical structures, as well as the description and extent of biological activities related to essential oils are detailed in this review. Finally, the text discusses the chemical and biological aspects of these studies in a comparatively way, also informing additional data such as yield, biome of origin and pharmacobotanical location.
... Regarding antiviral potential, the essential oil of the leaves of Piper aduncum L. obtained in the Amazon region of Pastaza-Ecuador acted efficiently in cell protection and inhibition of the replication of the West Nile virus (WNV), responsible for neuroinvasive manifestations (52). ...
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The genus Piper, belonging to the family Piperaceae, includes approximately 2000 species of plants. Many of them are producers of essential oils. Species of this genus are popularly used for the treatment of chronic diseases such as diabetes, atherosclerosis, and osteoporosis. In the Amazon, they are used to treat infections caused by parasites. This work describes information from the most recent articles on the chemical composition and biological activities of essential oils of Piper species collected in the Amazon, guiding researchers for future studies. The essential oils derived from Amazonian Piper species have major components that have demonstrated larvicidal activity, such as dillapiole, and others that may have antioxidant and anticholinesterase properties, such as γ-terpinene, p-cymene and α-terpinene.
... The existence of essential oil in ishpingo leaves has been previously reported [8]. Some important biological properties are attributed to the O. quixos essential oil, such as antioxidant activity [9], antifungal activity [10], larvicidal activity [11], antiviral activity [12], and phytotoxicity [13]. The presence of secondary metabolites in plant species changes due to variations in the intrinsic and extrinsic factors, which modifies the biological properties of Eos. ...
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Ocotea quixos (Lam.) Kosterm. is an aromatic tree native to Ecuador, whose leaves are used to prepare aromatic beverages to which different health benefits are attributed. In this study, Ocotea quixos leaves were collected in the Amazon region in different environmental conditions and subjected to hydrodistillation to isolate the essential oil. The collection variables used were type of soil, amount of shade, and height; in addition, the presence of twig and leaf age and moisture were used as variables. Chemical composition was analyzed by means of gas chromatography equipped with a flame ionization detector and gas chromatography coupled to mass spectrometry. A wide variety of chemical compositions were detected in the samples. In total, forty-seven compounds were identified, which represented between 97.17% and 99.89% of the total composition. The constituents were mainly grouped into aliphatic sesquiterpene hydrocarbons (33.03–55.89%), other compounds (8.94–47.83%), and oxygenated monoterpenes (1.97–39.66%). The main constituents were found to be (E)-cinnamyl acetate (5.96–41.65%), (E)-methyl cinnamate (0.38–37.91%), and trans-caryophyllene (8.77–37.02%). The statistical analysis suggested the existence of two essential oil chemotypes and a direct correlation between environmental conditions and chemical composition of the essential oils.
... The major component of P. aduncum essential oil, dillapiole exhibited antiviral against West Nile virus (WNV) is a mosquito-borne flavivirus (Radice et al. 2019) and poliovirus (Lohézic-Le Dévéhat et al. 2002). The ethanolic extracts of P. aduncum has a gastroprotective effect in mice and antisecretory effect in rats (Arroyo et al. 2013). ...
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Piper aduncum L., commonly known as 'spiked pepper', has various uses in traditional medicine that include treating wounds, skin boils, infections, and diarrhoea. Its properties as an anti-parasitic, antimicrobial, insecticidal, antitumor, and anticancer agent indicates that it could have further therapeutic potential in treating infections and cancers. The aim of this review was to provide a comprehensive summary of the traditional uses, phytochemistry and pharmacological properties of P. aduncum. Data were collected from electronic databases from 1978 to 2019. The plant is traditionally used for treating diarrhoea in Peru and for its wound-healing properties in Brazil and Papua New Guinea. Phenolics, monoterpenes, sesquiterpenes, and chromene have been found in the P. aduncum plant, and these bioactive compounds contribute to its anti-parasitic, antimicrobial, insecticidal, antitumor, and anticancer properties. Several pharmacological activities of P. aduncum have been reported, most notably in the treatment of infectious diseases and cancer. However, information regarding its safety and efficacy in humans is lacking. Further study is needed to examine the benefits of P. aduncum and its potential applications in a clinical setting. ABSTRAK Piper aduncum L., yang dikenali sebagai lada berduri, mempunyai pelbagai kegunaan dalam ubat tradisi termasuk merawat luka, bisul kulit, jangkitan dan cirit-birit. Sifatnya sebagai agen anti-parasit, antimikrob, insektisid, antitumor dan antikanser menunjukkan bahawa ia mempunyai potensi terapeutik dalam merawat jangkitan dan kanser. Tujuan kajian ini adalah untuk memberikan ringkasan komprehensif mengenai kegunaan tradisi, fitokimia dan sifat farmakologi P. aduncum. Data telah dikumpulkan dari pangkalan data elektronik dari tahun 1978 hingga 2019. Tumbuhan ini secara tradisinya digunakan untuk merawat cirit-birit di Peru serta menyembuhkan luka di Brazil dan Papua New Guinea. Fenol, monoterpena, sesquiterpena serta kromena telah dijumpai dalam tumbuhan P. aduncum, dan sebatian bioaktif ini menyumbang kepada sifatnya sebagai anti-parasit, antimikrob, insektisid, antitumor dan antikanser. Beberapa aktiviti farmakologi P. aduncum telah dilaporkan, terutamanya dalam rawatan penyakit berjangkit dan kanser. Walau bagaimanapun, maklumat mengenai keselamatan dan keberkesanannya pada manusia adalah kurang. Kajian lanjut diperlukan untuk mengkaji manfaat P. aduncum dan potensi pengaplikasiannya dalam persekitaran klinikal.
... The biological activity of the plants of the genus Piper, the largest genus of the family Piperaceae (Almeida et al. 2018), has been widely investigated by the scientific community because of its high pharmacological and chemical potential. Among the species of this genus, Piper aduncum, a shrub found in the Brazilian Amazon and popularly known as spiked pepper (Gaia et al. 2010), stands out given its antitumorigenic, anti-inflammatory, antioxidant (Arroyo-Acevedo et al. 2015), and antiviral properties (Radice et al. 2018). ...
Article
An efficient protocol is reported for in vitro plant regeneration through somatic embryogenesis in Piper aduncum, a Brazilian Amazon species with high economic potential. The species is important due to a variety of components found in its essential oil, with emphasis on dillapiole. Leaf explants from five accessions identified for high oil yield and levels of dillapiole were evaluated for their embryogenic potential. To induce embryogenic calli, the explants were cultivated in MS medium supplemented with 5 mg L −1 of 1-naphthaleneacetic acid (NAA) and 2.5 mg L −1 of N6-benzylaminopurine (BAP) for 80 d. For somatic embryo-genesis, the embryogenic calli were transferred to MS medium with 10 mg L −1 of NAA and 2.5 mg L 1 of BAP and incubated for 45 d. The obtained somatic embryos were germinated in MS medium without regulators by 45 d and the obtained plantlets were subjected to acclimatization. Somatic embryos and calli from this process were subjected to anatomical and histochemical analyses. Biochemical analyses (total soluble sugars, starch, total amino acids, and proteins) were also performed to identify markers for embryogenic competence acquisition. In addition, the germination of somatic embryos was evaluated in a semi-solid and liquid system (R.I.T.A.® temporary immersion bioreactors). The obtained plants were evaluated for genetic fidelity using ISSR markers. The present study indicate that the accessions did not differ in embryogenic potential, with a mean percentage of calli with somatic embryos of 82.4%. Anatomical analyses confirmed the occurrence of the embryogenic route and the histo-chemical analyses identified starch grains in somatic embryos at different developmental stages. The biochemical analyses showed high total soluble sugars and total amino acids in embryogenic calli, marks of the acquisition of the embryogenic competence of P. aducum. The R.I.T.A.® temporary immersion bioreactors were highly efficient in the regeneration of somatic plants, with 100% germination. The plants regenerated in the semi-solid and liquid systems showed high genetic homogeneity. The survival rate of the acclimatized plants was 100%.
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Nanoemulsions (NEs) of essential oil (EO) have significant potential to target microorganisms, especially viruses. They act as a vehicle for delivering antiviral drugs and vaccines. Narrowing of drug discovery pipeline and the emergence of new viral diseases, especially, COVID-19 have created a niche to use nanoemulsions (NEs) for augmenting currently available therapeutic options. Published literature demonstrated that EOs have an inherent broad spectrum of activity across bacterial, fungal, and viral pathogens. The emulsification process significantly improved the efficacy of the active ingredients in the EOs. This article highlights the research findings and patent developments in the last two years especially, in EO antiviral activity, antiviral drug delivery, vaccine delivery, viral resistance development, and repurposing EO compounds against SARS-CoV2.
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Abstract In the present study, cytotoxic (apoptotic and necrotic), antioxidant, prooxidant, genotoxic, and apoptosis-related genes expression modulatory effects of physodic acid (PA) isolated from Pseudevernia furfuracea on cancerous (HepG2) and healthy (THLE2) human liver cells were evaluated. Cytotoxic effects PA obtained from P. furfuracea on HepG2 and THLE2 cells were tested via MTT and LDH analyses. In order to determine genotoxic effect of PA, 8-OH-dG levels in the cells were measured. In addition, apoptotic or necrotic effects of PA on the hepatic cells were determined by qRT-PCR analysis. Cytotoxicity analyses revealed the cytotoxic effect of PA on HepG2 cells, which is much higher than THLE2 cells. Gene expression studies demonstrated the apoptotic effects of PA on HepG2 cells through both extrinsic and intrinsic pathways. PA increased the antioxidant capacity of THLE2 cells with increasing concentrations, which describes its antioxidant power. Additionally, none of the tested levels of PA caused oxidative DNA damage on THLE2 cells, but its high concentration increased genetic damages on HepG2. Taken together, P. furfuracea lichen and its metabolite, PA, would be a promising alternative for complementary treatments against liver cancer. Keywords: 8-hydroxy-2′-deoxyguanosine, lactate dehydrogenase, lichen, total antioxidant capacity, total oxidative stress
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The Flavivirus genus contains important pathogens, such as West Nile virus (WNV), Zika virus (ZIKV), and Dengue virus (DENV), which are enveloped plus-strand RNA viruses transmitted by mosquitoes and constitute a worrisome threat to global human and animal health. Currently no licensed drugs against them are available, being, thus, still necessary the search for effective antiviral molecules. In this line, a novel antiviral approach (economical, simple to use, and environmental friendly) is the use of natural compounds. Consequently, we have tested the antiviral potential of different polyphenols present in plants and natural products, such as wine and tea, against WNV, ZIKV, and DENV. So that, we assayed the effect of a panel of structurally related polyphenols [delphinidin (D), cyanidin (Cy), catechin (C), epicatechin (EC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG)] on WNV infection, and found that D and EGCG inhibited more effectively the virus production. Further analysis with both compounds indicated that they mainly affected the attachment and entry steps of the virus life cycle. Moreover, D and EGCG showed a direct effect on WNV particles exerting a virucidal effect. We showed a similar inhibition of viral production of these compounds on WNV variants that differed on acidic pH requirements for viral fusion, indicating that their antiviral activity against WNV is produced by a virucidal effect rather than by an inhibition of pH-dependent viral fusion. Both polyphenols also reduced the infectivity of ZIKV and DENV. Therefore, D and EGCG impair the infectivity in cell culture of these three medically relevant flaviviruses.
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Essential oil are very important and widely used since early times. The important role and mode of action of these naturally occurring products is discussed with regards to its bioactivity as antibacterial, antiviral, antioxidant and diabetic. Also its important role in chemoprevention and cancer suppression is discussed. The therapeutic properties of essential oils in aroma will be outlined. All these functions and its properties are due to the presence of certain constituents which are discussed in this review along with their chemical structures.
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In this study, we performed the chemical characterization of Myrcia splendens (Sw.) DC. (Myrtaceae) essential oil from Amazonian Ecuador and the assessment of its bioactivity in terms of cytotoxic, antibacterial, and antioxidant activity as starting point for possible applicative uses. M. splendens essential oil, obtained by hydro-distillation, was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID): the major components were found to be trans-nerolidol (67.81%) and α-bisabolol (17.51%). Furthermore, we assessed the cytotoxic activity against MCF-7 (breast), A549 (lung) human tumor cell lines, and HaCaT (human keratinocytes) non-tumor cell line through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test: promising results in terms of selectivity and efficacy against the MCF-7 cell line (IC50 of 5.59 ± 0.13 μg/mL at 48 h) were obtained, mainly due to α-bisabolol. Furthermore, antibacterial activity against Gram positive and negative bacteria were performed through High Performance Thin Layer Chromatography (HPTLC) bioautographic assay and microdilution method: trans-nerolidol and β-cedren-9-one were the main molecules responsible for the low antibacterial effects against human pathogens. Nevertheless, interesting values of Minimum Inhibitory Concentration (MIC) were noticeable against phytopathogen strains. Radical scavenging activity performed by HPTLC bioautographic and spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) approaches were negligible. In conclusion, the essential oil revealed a good potential for plant defense and anti-cancer applications.
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Background: The challenge in antimicrobial chemotherapy is to find safe and selective agents with potency that will not be compromised by previously developed resistance. Terrestrial plants could provide new leads to antibacterial, antifungal, or antiprotozoal activity. Methods: The essential oil (EO) of Piper aduncum L. (Piperaceae) from Cuba was analyzed by gas chromatography—mass spectrometry (GC-MS). A cluster analysis of P. aduncum EO compositions reported in the literature was carried out. The EO was screened against a panel of microorganisms (bacteria, fungi, parasitic protozoa) as well as for cytotoxicity against human cells. In addition, a review of scientific literature and a bibliometric study was also conducted. Results: A total of 90 compounds were identified in the EO, of which camphor (17.1%), viridiflorol (14.5%), and piperitone (23.7%) were the main components. The cluster analysis revealed at least nine different chemotypes. The EO did not show notable activity against bacteria or fungi, but was active against parasitic protozoa. Conclusions: The results from this study indicate P. aduncum from Cuba is a unique chemotype, support the importance of P. aduncum EOs as medicines, and demonstrate the promise of Cuban P. aduncum EO as a chemotherapeutic agent against parasitic protozoal infections.
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Essential oils in leaves and spikes of Piper aduncum L. (Piperaceae) were examined in two Costa Rican locations by Gas Chromatography-Mass-Spectrometry (GC-MS) methods. Yields were 0.4-1.3% and oil composition was slightly different in each location. The main constituents were the phenylpropanoid dilapiole (32.9-61.8%), the monoterpenoids piperitone (2.2-13.5%), 1,8-cineole (0.1-8.6%), and 4-terpineol (1.6-5.4%) and the sesquiterpene β-caryophyllene (4.0-5.3%). Fourty five constituents not previously reported in this species were identified. The characterization of dilapiole was performed mainly on detailed spectroscopic analyses and via chemical transformation.
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Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. In this study the effect of three EOs, clove, oregano and zataria, was evaluated on the infectivity of norovirus surrogates, i.e. feline calicivirus (FCV) and murine norovirus (MNV).Different concentrations of EOs were individually mixed with each virus at titers of ca. 7–8 log TCID50/ml and incubated for 2 h at 4 °C and 37 °C. The infectivity of the recovered viruses after triplicate treatments was evaluated by cell-culture assays. 2% of oregano EO at 37 °C decreased the FCV titers by 3.75 log TCID50/ml, with decreasing effects at lower concentrations, and also decreased MNV titers by 1.04–1.62 log TCID50/ml, with respect to the concentration used. Clove and zataria EO effects on FCV showed similar trends in titer reductions to those obtained with oregano EO, achieving the maximum titer reduction when FCV was treated at 37 °C with 0.1% of zataria EO. These results represent a step forward in the understanding of EOs as antimicrobials and their possible application in the food industry as alternative natural compounds to reduce viral contamination and, therefore enhancing food safety.
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The essential oil of the leaves of six samples of Piper aduncum L. growing wild in two different region of Pernambuco, northeast of Brazil (Brejo da Madre de Deus—samples BMD1—3 and Serra Negra—samples SN1—3), were investigated by a combinations of GC and GC/M S. The characterized oil samples from different localities show a high percentage of oxygen-containing sesquiterpenes in both places (BMD1—3: 90.1–90.8% and SN1—3: 95.6–97.5%). Oxygen-containing monoterpenes represented less than 6% in BMD1—3 and less than 1% in SN1—3. Twelve compounds were identified representing more than 95% of each of the oil samples. (E)-Nerolidol was found to be the main component in all the samples being present in BMD1—three in the range of 81.5 to 82.5% while in SN 1—3 79.2–81.2% were found. Despite the fact that some minor components are not present in each oil sample, this investigation showed the same compositions of the major components of the essential oils of these P. aduncum from different region of Pernanambuco. Longipinanol (BMD1—3: 2.4–5.6%; SN1—3: 11.1–13.6%) and epi-logipinanol (BMD1—3: 2.1–2.2%; SN1—3: 2.2–0.3%) are unusual components present in the samples, which could be identified for the first time in the P. aduncum species.
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Natural essential oil constituents play an important role in cancer prevention and treatment. Essential oil constituents from aromatic herbs and dietary plants include monoterpenes, sesquiterpenes, oxygenated monoterpenes, oxygenated sesquiterpenes, phenolics and others. Various mechanisms like antioxidant, antimutagenic, anti-proliferative, enhancement of immune functions and surveillance, enzyme induction and enhancing detoxification, modulation of multi-drug resistance and synergistic mechanism of volatile constituents are responsible for their chemo preventive properties. This review covers the most recent literature to summarize structural categories and molecular anticancer mechanisms of constituents from aromatic herbs and dietary plants.
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Food-borne viruses such as human Noroviruses (NoVs), hepatitis A virus (HAV), Rotaviruses (RoVs) are a public health concern worldwide. Biochemical substances, which occur naturally in plants, animals or microorganisms, might possess considerable antimicrobial properties. In this study, the reported effects of biochemical substances on food-borne viruses are reviewed. The biochemical substances are grouped into several categories including (i) polyphenols and proanthocyanins, (ii) saponin, (iii) polysaccharides, (iv) organic acids, (v) proteins and polypeptides, (vi) essential oils. Although not fully understood, the mechanism of action for the antiviral activity of the natural compounds is presented. Generally, it is thought to be the prevention of the viral attachment to host cells, either by causing damage on the viral capsids or change of the receptors on the cell membranes. It is recommended that further studies are undertaken not only on the wide-range screening for novel antiviral substances, but also on the mechanism in-depth as well as the exploration for their potential application in controlling virus contamination in foods or food processing.
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In 2011, from 26 September to 16 October, a small outbreak of West Nile virus (WNV) disease occurred on the island of Sardinia (Italy). According to the national case definition, six cases with acute neurological disease were confirmed in hospitalized patients, and four of them died; one of these was only 34years old. In two case, WNV RNA was detected in urine, suggesting renal involvement. Sequence analysis showed lineage 1 and 2 circulation. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.
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The essential oil extracted by steam distillation from the leaves of wild Ocotea quixos (Lauraceae) was analysed by GC and GC-MS. Sixty-one compounds were identified, representing 93.6% of the total detected. The main components were P-caryophyllene (15.1%), cinnamyl acetate (11.4%), sabinene (7.6%), geranial (5.6%) and trans-cinnamaldehyde (5.1%). Remarkable differences were noted with respect to the essential oil previously obtained from the flower calices of the same plant. Copyright (c) 2006 John Wiley & Sons, Ltd.
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The essential oil of Ishpingo (Ocotea quixos, Lauraceae) fruit calices was analysed by GC (gas chromatography) and GC–MS (gas chromatography–mass spectrometry). Fourty-four compounds were identified. The main components detected were trans-cinnamaldehyde (27.9%), methylcinnamate (21.6%), 1,8-cineole (8.0%), benzaldehyde (3.6%), and β-selinene (2.1%). In vitro antioxidant properties of the essential oil, obtained by DPPH (1,1-diphenyl-2-picrylhydrazyl) and β-carotene bleaching assays, were also evaluated. The oil exerted a relatively good capacity to act as a non-specific donor of hydrogen atoms or electrons when checked by the diphenylpicrylhydrazyl assay, quenching 52% of the radical. On the other hand, it showed weak effects in inhibiting oxidation of linoleic acid when assayed by the β-carotene bleaching test. Antibacterial activity of the essential oil was also checked against gram positive (Enterococcus foecalis, Staphylococcus aureus) and gram negative strains (Escherichia coli, Pseudomonas aeruginosa). The oil also showed a dose-dependent antifungal activity against Candida albicans, Saccharomyces cerevisiae, phytopathogen Pythium ultimum and dermatophyte Trichophyton mentagrophytes.
Article
Setting conservation priorities with a geographic vision is essential in developing countries due to limited resources for conservation and the urgency of establishing new reserves. Priorities should reflect the risk ecosystems are facing and how represented they are in current national reserve networks (NRN). Conservation planning, research, and reserve design should focus on priority ecosystems. This study identified the priority ecosystems for the conservation of continental Ecuador’s biodiversity using a multi-criteria model developed in collaboration with Ecuador’s Ministry of the Environment. This model ranked ecosystems based on four conditions: representativeness in the current reserve network, human pressure, habitat loss, and species-level value based on bird species data. Results show that although Ecuador’s reserve network covers approximately 14% of the country, several ecosystems are poorly represented using a conservative 10% level benchmark, while others are not included at all. In general, highly diverse, humid environments are well represented while low-diversity, dry environments, are poorly represented. These low-diversity, low-representation ecosystems are also the most threatened. Conservation programs and new reserves design in continental Ecuador should concentrate on these ecosystems until the desired representativeness is attained. These results were formally presented to Ecuador’s Ministry of the Environment in 2000. This study shows that an ecosystem-risk-representativeness approach can be implemented efficiently at scales that take advantage of limited resources yet are effective for policy and planning. Current technologies can be used effectively to map major ecosystems, and to assess various forms of risk and representativeness in reasonable time for a country or a large region.
Article
Essential oils from aerial parts of Piper aduncum (Matico) and Piper obliquum (Anis del Oriente) of ecuadorian origin were analyzed by GC-FID, GC-MS, (13)C NMR and their biological and pharmacological activities were assessed. Chemical composition proved to be unusually different from previous reports for safrole-rich P. obliquum (45.8%), while P. aduncum main constituent was dillapiol (45.9%). No genotoxic activity was found in the Ames/Salmonella typhimurium (TA98 and TA100) assay, either with or without S9 activation. Mutagen-protective properties, evaluated using sodium azide, 2-nitrofluorene and 2-aminoanthracene as mutagens/promutagens, was observed against promutagen 2-aminoanthracene, likely in consequence of microsomial deactivation. Antimicrobial assays have been performed on Gram+/Gram- bacteria, dermatophyte and phytopathogenic fungi and best results were provided by P. aduncum against fungal strains with complete inhibition at 500μg/ml. Preliminary analgesic and antithrombotic activities evidenced the absence of the former in hot plate and edema assays and a limited antiplatelet action against three different agonists (ADP, AA and U46619). Both oils have a very limited antioxidant capacity.
Article
The essential oils of the specimens of Piper aduncum that occur in deforested areas of Brazilian Amazon, North Brazil, are rich in dillapiole (35-90%), a derivative of phenylpropene, to which are attributed biological properties. On the other hand, the oils of the specimens with occurrence in the Atlantic Forest, and Northeastern and Southeastern Brazil, do not contain dillapiole, but only terpene compounds such as (E)-nerolidol and linalool. One specimen existing in the Amazon was hydrodistilled. The obtained oil was fractioned on a silica chromatographic column, resulting in fractions rich in dillapiole (95.0-98.9%) utilized for analyses by GC and GC/MS, structural characterization by NMR, confirmation of their biological properties, and to obtain the isomer isodillapiole. Dillapiole showed a fungicide action against the fungus Clinipellis perniciosa (witches' broom) by inhibition of its basidiospores, in concentrations ranging from 0.6 to 1.0 ppm. The larvicide and insecticide actions of dillapiole were tested against the larvae and the adult insects of Anopheles marajoara and Aedes aegypti (malaria and dengue mosquitoes), resulting in mortality of the larvae (48 h, 100%) at a concentration of 100 ppm, and mortality of the insects (30 min, 100%) at a concentration of 600 ppm. The isomeric isodillapiole showed no significant activity in the same biological tests.
Article
Essential oils are complex natural mixtures, their main constituents, e.g. terpenes and phenylpropanoids, being responsible for their biological properties. Essential oils from eucalyptus, tea tree and thyme and their major monoterpene compounds alpha-terpinene, gamma-terpinene, alpha-pinene, p-cymene, terpinen-4-ol, alpha-terpineol, thymol, citral and 1,8-cineole were examined for their antiviral activity against herpes simplex virus type 1 (HSV-1) in vitro. These essential oils were able to reduce viral infectivity by >96%, the monoterpenes inhibited HSV by about >80%. The mode of antiviral action has been determined, only moderate antiviral effects were revealed by essential oils and monoterpenes when these drugs were added to host cells prior to infection or after entry of HSV into cells. However, both essential oils and monoterpenes exhibited high anti-HSV-1 activity by direct inactivation of free virus particles. All tested drugs interacted in a dose-dependent manner with herpesvirus particles thereby inactivating viral infection. Among the analysed compounds, monoterpene hydrocarbons were slightly superior to monoterpene alcohols in their antiviral activity, alpha-pinene and alpha-terpineol revealed the highest selectivity index. However, mixtures of different monoterpenes present in natural tea tree essential oil revealed a ten-fold higher selectivity index and a lower toxicity than its isolated single monoterpenes.
Article
Among the three South American Lauraceae with cinnamon odours, Ocotea quixos Lam. is distinguished with the richest historical legacy. Cinnamaldehyde, its odoriferous principle, occurs besides o-methoxycinnamaldehyde, cinnamic acid and methyl cinnamate in the fruit calyx. In contradistinction, 1-nitro-2-phenylethane is responsible for the cinnamon odour of bark and leaves of Aniba canelilla (H..B.K.) Mez and Ocotea pretiosa (Nees) Mez.
Article
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Article
Ten methanolic extracts from eight Indonesian medicinal plants were phytochemically screened and evaluated for antiviral (HSV-1 and Poliovirus) and cytotoxic activities on murine and human cancer lines (3LL, L1210, K562, U251, DU145, MCF-7). Besides Melastoma malabathricum (Melastomataceae), the Indonesian Loranthaceae species among which Elytranthe tubaeflora, E. maingayi, E. globosa and Scurrula ferruginea exhibited attractive antiviral and cytotoxic activities. Piper aduncum (Piperaceae) was found active on Poliovirus. S. ferruginea was selected for further studies because of its activity on the U251 glioblastoma cells.
Article
The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.
Article
Essential oils obtained from eight aromatic plants of San Luis Province, Argentina, were screened for virucidal activity against herpes simplex virus type 1 (HSV-1), Junin virus (JUNV) and dengue virus type 2 (DEN-2). The most potent inhibition was observed with the essential oil of Lippia junelliana and Lippia turbinata against JUNV with virucidal concentration 50% (VC(50)) values in the range 14-20 ppm, whereas Aloysia gratissima, Heterotheca latifolia and Tessaria absinthioides inhibited JUNV in the range 52-90 ppm. The virucidal activity was time- and temperature-dependent. The essential oils of A. gratissima, Artemisia douglasiana, Eupatorium patens and T. absinthioides inactivated HSV-1 at 65-125 ppm. However, only A. douglasiana and E. patens had any discernible effect on DEN-2 infectivity with VC(50) values of 60 and 150 ppm, respectively.
Article
Essential oils extracted from different plants (Anthemis nobilis L., Artemisia dracunculus L., Cannabis sativa L., Cupressus sempervirens L., Cymbopogon citratus (DC.) Stapf., Curcuma longa L., Foeniculum vulgare L., Hypericum perforatum L., Hyssopus officinalis L., Mentha spicata L., Monarda didyma L., Ocimum basilicum L., Ocotea quixos Kosterm., Origanum vulgare L., Pinus nigra J.F. Arnold, Pinus silvestris L., Piper crassinervium Kunth., Rosmarinus officinalis L., Salvia officinalis L., Salvia sclarea L., Santolina chamaecyparissus L., Thymus vulgaris L., Zingiber officinaie L.) were screened in guinea pig and rat plasma in order to assess antiplatelet activity and inhibition of clot retraction. The oils were chemically analysed and a relationship between components and ability to affect hemostasis was evidenced. O. quixos, F. vulgaris, and A. dracunculus showed the highest antiplatelet activity against ADP, Arachidonic Acid and the Thromboxane A2 agonist U46619 (IC50, 4-132 microg ml(-1)), and a good ability to destabilize clot retraction (IC50, 19-180 microg ml(-1)). For these oils a significant correlation between antiplatelet potency and phenylpropanoids content (54-86%) was evidenced thus suggesting a key role for this moiety in the prevention of clot formation. These findings provide the rationale to take in account the antiplatelet activity in the pharmacological screening of natural products containing phenylpropanoids.
Article
Ocotea quixos essential oil was shown to possess significant inhibitory activity of platelet aggregation and clot retraction in rodent plasma. This study is aimed at fully characterizing the antiplatelet activity of the whole essential oil and its main components trans-cinnamaldehyde and methyl cinnamate also in human plasma, at investigating the mechanism underlying such activity and at evaluating the potential antithrombotic activity of subacute treatment of mice with Ocotea essential oil. In vitro Ocotea essential oil and trans-cinnamaldehyde inhibited arachidonic acid-, U46619-, ADP-, phorbol12-myristate13-alcetate-, collagen-induced platelet aggregation and thrombin-induced clot retraction in human and rodent plasma; Ocotea oil and trans-cinnamaldehyde competitively antagonized contractions induced by thromboxane A2 receptor agonist U46619 in rat isolated aortic ring (K(B) = 18 and 3.2 microg ml(-1), respectively). In vivo Ocotea oil, orally administered in a subacute treatment (30-100 mg kg(-1) day(-1) for 5 days) to mice, prevented acute thrombosis induced by collagen-epinephrine intravenous injection. This antithrombotic activity was not accompanied by pro-haemorragic side effect, as detected by the inactivity in bleeding test, thus showing a favourable safety profile compared to the conventional antiplatelet agent, acetylsalicylic acid. Present findings indicate that Ocotea essential oil possesses potent and safe antithrombotic activity attributable to its antiplatelet and vasorelaxant effects. The main constituent trans-cinnamaldehyde seems to be the primary responsible for this activity through a putative mechanism involving the inhibition of thromboxane A2 receptors.
Article
This review is an updated and expanded version of two prior reviews that were published in this journal in 1997 and 2003. In the case of all approved agents the time frame has been extended to include the 251/2 years from 01/1981 to 06/2006 for all diseases worldwide and from 1950 (earliest so far identified) to 06/2006 for all approved antitumor drugs worldwide. We have continued to utilize our secondary subdivision of a "natural product mimic" or "NM" to join the original primary divisions. From the data presented, the utility of natural products as sources of novel structures, but not necessarily the final drug entity, is still alive and well. Thus, in the area of cancer, over the time frame from around the 1940s to date, of the 155 small molecules, 73% are other than "S" (synthetic), with 47% actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the antiinfective area being dependent on natural products and their structures. Although combinatorial chemistry techniques have succeeded as methods of optimizing structures and have, in fact, been used in the optimization of many recently approved agents, we are able to identify only one de novo combinatorial compound approved as a drug in this 25 plus year time frame. We wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore we consider that this area of natural product research should be expanded significantly.
Article
The activity of Eucalyptus globulus essential oil was determined for 120 isolates of Streptococcus pyogenes, 20 isolates of S. pneumoniae, 40 isolates of S. agalactiae, 20 isolates of Staphylococcus aureus, 40 isolates of Haemophilus influenzae, 30 isolates of H. parainfluenzae, 10 isolates of Klebsiella pneumoniae, 10 isolates of Stenotrophomonas maltophilia and two viruses, a strain of adenovirus and a strain of mumps virus, all obtained from clinical specimens of patients with respiratory tract infections. The cytotoxicity was evaluated on VERO cells by the MTT test. The antibacterial activity was evaluated by the Kirby Bauer paper method, minimum inhibitory concentration, and minimum bactericidal concentration. H. influenzae, parainfluenzae, and S. maltophilia were the most susceptible, followed by S. pneumoniae. The antiviral activity, assessed by means of virus yield experiments titered by the end-point dilution method for adenovirus, and by plaque reduction assay for mumps virus, disclosed only a mild activity on mumps virus.
Constituents of the essential oil of Piper aduncum L. growing wild in the Amazon region
  • Jgs Maia
  • Mdgb Zohhbi
  • Eha Andrade
  • A S Santos
  • Mhl Da Silva
  • Air Luz
  • C N Bastos
Maia JGS, Zohhbi MDGB, Andrade EHA, Santos AS, da Silva MHL, Luz AIR, Bastos CN. 1998. Constituents of the essential oil of Piper aduncum L. growing wild in the Amazon region. Flavour Fragr J. 13(4): 269-272.
Megadiversity: Earth’s biologically wealthiest nations
  • R A Mittermeier
  • C G Mittermeier
  • P R Gil
Mittermeier RA, Mittermeier CG, Gil PR. 1999. Megadiversity: Earth's biologically wealthiest nations. Chelonian Conserv Biol. 3: 537-537.