Marcos J. Nakamura

Essential Oils From Myrtaceae Species of the Brazilian Southeastern Maritime Forest (Restinga)

Abstract The leaf oils from five Myrtaceae species growing in the restinga forest of Carapebus, on the southeastern Brazilian coast, were obtained by hydrodistillation and analyzed by GC/MS. Eugenia punicifolia oil contained only sesquiterpenes, with predominance of β-elemene (22.1%) followed by β-caryophyllene (8.5%) and components of the selinane (24.8%) and cadinane (14.0%) skeletal-types. Eugenia sulcata oil had the highest content of monoterpenes, among which α-pinene (12.2%), β-pinene (10.8%) and 1,8-cineole (19.6%) predominated. This profile was completed with 23.5% of caryophyllane and humulane derivatives. α-Pinene also appeared significantly (18.8%) in Eugenia rotundifolia, followed by small amounts of α-terpineol (4.4), while limonene (4.7%) and 1,8-cineole (6.8%) were the main monoterpenes in Myrciaria floribunda. The chemical profile of Neomitranthes obscura was also dominated by selinane-type sesquiterpenes: α-cadinene (23.4%), selina-3,7(11)-diene (13.9%), β-selinene (8.5%) and α-selinene (5.5%), followed by traces of pinenes, limonene and 1,8-cineole (2.4%). Total cyclic sesquiterpene alcohols, esters and epoxides decreased from 55.5% in E. rotundifolia to 8.6% in M. floribunda. However, the latter species contained 26.9% of (E)-nerolidol in the oil composition. Traces of ketones (up to 3.1%) are present in E. punicifolia, N. obscura and M. floribunda.

Aplicação de cromatografia centrífuga de contra-corrente na purificação de ácido ursólico das folhas de Eugenia brasiliensis Lam.

Triterpene acids are common metabolites in the Myrtaceae family, especially in the genus Eugenia. Ursolic acid was found in Eugenia brasiliensis collected in Southeastern Brazil. A previous solvent partition of the ethanol or chloroform extracts of the leavesof E. brasiliensis, followed by rapid high-speed counter-current chromatography (HSCCC) afforded ursolic acid in high purity (> 97%). This compound was also purified apart by conventional column chromatography (yield of 0.22% from the ethanolic extract) and characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique, usually applied to triterpene glycosides, was here applied successfully to an aglycone, to which examples are rarely described. The mobile and stationary phase for the HSCCC experiment were derived from the two-phase solvent system composed by n-hexane : ethyl acetate : methanol : water in the proportion of 10:5:2.5:1. The choice of the developing solvent system for optimum HSCCC separation was determined by TLC coupled to densitometric measurements of ursolic acid in both stationary and mobile phase, generated by the upper and lower layer of the system above. Commercial ursolic acid was used as standard.

Chemical composition of South American Burseraceae non-volatile oleoresins and preliminary solubility assessment of their commercial blend.

INTRODUCTION Non-volatile oleoresins from neotropical Burseraceae are traditionally used for craft, technological and medicinal purposes. The crude resin is usually sold in popular markets of the forest communities. Adding value to this rainforest raw material requires establishing its composition. OBJECTIVE To analyse the resin composition from different Burseraceae species and establish a minimally reproducible profile by gas chromatography, in order to parameterise its quality control. METHODOLOGY Crude oleoresin samples of 10 Protium and Trattinnickia species and a commercial blend were subjected to hydrodistillation to remove volatile compounds. The chloroform-soluble residues were methylated, analysed by GC-FID (flame ionisation detection), and individual components were identified by analysing their mass fragmentation pattern in GC-MS and comparison with data from the literature. The blend solubility was assayed in 30 non-chlorinated solvents at three different proportions. RESULTS The resins consisted exclusively of triterpenes, showing a common predominance of four major compounds in all the samples, corresponding to α-amyrin, β-amyrin, α-amyrenone and β-amyrenone. This profile was complemented with minor amounts of the tetracyclic β-elemolic and α-elemolic acids, maniladiol, brein and other oxidised trace compounds. The better solvents for the resin were those chemically bearing more than four carbon atoms, as n-butyl acetate, 2-ethoxyethanol and isopropanol. The crude resin blend sold contained around 10% of insoluble material that was constituted by up to 70% inorganic residues mixed with humic acid derivatives, as attested by ash analysis and IR spectroscopy, respectively. CONCLUSION The experimental results, complemented by a general inspection of the literature, demonstrated a systematically reproducible triterpene profile in Protium and Trattinnickia species.

Chemical composition and anti-inflammatory activity of the hydrodistillate from Mariscus pedunculatus

Os rizomas de Mariscus pedunculatus, quando submetidos a hidrodestilacao, forneceram uma mistura do diidrobenzofurano remirol e do benzopirano iso-evodionol, na proporcao de 5.7:1. Estas substâncias foram separadas e purificadas por intermedio de sucessivas cromatografias de camada delgada preparativa. As estruturas foram confirmadas por metodos espectroscopicos, incluindo tecnicas bidimensionais de RMN, e pelo produto de hidrogenacao, no caso do remirol. Entre as substâncias volateis, foram identificados tracos de outros oito sesquiterpenos por CG-EM associada aos calculos de indice de retencao. O hidrodestilado bruto, assim como as substâncias purificadas foram testadas quanto a atividade antiinflamatoria, no modelo de pleurisia induzida em camundongos por lipopolissacarideo (LPS, 12,5 ng/cavidade). Tanto o extrato bruto quanto o remirol puro (100 e 200 mg kg-1, via oral) demonstraram uma capacidade dose-dependente de inibir o acumulo de neutrofilos e eosinofilos na cavidade pleural dos animais. O iso-evodionol nao apresentou efeito antiinflamatorio.

Selective and cost effective protocol to separate bioactive triterpene acids from plant matrices using alkalinized ethanol: Application to leaves of Myrtaceae species

Background: Triterpenes as betulinic (BA), oleanolic (OA) and ursolic acids (UA) have increasingly gained therapeutic relevance due to their wide scope of pharmacological activities. To fit large-scale demands, exploitable sources of these compounds have to be found and simple, cost-effective methods to extract them developed. Leaf material represents the best plant sustainable raw material. To obtain triterpene acid-rich extracts from leaves of Eugenia, Psidium and Syzygium species (Myrtaceae) by directly treating the dry plant material with alkalinized hydrated ethanol. This procedure was adapted from earlier methods to effect depolymerization of the leaf cutin. Materials and Methods: Extracts were prepared by shaking the milled dry leaves in freshly prepared 2% NaOH in 95% EtOH solution (1:4 w/v) at room temperature for 6 h. Working up the product in acidic aqueous medium led to clear precipitates in which BA, OA and UA were quantified by gas chromatography. Results: Pigment-free and low-polyphenol content extracts (1.2–2.8%) containing 6–50% of total triterpene acids were obtained for the six species assayed. UA (7–20%) predominated in most extracts, but BA preponderated in Eugenia florida (39%). Carried out in parallel, n-hexane defatted leaves led to up to 9% enhancement of total acids in the extracts. The hydroalcoholate treatment of Myrtaceae species dry leaves proved to be a cost-effective and environmentally friendly method to obtain triterpene acids, providing them be resistant to alkaline medium. These combined techniques might be applicable to other plant species and tissues.

Efficiency and selectivity of triterpene acid extraction from decoctions and tinctures prepared from apple peels

Background: This study assessed the extraction efficiency of ursolic (UA) and oleanolic acids (OA), as well as the total phenols in aqueous and hydroethanolic extracts of dry apple peels at room temperature. Materials and Methods: After running preliminary assays on decoctions and tinctures (ethanol: water 7:3 v/v), the extracts from dried apple (cv. Fuji) peels were obtained by static maceration over varied intervals (2 to 180 days). The UA and OA content in the extracts was quantified by High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) with a reversed phase column and isocratic elution (CH3CN/H2O/H3PO4) against calibration curves (R2 > 0.9995). The total phenol content in the extracts was evaluated spectrophotometrically at 760 nm using the Folin-Ciocalteau method referencing gallic acid. Results: UA and OA in the hydroethanolic extracts ranged from 3.63-6.12 mg/g and 2.12-3.30 mg/g, corresponding to 1.72-3.07 and 1.00-1.66 mg/g in the raw material, respectively. Higher values of triterpene acid content corresponded to maceration periods of 10 or 30 days. The residual phenol and polyphenol content ranged from 6.97 to 11.6 mg/g. The UA and OA yields, as well as the total phenol content, versus the maceration time were plotted in Control Charts within confidence intervals (95%) and were unaffected during the assayed period. Conclusion: Apple peel tinctures from 10% solids obtained at room temperature exhibited the highest content of triterpene acids when employing a maceration period of 10 to 30 days. Extracts prepared using this procedure contained an average of 7.33 mg/g of total triterpene acids and 10.6 mg/g phenolic compounds. These results establish supporting data for apple peel tinctures and their derived phytopharmaceuticals that are standardized on the ursolic-oleanolic acid content.

Efficient and selective method to separate triterpene acids by direct treatment of apple peels with alkaline ethanol

ABSTRACT Triterpene acid extraction from dried apple peels of Fuji and Gala varieties in 2%NaOH/ethanol at room temperature, followed by filtration and acidification (pH 3), was investigated, comparing to a classical exhaustive extraction (Soxhlet, 2 h, with ethanol and methanol, >97% recovery). Alkali-ethanol process yielded 4.35–6.86% (w/w) of whitish extracts, up to 15 times less than Soxhlet but with same recovery (P > 0.05) of ursolic and oleanolic acids. Alkaline extraction of previously defatted peels led to same recovery (P > 0.05), but the yield increased slightly (P < 0.01). The cost-effective and environmental-friendly procedure prepares triterpene-rich extracts from apple peels at high recovery, directly eliminating the cutin and avoiding prior treatments with hazardous organic solvents.

Development, Optimization and Validation of a GC Method by Polarity Phase Constants and Statistical Design of Experiments for the Determination of Monoterpenes in Alpinia zerumbet Essential Oil

In this report, a rapid and selective analytical method for the determination of monoterpenes in Alpinia zerumbet essential oil (AZEO) by gas chromatography (GC) flame ionization detection (FID) was developed, optimized and validated. The suitability of six different capillary columns was investigated by polarity phase constants calculated based on the methodology proposed by Rohrschneider and McReynolds. The most suitable column was then used in a central composite design applying the following GC factors to investigate responses based on peak resolutions and analysis time: initial oven temperature, heating rate and carrier gas flow rate. The optimized method has initial oven temperature of 60 C, heating rate of 5.1 C min and flow rate of 1.0 mL min using the DB-35 capillary column. The validation acceptance criteria were met in all cases. The method was successfully applied for the quantification of major monoterpenes found in AZEO in different samples of Alpinia zerumbet.

Copaiba Oil: Chemical Composition and Influence on In-vitro Cutaneous Permeability of Celecoxib

BACKGROUND Use of topical or transdermal administration of Celecoxib (Cxb) is an interesting strategy in cutaneous treatments since it reduces or avoids side effects of the oral route. However, Cxb´s high lipophilicity and the stratum corneum (SC) barrier impair cutaneous penetration. OBJECTIVE Evaluation of copaiba oil (C.O) as a potential skin penetration enhancer (P.E) for Cxb. METHODS The chemical composition of C.O was evaluated by GC-MS. Both in-vitro release and permeability assay of Cxb in Polyethylene glycol 400/ propylene glycol (PEG 400/PG) vehicle associated to C.O (1-50% w/w) were determined in a modified diffusion cell fitted with a synthetic hydrophobic membrane and pig ear skin as model, respectively. RESULTS GC-MS analysis of C.O showed that it is composed of sesquiterpenes (68.65%) and diterpenes (22.26%). Formulations containing 25% C.O (F4) and 50% C.O (F5) have shown in-vitro burst release in the first 2 h, but only F4 released 100% of drug after 24 h. The highest Cxb permeation across skin was obtained from F4 and the highest skin retentions for F4 and F5 in the stratum corneum and epidermis plus dermis. CONCLUSION The increased Cxb permeability through skin and its retention for an extended time (24h) at 25% C.O suggest that it could be a promising adjuvant for the development of transdermal formulations of Cxb.

Leaf Essential Oil from Eugenia luschnathiana and Myrciaria tenella (Myrtaceae) from Two Different Accesses in Southeastern Brazil

Abstract The leaf essential oils from Eugenia luschnathiana Klotzsch ex B.D. Jacks. and Myrciaria tenella (DC.) O. Berg growing in two different locations in southeastern Brazil were obtained by hydrodistillation and analyzed by GC and GC-MS. The oil profiles of individual accesses of E. luschnathiana from each location were characterized by the virtual absence of monoterpenes. The sesquiterpenes were of the caryophyllane (32%), guaiane (27%) and cadinane (19%) types in the São Paulo State access and caryophyllane (43%) and cadinane (28%) types in the Rio de Janeiro access. This is the first report on the composition of E. luschnathiana oil. Monoterpenes were predominant in both accesses of M. tenella (57-65%), and sesquiterpenes were mostly represented by the guaiane-eudesmane (16%) or caryophyllene-guaiane types (17%), respectively, in the São Paulo and Rio de Janeiro accesses. In monoterpene composition, the M. tenella oils qualitatively and quantitatively resembled that from a previous access from southern Brazil, whereas the sesquiterpene accumulation showed no similarity to any previous collection in Brazil regarding any favored skeleton type.