A Comparative Study on Chemical Compositions and Biological Activities of Four Amazonian Ecuador Essential Oils: Curcuma longa L. (Zingiberaceae), Cymbopogon citratus (DC.) Stapf, (Poaceae), Ocimum campechianum Mill. (Lamiaceae), and Zingiber officinale Roscoe (Zingiberaceae).

Essential oils (EOs) and their vapour phase of Curcuma longa (Zingiberaceae), Cymbopogon citratus (Poaceae), Ocimum campechianum (Lamiaceae), and Zingiber officinale (Zingiberaceae) of cultivated plants grown in an Amazonian Ecuador area were chemically characterised by Gas Chromatography-Flame Ionization Detector (GC-FID), Gas Chromatography-Mass Spectrometry (GC-MS), and Head Space-Gas Chromatograph-Flame Ionization Detector-Mass Spectrometry (HS-GC-FID-MS).figure The EOs analyses led to the identification of 25 compounds for C. longa (99.46% of the total; ar-turmerone: 23.35%), 18 compounds for C. citratus (99.59% of the total; geraniol: 39.43%), 19 compounds for O. campechianum (96.24% of the total; eugenol: 50.97%), and 28 for Z. officinale (98.04% of the total; α-Zingiberene: 15.45%). The Head Space fractions (HS) revealed C. longa mainly characterised by limonene and 1,8-cineole (37.35%) and α-phellandrene (32.33%); Z. officinale and C. citratus showed camphene (50.39%) and cis-Isocitral (15.27%) as the most abundant compounds, respectively. O. campechianum EO revealed a higher amount of sesquiterpenes (10.08%), mainly characterised by E-caryophyllene (4.95%), but monoterpene fraction remained the most abundant (89.94%). The EOs were tested for antioxidant, antimicrobial, and mutagen-protective properties and compared to the Thymus vulgaris EO as a positive reference. O. campechianum EO was the most effective in all the bioactivities checked. Similar results emerged from assaying the bioactivity of the vapour phase of O. campechianum EO. The antioxidant and antimicrobial activity evaluation of O. campechianum EO were repeated through HP-TLC bioautography assay, pointing out eugenol as the lead compound for bioactivity. The mutagen-protective evaluation checked through Ames's test properly modified evidenced a better capacity of O. campechianum EO compared with the other EOs, reducing the induced mutagenicity at 0.1 mg/plate. However, even with differences in efficacy, the overall results suggest important perspectives for the functional use of the four studied EOs.

Biological activity of new bioactive steroids deriving from biotransformation of cortisone.

Cortisone is a metabolite belonging to the corticosteroid class that is used pharmaceutically directly as a drug or prodrug. In addition to its large consumption, its use is linked to several side effects, so pharmaceutical research aims to develop effective drugs with low or no side effects, alternative compounds to cortisone are part of an active investment in ongoing research on drug discovery. Since biotransformation can be considered a source of new molecules with potential therapeutic use, the present work focuses on a preliminary in vitro study aimed at evaluating the mutagenic, anti-inflammatory, antioxidant and neuroprotective activity of SCA and SCB molecules obtained from the biotransformation of cortisone using Rh. Rhodnii strain DSM 43960. The results obtained are very encouraging due to the safety of biotransformed compounds with reference to genotoxicity checked by Ames test, to the very high antioxidant capacity and to the anti-inflammatory activity. In fact, thecompounds inhibited both the TNFα-stimulated expression and secretion of NFkB target cytokines, and COX activity, and can activate the glucocorticoid receptor. Finally SCA and SCB exhibited neuroprotective properties.

Biotransformation of Waste Bile Acids: A New Possible Sustainable Approach to Anti-Fungal Molecules for Crop Plant Bioprotection?

Phytopathogenic fungi are among the main causes of productivity losses in agriculture. To date, synthetic chemical pesticides, such as hydroxyanilides, anilinopyrimidines and azole derivatives, represent the main treatment tools for crop plant defence. However, the large and uncontrolled use of these substances has evidenced several side effects, namely the resistance to treatments, environmental damage and human health risks. The general trend is to replace chemicals with natural molecules in order to reduce these side effects. Moreover, the valorisation of agri-food industry by-products through biotransformation processes represents a sustainable alternative to chemical synthesis in several sectors. This research is aimed at comparing the anti-phytopathogenic activity of waste bovine and porcine bile with secosteroids obtained by biotransformation of bile acids with Rhodococcus strains. The ultimate goal is to apply these natural products on food crops affected by phytopathogenic fungi.

Wild Italian Hyssopus officinalis subsp. aristatus (Godr.) Nyman: From Morphological and Phytochemical Evidences to Biological Activities.

Three specimens of H. officinalis subsp. aristatus were collected in three areas of the Abruzzo region (Italy) and subjected to macroscopic and microscopic observation to support their botanical identification. The essential oils (EOs) obtained from the aerial parts of the samples were characterized with the object to define their phytochemical and pharmaceutical biology profile. They highlight three different chemotypes, including one never seen in previous literature (CIV17-EO, distilled from sample harvested in 2017 at Civitaretenga), that showed a fingerprinting with the predominance of (-)-limonen-10-yl-acetate (67.9%). In 2017 European Food Safety Authority (EFSA) reported the genotoxicity of similar compounds, therefore, to dismiss any safety concern for the CIV17-EO use as flavouring substance, the Ames test was performed with no evidence of mutagenic activity. Safety of use coupled with chemical characterization of this new chemotype set the stage for a better standardization of H. officinalis EOs. The ethanolic extracts, on the other hand, with qualitatively similar chemical profiles in which caftaric, chlorogenic and rosmarinic acid were the main molecules, showed interesting antioxidant activity and a slight cytotoxicity towards the A549 cell line that could indicate a starting point for the evaluation of an additional preventive tool for maintaining health status.

Ocimum campechianum Mill. from Amazonian Ecuador: Chemical Composition and Biological Activities of Extracts and Their Main Constituents (Eugenol and Rosmarinic Acid).

The essential oil (EO), the methanolic (MeOH), and the 70% ethanolic (70% EtOH) extracts obtained from the aerial parts of Ocimum campechianum Mill. (Ecuador) were chemically characterized through gas-chromatography coupled to mass spectrometry detector (GC-MS), high-performance liquid chromatography coupled to diode array-mass spectrometry detectors (HPLC-DAD-MS) and studied for their in vitro biological activity. The radical scavenger activity, performed by spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, highlighted significant IC50 values for the EO, extracts and their main constituents (eugenol and rosmarinic acid). EO (and eugenol) showed noteworthy activity against Pseudomonas syringae pv. syringae and a moderate effect against clinical Candida strains, with possible synergism in association to fluconazole against the latter microorganisms. The extracts and pure molecules exhibited weak cytotoxic activity against the HaCat cell line and no mutagenicity against Salmonella typhimurium TA98 and TA100 strains, giving indication of safety. Instead, EO showed a weak activity against adenocarcinomic human alveolar basal epithelial cells (A549). The above-mentioned evidence leads us to suggest a potential use of the crude drug, extracts, and EO in cosmetic formulation and food supplements as antioxidant agents. In addition, EO may also have a possible application in plant protection and anti-Candida formulations.

Chemical Composition and Biological Activity of Five Essential Oils from the Ecuadorian Amazon Rain Forest.

The chemical composition and biological activity of essential oils isolated from the leaves of Siparuna aspera, Siparuna macrotepala, Piper leticianum, Piper augustum and the rhizome of Hedychium coronarium were evaluated. These species are used medicinally in different ways by the Amazonian communities that live near the Kutukú mountain range. Chemical studies revealed that the main components for the two Siparuna species were germacrene D, bicyclogermacrene, α-pinene, δ-cadinene, δ-elemene, α-copaene and ß-caryophyllene; for the two Piper species ß-caryophyllene, germacrene D, α-(E,E)-farnesene, ß-elemene, bicyclogermacrene, δ-cadinene and for H. coronarium 1,8-cineole, ß-pinene, α-pinene and α-terpineol. The antioxidant activity of all essential oils was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), photochemiluminescence (PCL) quantitative assays, and DPPH and ABTS bioautographic profiles, with different results for each of them. Antimicrobial activity studies were carried out on three yeasts, six Gram positive and four Gram negative bacteria, by means of the disc diffusion method. The essential oil of H. coronarium showed the most relevant results on L. grayi, K. oxytoca and S. mutans, P. augustum and P. leticianum on S. mutans. An antibacterial bioautographic test for H. coronarium was also carried out and highlighted the potential activity of terpinen-4-ol and 1,8-cineole.

Rediscovering Medicinal Amazonian Aromatic Plants: Piper carpunya (Piperaceae) Essential Oil as Paradigmatic Study.

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 ß-phellandrene (5.6%). In vitro DPPH antioxidant assay showed a weak radical scavenging ability (IC50) 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.

Flavonoids in Ecuadorian Oreocallis grandiflora (Lam.) R. Br.: Perspectives of Use of This Species as a Food Supplement.

Oreocallis grandiflora (Lam.) R. Br. is an Ecuadorian species belonging to the Proteaceae family, commonly known as cucharillo (Loja and Zamora provinces), cucharilla (Sierra region), gañal (Bolívar province), and algil (Chimborazo province). Its leaves and flowers, collected during blooming, are traditionally used for oral administration to treat liver diseases, vaginal bleeding, and ovary/uterus inflammation and as digestive, diuretic, and hypoglycemic remedy. Related literature does not report any scientific evidences regarding the chemical composition of the used parts of this species (leaves and flowers), while few indications are reported about the healthy properties of their preparations. Based on these premises, the present research was performed with the objectives to fill the gaps of the chemical and biological knowledge about this species, enriching the knowledge related to the plant biodiversity of Amazonian Ecuador and to the ethnobotanical tradition of Andean communities. Chemical and biological investigation (in vitro antioxidant and anti-inflammatory activity) of flower and leaf hydroalcoholic extracts shed a light on the functional metabolites putatively involved in healthy properties of the O. grandiflora traditional preparations. The chemical fingerprinting achieved by HPTLC and 1HNMR analyses showed the presence of flavonoids, subsequently quantitatively estimated by AlCl3 complexation assay and HPLC-DAD. Silica gel chromatography allowed the isolation of the main compounds of the flower extract: quercetin 3-O-ß-glucuronide and myricetin 3-O-ß-glucuronide. RP-HPLC-DAD-MS analyses showed the presence of quercetin 3-O-rutinoside and isorhamnetin 3-O-rutinoside, in addition to the above-mentioned molecules, in the leaf extract. Regarding the antioxidant (DPPH test, a radical scavenging assay) and anti-inflammatory (WST-1 assay, an oxidative burst test) activities, leaf extract showed the most promising results when compared to the positive controls. The same extract, however, exhibited a higher cytotoxicity compared to the flower extract, indicating the latter preparation as the most interesting anti-inflammatory crude drug.

Myrcia splendens (Sw.) DC. (syn. M. fallax (Rich.) DC.) (Myrtaceae) Essential Oil from Amazonian Ecuador: A Chemical Characterization and Bioactivity Profile.

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 ß-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.

Nanostructured lipid carriers (NLC) for the delivery of natural molecules with antimicrobial activity: production, characterisation and in vitro studies.

This study describes the preparation, characterisation and in vitro activity of nanostructured lipid carriers (NLCs) encapsulating natural molecules with antimicrobial activity, such as plumbagin, hydroquinon, eugenol, alpha-asarone and alpha-tocopherol. NLCs were prepared by melt and ultrasonication method, characterised by Cryo-TEM for morphology and SdFFF for dimensional distribution and active encapsulation yields. In vitro tests were conducted on bacteria, fungi and human cell cultures. In vitro tests demonstrated that plumbagin is strongly toxic towards F. oxysporum especially when active molecules are loaded on NLC. Plumbagin was completely non toxic on cyanobacterial model strain up to a threshold over which cell viability was completely lost. NLC loaded with active molecules showed a lower toxicity as compared to their free form on human cultured cells. Although further studies need to be performed, these systems can be potentially proposed to control phytopathogenic organisms.

In vitro activity of plant extracts against biofilm-producing food-related bacteria.

The identification of effective antimicrobial agents also active on biofilms is a topic of crucial importance in food and industrial environment. For that purpose methanol extracts of Turkish plants, Ficus carica L., Juglans regia L., Olea europaea L., Punica granatum L. and Rhus coriaria L., were investigated. Among the extracts, P. granatum L. and R. coriaria L. showed the best antibacterial activity with minimum inhibitory concentrations (MIC) of 78-625µg/ml for Listeria monocytogenes and Staphylococcus aureus and 312-1250µg/ml for Escherichia coli and Pseudomonas aeruginosa. SubMICs produced a significant biofilm inhibition equal to 80-60% for L. monocytogenes and 90-80% for S. aureus. The extracts showed also the highest polyphenol content and the strongest antioxidant activity. Bioassay-guided and HPLC procedures demonstrated the presence of apigenin 4'-O-ß-glucoside in P. granatum L. and myricetrin and quercitrin in R. coriaria L. Antigenotoxicity of plant extracts was also observed The present findings promote the value-adding of P. granatum L. and R. coriaria L. leaves as natural antimicrobial/antioxidant agents for control of food-related bacterial biofilms.

Cytotoxic Effect and TLC Bioautography-Guided Approach to Detect Health Properties of Amazonian Hedyosmum sprucei Essential Oil.

Bioautography has been used as rapid and easy strategy to detect and identify bioactive fractions/molecules in the never before investigated Hedyosmum sprucei Solms (Chloranthaceae) essential oil (EO). The antioxidant activity, performed through DPPH bioautographic assay and spectrophotometric evaluations (IC50 = 230 ± 10 µg/mL), seemed to be mainly due to α-cadinol and α-muurolol. (HP)TLC bioautography, focused on antimicrobial capacities, pointed out α-cadinol, α-muurolol, τ-muurolol, caryophyllene oxide, and methyleugenol as the most effective compounds against Staphylococcus aureus, considered as testing strain. Moreover, the microdilution method, assessed among a wide panel of microorganisms, revealed Listeria grayi and Staphylococcus aureus as the most sensitive among human tested strains and Clavibacter michiganensis among phytopathogens. GC-MS chemical profile showed that bioactive molecules represented only a small quantity of the whole EO: germacrene D (23.16%), ß-caryophyllene (15.53%), δ-cadinene (5.50%), α-copaene (5.08%), and α-phellandrene (3.48%) were the main compounds, highlighting an uncommon composition among the genus Hedyosmum. Finally, H. sprucei EO was checked for cytotoxic potential against A549 (lung cancer) and MCF-7 (breast cancer) cell lines showing promising cytotoxic effects against both cell lines after 48 h (IC50 A549 = 44.05 ± 2.35 µg/mL; IC50 MCF-7 = 32.76 ± 4.92 µg/mL) and 72 h (IC50 A549 = 43.55 ± 2.80 µg/mL; IC50 MCF-7 = 33.64 ± 0.43 µg/mL).

Phytotoxic Effects and Phytochemical Fingerprinting of Hydrodistilled Oil, Enriched Fractions, and Isolated Compounds Obtained from Cryptocarya massoy (Oken) Kosterm. Bark.

The hydrodistilled oil of Cryptocarya massoy bark was characterized by GC-FID and GC/MS analyses, allowing the identification of unusual C10 massoia lactone (3, 56.2%), C12 massoia lactone (4, 16.5%), benzyl benzoate (1, 12.7%), C8 massoia lactone (3.4%), δ-decalactone (5, 1.5%), and benzyl salicylate (2, 1.8%) as main constituents. The phytotoxic activities of the oil, three enriched fractions (lactone-rich, ester-rich, and sesquiterpene-rich), and four constituents (compounds 1, 2, 5, and δ-dodecalactone (6)) against Lycopersicon esculentum and Cucumis sativus seeds and seedlings were screened. At a concentration of 1000 µl/l, the essential oil and the massoia lactone-rich fraction caused a complete inhibition of the germination of both seeds, and, when applied on tomato plantlets, they induced an 85 and 100% dieback, respectively. These performances exceeded those of the well-known phytotoxic essential oils of Syzygium aromaticum and Cymbopogon citratus, already used in commercial products for the weed and pest management. The same substances were also evaluated against four phytopathogenic bacteria and ten phytopathogenic fungi, providing EC50 values against the most susceptible strains in the 100-500 µl/l range for the essential oil and in the 10-50 µl/l range for compound 6 and the lactone-rich fraction. The phytotoxic behavior was related mainly to massoia lactones and benzyl esters, while a greater amount of 6 may infer a good activity against some phytopathogenic fungi. Further investigations of these secondary metabolites are warranted, to evaluate their use as natural herbicides.

Biotransformations of terpenes by fungi from Amazonian citrus plants.

The biotransformations of (RS)-linalool (1), (S)-citronellal (2), and sabinene (3) with fungi isolated from the epicarp of fruits of Citrus genus of the Amazonian forest (i.e., C. limon, C. aurantifolia, C. aurantium, and C. paradisiaca) are reported. The more active strains have been characterized, and they belong to the genus Penicillium and Fusarium. Different biotransformation products have been obtained depending on fungi and substrates. (RS)-Linalool (1) afforded the (E)- and (Z)-furanlinalool oxides (7 and 8, resp.; 39 and 37% yield, resp.) with Fusarium sp. (1D2), 6-methylhept-5-en-2-one (4; 49%) with F. fujikuroi, and 1-methyl-1-(4-methypentyl)oxiranemethanol (6; 42%) with F. concentricum. (S)-Citronellal (2) gave (S)-citronellol (12; 36-76%) and (S)-citronellic acid (11; 5-43%) with Fusarium species, while diastereoisomeric p-menthane-3,8-diols 13 and 14 (20 and 50% yield, resp.) were obtained as main products with Penicillium paxilli. Finally, both Fusarium species and P. paxilli biotransformed sabinene (3) to give mainly 4-terpineol (19; 23-56%), and (Z)- and (E)-sabinene hydrates (17 (3-21%) and 18 (11-17%), resp.).

Metabolite profiling of polyphenols in a Terminalia chebula Retzius ayurvedic decoction and evaluation of its chemopreventive activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of Terminalia chebula fruit is an ayurvedic remedy whose prolonged oral administration is prized as a generic intestinal and hepatic detoxifying agent. Its administration is suggested also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. AIM OF THE STUDY: To evaluate the phytochemical profile and the chemopreventive potential of Terminalia chebula fruit decoction prepared according to the ayurvedic decoction recipe. MATERIALS AND METHODS: The quali- and quantitative metabolite profiling of polyphenols was obtained using HPLC-UV/DAD and HPLC-ESI-MS. The crude decoction and purified compounds were tested for their capability to interact with the EphA2-ephrin-A1 system and for their antimutagenic properties against dietary and environmental mutagens (AA, 2-NF, NaN3, and heterocyclic amines IQ, MeIQ, MeIQx, Glu-P1, Glu-P2,) in the Ames-Salmonella/microsome assay, with and without enzymatic induction. RESULTS: The decoction was found to contain 3,4,6-tri-O-galloyl-d-glucose (55.87 mg/g), chebulic acid (54.03 mg/g), ß-punicalagin (41.25mg/g), corilagin (40.31 mg/g), α-punicalagin (35.55 mg/g), chebulagic acid (29.09 mg/g), gallic acid (27.96 mg/g) 1,3,4,6-tri-O-galloyl-ß-d-glucose (24.25mg/g) chebulinic acid (20.23 mg/g), 1,2,3,4,6-penta-O-galloyl-d-glucose (13.53 mg/g), ellagic acid (8.00 mg/g), 1,6-di-O-galloyl-d-glucose (4.16 mg/g). An inhibitory effect was recorded in both Salmonella typhimurium TA98 and TA100 strains against the mutagenic activity of heterocyclic amines (22-61%), promutagen AA (91-97%) and directly acting mutagen 2-NF (52%) with but not against NaN3 (7%). Galloyl derivatives allowed an inhibition of mutagenicity induced by MeIQ above 80% at 0.01 mol/plate. Both decoction and purified compounds were able to modulate the EphA2-ephrinA1 system, suggesting a potential multiple chemopreventive mechanism. CONCLUSIONS: The traditional ayurvedic decoction of Terminalia chebula may harbour a potential as a safe and low-cost chemopreventive agent at the intestinal level, if administered according to the ayurvedic specifications. Moreover, its recourse may enhance the presence of some polyphenolic constituents.

Clotrimazole nanoparticle gel for mucosal administration.

In this study a formulation suitable to be applied on oral and/or vaginal mucosa has been developed for the treatment of fungal infections. The aim of the research is a comparison between clotrimazole (CLO) containing semisolid formulations based on monoolein aqueous dispersion (MAD) or nanostructured lipid carrier (NLC). MAD and NLC have been characterized in terms of morphology and dimensional distribution by cryogenic Transmission Electron Microscopy (cryo-TEM) and Photon Correlation Spectroscopy (PCS). CLO was encapsulated with high entrapment efficiency both in MAD and in NLC, according to Sedimentation Field Flow Fractionation (SdFFF) combined with HPLC. CLO recovery in MAD and NLC has been investigated by time. In order to obtain formulations with suitable viscosity for mucosal application, MAD was diluted with a carbomer gel, while NLC was directly viscosized by the addition of poloxamer 407 in the dispersion. The rheological properties of MAD and NLC after viscosizing have been investigated. Franz cell has been employed to study CLO diffusion from the different vehicles, evidencing diffusion rates from MAD and NLC superimposable to that obtained using Canesten(®). An anticandidal activity study demonstrated that both CLO-MAD and CLO-NLC were more active against Candida albicans with respect to the pure drug.

7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid.

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).