Canadian Journal of MicrobiologyApril 11, ; Accepted transformwtion Apr 25, ; Published date: J Drug Metab Toxicol 7: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, microbial transformation of steroids and sterols pdf reproduction in any medium, provided the original author and source are credited. The aim of the paper is to present microbial transformation reactions as a step in the preparation of drugs or their key intermediates from plant derived compounds. From ancient times to stanozolol and anavar cycle days plants have been subjected to different manipulations to obtain biologically active compounds for medical sterolx.
Advances in microbial steroid biotransformation - ScienceDirect
April 11, ; Accepted date: Apr 25, ; Published date: J Drug Metab Toxicol 7: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The aim of the paper is to present microbial transformation reactions as a step in the preparation of drugs or their key intermediates from plant derived compounds. From ancient times to recent days plants have been subjected to different manipulations to obtain biologically active compounds for medical purposes. Methods applied range from traditional extractions to environmental friendly techniques including extraction with ionic liquids, microwave-assisted and ultrasound-assisted extraction, solid phase extraction and supercritical fluid extraction [ 1 - 3 ].
Additionally, acid or enzymatic hydrolysis may be applied in order to make the active ingredients available for the intended clinical applications [ 4 , 5 ]. The plant derived compounds are also subjected to processes of microbial transformation which are nowadays considered as promising technologies for drug development and improvement [ 6 - 9 ]. The microbial transformations are carried out in mild conditions, proceed with high regio- and stereo-specificity and give rise to derivatives which are either difficult to be prepared by chemical means or not economically reasonable.
In the recent years the interest in microbial transformations of plant derived biologically active compounds affected even the alternative medicine. Some traditional Chinese medicinal herbs and their ingredients were subjected to biotransformations as well [ 10 , 11 ]. Several requirements were further on added to this definition aiming to distinguish the microbial transformation processes from these of bioconversion and biodegradation , i.
Actually, the definition of the microbial transformation process was refined primarily based on studies dealing with microbial transformations of steroid compounds and these processes gained their recent importance due to their successful application in steroid drug manufacturing [ 14 , 15 ]. Microbial transformation reactions of phytosterols include as a first step their side-chain cleavage as well as processes of hydroxylation and dehydrogenation of the steroid ring structure and isomerization of the double bonds.
It is important to highlight that any of the positions of the steroid ring is prone to microbial attack, either bacterial or fungal. In general, bacteria are more active in complete steroid structure degradation while fungi are much more active in multiple steroid hydroxylations. However, despite of the attempts that have been made to find out which microorganisms are best in performing each type of the transformation reaction, no proper correlation between the type of substrate, taxonomic position of the microorganism and the reaction performed has been established.
The same investigation was marked as a starting point in studies on a large group of microbial transformation reactions proceeding with cleavage of carbon-carbon bonds and leading to partial or full splitting of the steroid molecule [ 16 ].
Later on huge amounts of data on diversity of microorganisms revealing steroid transformations activities were collected and made available [ 17 - 25 ]. In the Proceeding of The International Symposium on the History of Steroid Chemistry held in New York City in , representatives of the pharmaceutical companies involved in the early stages of steroid drug manufacturing like Upjohn [ 27 ], Merck [ 28 ], Schering [ 29 ], Searle [ 30 ], Squibb [ 31 ], Syntex [ 32 ] presented their point of view.
As seen from what participants in development of the steroid drug story told firsthand, a lot of knowledge had been accumulated and lot of specialists had been involved as the steroid drug manufacturing to become possible. Among these Nobel Prizes, the one in Medicine and Physiology from was awarded for discoveries regarding the hormones of the adrenal cortex, their structure, and biological effects [ 34 ].
Thus, through discovery of cortisone and its activity against the rheumatoid arthritis, the chemist Edward Kendall, the clinic physician Philip Hench and the professor of pharmaceutical chemistry Tadeusz Reichstein paved the way towards the forthcoming successful steroid drug story.
Of key importance in this story was to find out an available and cheap raw material to serve as precursor in large scale production. The breakthrough was made by Russel Marker, a brilliant chemistry professor conducting research on sapogenins who concentrated his attention on the chemistry of the steroid sapogenin diosgenin , present in certain inedible yams growing wild in Mexico.
His efforts in the area led to the formation in of the Syntex Company to produce progesterone from diosgenin in Mexican yams by a four-step process now known as Marker degradation [ 35 ].
Almost at the same time, in the early s Glaxo started in UK production of cortisone from hecogenin from Agave family plants [ 36 ] while Upjohn concentrated on production of progesterone from sitosterol [ 37 ]. Simultaneously, the enzymatic transformations of steroids evolved as a particularly important new dimension in the steroid research at Upjohn during [ 27 ] where the microbiologist Herbert C. Murrey and the biochemist Durey H. Peterson made together a history-making discovery: This process leads to formation of androstenedione AD and androstadienedione ADD which are key intermediates in steroid drug manufacturing [ 38 , 39 ].
The process has been carried out with immobilized cells [ 40 - 42 ], with micronized substrate [ 43 ], in organic medium [ 44 , 45 ], in the presence of water-miscible solvents [ 46 ], in microemulsions [ 47 ], in liquid polymer medium, e. The introduction of hydroxyl function at C9 unlocks the way to opening the ring B of the steroid structure. Both enzymes normally exist in bacteria and due to their activity steroid ring is cleaved and further degraded.
As in any of the cases both chemical and microbial steps are involved, the decision on which way to choose depends on a large extent on the activity of microorganisms. To underline the importance of the conditions at which microbial transformation processes are performed pH, temperature, aeration etc. Employed are free and immobilised [ 78 - 82 ], growing and resting cells [ 75 - 77 , 83 ], reactions are performed in microchannels [ 84 ] etc.
Reported in the literature are details regarding inducibility of steroid hydroxylases [ 85 ], application of cyclodextrins [ 55 ] and crystalline substrates [ 86 ] in processes aimed at improving their effectiveness. The importance of the oxygen function at 11position is also due to the possibility which it opens for introducing by chemical means of fluorine in the ring B which further enhances anti-inflammatory activity of the obtained steroid [ 12 ]. The introduction of double bond in ring A of hydrocortisone and cortisone creates derivatives with improved anti-inflammatory properties and reduced undesirable side effects.
Arthur Nobile and his team from Schering Corporation discovered that cortisone can be oxidized to prednisone by the bacterium Corynebacterium simplex [ 87 ]. Prednisolone was also synthesized at Schering. Both, prednisone and prenisolone revealed antiarthritic activity and absence of significant associated salt retention [ 29 ].
Leader in studying and development of substituted corticoids was Lederle with its research on oxygenated steroids culminating in the synthesis and therapeutic use of triamcinolone and related compounds [ 89 ]. The merit for the discovery of fluorosteroids belongs to Squibb chemists and a patent was issued in [ 94 ]. As he mentioned, nobody at Squibb really believed that this would be of great interest, since no fluorine-containing drug had ever reached the market, even more fluoroacetate was a highly toxic enzyme inhibitor.
Pregnane skeleton and some steroid drugs which manufacturing involves microbial steps like hydroxylation at C9, C11 and C16, and dehydrogenation at C1-C2. It is important to notice that they are still irreplaceable in medicine despite of being denied and blamed for different reasons. Terpenes have always been in the focus of investigations due to their wide applications in the flavor and fragrance industry, as well as because of their potential for further biotechnological developments as pharmaceutical agents and insecticides [ 95 ].
Terpenes have a variety of roles in mediating antagonistic and beneficial interactions among organisms [ 96 ]. There are a lot of data in the literature describing the ability of specific microorganisms to perform microbial transformations of terpenes like Mucor sp. There are reviews on microbial transformation of monoterpenes [ 99 ] and triterpenes [ - ]. The microbial transformations of terpenoids applied in folk medicine and of interest for pharmacy are also reviewed in the literature like antimalarial ones [,], ent -kaurane diterpenes [ ], Schisandraceae [ ] and oleanane triterpenoids [ ], taxanes [ ], etc.
Here we will discuss microbial transformations of terpenes which afford compounds with improved biological activity. Taxol is a naturally occurring diterpenoid widely applied as a powerful anti-cancer drug. MA was selected due to its ability for formation of hydroxy-derivatives with significantly enhanced characteristics against human tumor cell lines than the respective substrates [ ]. Two taxadienes obtained from sinenxan A by chemical synthesis were transformed by filamentous fungi Cunninghamella echinulata and Aspergillus niger and actinomycete strains Streptomyces griseus and Nocardia purpurea into twenty one derivatives.
When subjected oleanoic acid to transformation with the filamentous fungus Fusarium lini , Choudhary et al. In , Martinez et al. Subjected to transformation by Cunninghamella blakesleeana cycloastragenol gave a metabolite with an interesting triterpenic skeleton derived due to an exceptional transformation involving ring cleavage and methyl group migration [ ].
Steviol is an aglycone of stevioside, the major sweet component isolated from leaves of Stevia rebaudiana Bertoni Bertoni Compositae. The group of de Olivera et al. The transformation of isosteviol by Glomerella cingulata afforded hydroxyisosteviol and resulted in 7- oxoisosteviol when Mortierella elongate was employed.
Importantly, all five hydroxylated metabolites exhibited more potent inhibitory effects on tumor promoters than parent diterpenes [ ]. Importantly, the amounts of the derivative were enough to provide a technical basis for studying its mechanism of action as well as its pharmacological and toxicological effects [ ].
Aspergillus niger and Fusarium moniliforme are found capable of increasing polarity of Isodon and Rabdosia diterpenoids, known as antimicrobial and antitumor compounds, by hydroxylating nonactivated positions. The activity of the polyhydroxylated derivatives is dependent on the number and the position of hydroxyl groups in the molecule [ ]. Ent -8 14 ,primaradienol pimarane-type diterpene was obtained by fungal transformation and showed very promising Ent -8 14 ,primaradienol pimarane-type diterpene was obtained by fungal transformation and showed very promising minimal inhibitory concentration value against the main microorganisms responsible for dental caries [ ].
Diterpenes stemodin, stemodinone, stemarin are isolated from the shrub Stemodia maritime. All three compounds were transformed by Aspergillus niger ATCC and gave three hydroxylated compounds, two known analogues and one novel metabolite. Stemodione was hydroxylated to two known analogues while stemarin gave four new compounds [ ].
When transformed by Phanerochaete chrysosporium three three-hydroxylated products of stemodin and one dihydroxylated product of stemodin were produced [ ]. The transformation with Cunninghamella echinulata resulted in three three-hydroxylated products of stemodin: Rhizopus nigricans gave rise to hydroxy-8,epoxyderivative.
The main products obtained by transformation with Cunninghamella echinulata were identified as mycophenolic acid and its 3-hydroxy derivative. Microbial transformation of the two 8,9-unsaturated lactonic drimane derivatives confertifolin and isodrimenin isolated from the bark of Drymus winteri Forst, Winteraceae , a South American tree commonly found in Chile and Argentina with Mucor plumbeus , Aspergillus niger and Rhizopus arrhizus has been reported.
In the case of incubation of isodrimenin with R. Such regio- and stereoselectively functionalized compounds are of interest because they often correspond to minor natural products usually isolated in very small amounts [ ]. Limonoids, chemically classified as tetranortriterpenoids, have been found to possess anti-cancer, anti-malarial, anti-HIV , antimicrobial and several other pharmacological activities. The maslinic acid was transformed by Cunninghamella blakesleeana.
Licorice Glycyrrhiza grabra has well known pharmacological properties [ ]. When transformed by Cunninghamella blakesleeana , its active component, the glycyrrhetinic acid, affords six metabolites, two of them being major ones and revealing considerable activities against the drug-resistant Enterococcus faecalis [ ].
Betulin lupane-type triterpene obtained from the bark extract of white birch, Betula platyphylla Sukatshev var. All compounds betulin, betulonic acid and their metabolites showed potent inhibitory effects on tumor promotion. Biotransformatio n products that underwent ring-opening and hydroxylation exhibited more potent activity than their corresponding precursors [ ]. Betulin was transformed also by Cunninghamella blakesleeana cells with formation of at least five products among which betulinic acid was the most important one due to its antiretroviral, antimalarial and anti-inflammatory properties.
Recently, betulinic acid was described as potential anticancer agent as well. This transformation reaction provides an attractive alternative approach to chemical synthesis, because is less time-consuming and more environmentally friendly [ ]. Gentiopicroside is a principal bitter substance found in many gentianaceous plants which are widely used as medicinal herbs in China and Europe due to the variety of pharmacological activities they exhibit.
When transformed by the endophytic fungus Penicillium crustosum it gave several metabolites, three of them showing potent protective effects against HL cell injury induced by hydrogen peroxide in the in vitro bioassay, while the substrate exhibited no activity at the tested concentrations [ ]. It has been included in clinical trials as mono therapy and in combination with other cytotoxic drugs. Alkaloids represent a diverse group of plant natural products with variable chemical structure.
They are used for centuries because of the wide variety of their physiological effects [ ]. The interactions of microorganisms with alkaloids are of special interest. The data on microbial transformations of alkaloids accumulated up to were reviewed by Abraham and Spassov [ ] while at the same time Rathborne and Bruce emphasized in their review paper on the engineering biocatalytic routes for production of semisynthetic opiate drugs [ ].
Demethylations, oxidations and reductions of morphine alkaloids were performed with different fungal strains, Cunninghamella echinulata being the most effective one [ ]. Rhizobium radiobacter was reported to hydroxylate codeine to its C derivative. This transformation reaction is of importance for the production of drugs displaying analgesic, antitussive and narcotic antagonist characteristics like oxycodone [ ].