Enzymatic biotransformation of terpenes as bioactive agents

Abstract

The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18β glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18β glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3β-hydroxy sclareol 18α-hydroxy sclareol, 6α,18α-dihydroxy sclareol, 11S,18α-dihydroxy sclareol, and 1β-hydroxy sclareol and 11S,18α-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1β-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases.

Keywords::

• Biotransformation
• biological significance
• bioactive cyclic terpenoid compounds

Introduction

In plants, terpenoids represent a chemical defence against environmental stress and provide a repair mechanism for wounds and injuries. Interestingly, effective ingredients in several plant-derived medicinal extracts are terpenoid compounds of monoterpenes, sesquiterpenoids, diterpenoids and triterpenoid groups. Terpenes have a widespread occurrence and can be found in all organisms, i.e., in prokaryotic as well as eukaryotic systems. However, most of the bioactive terpenes have been detected in higher plants. Whereas mono- and sesquiterpenes are predominantly found in essential oils of plant raw material, the higher terpenes, such as triterpenes, are mainly found in balsams and resins^1–4.

From a biological perspective, it is assumed that the most important triterpenoid structures are the oleanane (2), ursane (1), lupine (3) and cycloartane–euphane (58a-58n) carbon skeletons. The global market for plant-derived drugs has been estimated to be approximately 30.69 billion USD for the last decade, with phytochemicals such as terpenes and steroids representing the most significant fraction with estimated annual sales of 12.4 billion USD⁵. A large number of triterpenoids have been synthesized by structural modification of natural compounds for optimization of bioactivity. An increasing number of triterpenoids have been reported to exhibit cytotoxicity against a variety of cancer cells without manifesting any toxicity in normal cells^6–8. They also demonstrate antitumour efficacy in preclinical animal models of cancer^7,8. Ursolic acid (1) inhibits growth of tumor cells both in vitro and in vivo by decreasing proliferation of cells and inducing apoptosis⁹. It was converted into oleanolic acid methyl ester via two intermediates, oleanolic acid, and ursolic acid methyl ester¹⁰.

Cucurbitacin I¹⁰ has been found to inhibit the proliferation of MDA-MB-468 human breast cancer cells that express constitutively activated STAT3¹¹ and also reduced the levels of phosphotyrosine of constitutively activated STAT3 and induced apoptosis in the same tumour model¹². Cucurbitacin Q induces apoptosis more potently in MDA-MB-435 human breast cancer cells. Cucurbitacin D analog, 2-deoxycucurbitacin D as well as cucurbitacin D and 25-acetylcucurbitacin have exhibited potent cytotoxic activity against MCF-7 human breast cancer cells¹³. Cucurbitacin B, D, E and I, displayed antitumour effects with specific cyclooxygenase-2 (COX-2) enzyme inhibition; however, cucurbitacin B has been found to be the most potent compound¹⁴.

23,24-Dihydrocucurbitacin F and 23,24-dihydro-25-acetylcucurbitacin F have shown moderate cytotoxic activity against MCF-7 cells¹⁵. 23,24-Dihyderocucurbitacin B, possesses anti-cancer activity in a dose- and time-dependent manner against human breast cancer cell lines including Bcap37 and MCF-7. This triterpenoid exerts cell cycle arrest at G₂/M phase and induces apoptosis in Bcap37 cells through mitochondria-dependent pathway^16,17.

Cucurbitacin B exhibited antiproliferative effects against all the cell lines tested. MDA-MB-435 was found to be the most sensitive¹⁸. Balsaminapentanol, balsaminol A, balsaminol B and cucurbalsaminol B have suppressed the growth of MCF-7 cells with varying potency¹⁹. Four dammarane triterpenoids showed weak anti-breast cancer activity in vitro²⁰. Three ergostane-type triterpenoids²¹ displayed potent cytotoxic effects against MCF-7 as well as MDA-MB-231 cells²². Tingenone and netzahualcoyonol, exhibited cytotoxicity against MDA-MB-231 cells²³. Triterpenoids, celastrol and its methyl ester pristimerin are significantly active against the proliferation of MCF-7 cell lines²⁴. Mechanistic studies have revealed that pristimerin induces rapid apoptosis characterized by caspase activation, decrease of mitochondrial membrane potential and a rapid release of cytochrome²⁵.

Celastrol triterpenoid has shown to inhibit the growth and induce apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. It also abolished interleukin-1β and tumour necrosis factor-α (TNF-α)-induced IκB phosphorylation, IκB kinase activation and prevented nuclear translocation of nuclear factor-kappa B (NF-κB) and DNA binding reduced the expression of matrix metalloproteinase-9 (MMP-9) and urinary plasminogen activator leading to inhibition of migration of W256 cells²⁶. It has been reported to potentiate tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induced cytotoxicity and apoptosis of T47D and MDA-MB-231 cells. It also downregulated the expression of cell survival proteins, namely cFILP, IAP-1, Bcl-2, Bcl-xL, survivin and XIAP and upregulated Bax expression. The cell surface expression of both the tumour necrosis factor-related apoptosis-inducing ligand receptors–death receptor 4 (DR4) and (DR5) have also been found to be induced by celastrol treatment. Sulphurenic acid and its analog 15α-acetyl-dehydrosulphurenic acid have also shown antitumour efficacy with both compounds being more potent against MDA-MB-231 cells as compared with MCF-7 cells. Limonoid was found to be cytotoxic against MCF-7 cells^27,28. Other limonoids, melianin B, melianin C and meliavolkinin are active in killing MCF-7 cells²⁹.

Betulinic acid has shown antiproliferative activities toward MCF-7 as well as MDA-MB-231 cells^30,31. It also exhibited a remarkable cytotoxic effect against T47D cells with a simultaneous induction of apoptosis, decrease of Bcl-2 and cyclin D1 and increase of Bax gene expression³². Basu and colleagues³³ have demonstrated lethal effects of betulinic acid in another breast cancer cell line SKBR3. Betulinic acid has been shown to induce early apoptotic events accompanied by flopping of phosphatidylserine on the outer lamella of the plasma membrane as evidenced by elegant fluorescent microscopy. The mitochondrial signalling pathway has been implicated in the observed apoptosis-inducing property of betulinic acid. Using nine breast cancer cell lines³⁴ have confirmed the antitumour effects of betulinic acid against mammary carcinoma with various degree of sensitivity. Betulin (53) inhibits the proliferation of T47D breast carcinoma cells³⁵. Lupeol (3) has caused significant inhibition of growth of MDA-MB-231 cells. It has also been able to induce ERα expression in this ERα-negative breast cancer cell line³⁶.

The extensive literature on the biotransformation of different types of terpenoid molecules were reviewed, which have verified that biotransformed metabolites suppressed the process of inflammation and cancer. Terpenoids have been a very promising chemotherapeutic agent for the treatment of HIV infection, cancers and other pharmacological studies³⁷. Bioconversion of bioactive natural products produces metabolites with an enhanced bioactivity and low toxicity^38–40. Apart from the previous study, there are no detailed available data on the biotransformation of terpenes. This review examines the potential role of natural terpenoids and their biotransformed products. The substrates discussed in this review were xenobiotic in nature, which enhances the substrate flexibility towards the microbial enzymatic system.

Biotransformation

Biotransformation is defined as the use of biological systems to bring about structural changes in chemical compounds that are not their natural substrates. This distinguishes biotransformation from biosynthesis, which is concerned with the synthetic ability of biological systems in their normal habitat.

Classification of biotransformation

Biotransformation may be classified into two different categories as under:

1. The first type of transformation is “xenobiotic transformation” in which the substrate is completely alien to the microorganism.

2. The second type of transformation is “biogenetically directed”, and is also known as “analogue biosynthesis”, in which the substrate is a structural analogue of a biosynthetic intermediate. This biotransformation relies on the substrate flexibility of the enzymatic pathway responsible for the secondary metabolic biosynthesis.

Advantages of biotransformation

Biotransformation has a number of advantages when compared with the corresponding chemical methods. Many biotransformation not only are regio- and stereo specific, but are also enantiospecific, allowing the production of chiral products from racemic mixtures. The conditions for biotransformations are mild and in the majority of cases, they do not require the protection of pre-existing functional groups.

Furthermore, the features governing their regiospecificity differ from those controlling the chemical specificity, and indeed it is possible to obtain structural transformation at positions that are chemically unreactive. Biosynthetically-directed transformations are also used to obtain biosynthetic informations, the design of selective biosynthetic inhibitors. The dominant feature in any biotransformation is the topological relationship between the substrate and the active site of the enzyme. From a commercial point of view, some biotransformations can be cheaper and more direct than their chemical analogues, while the transformations proceed under conditions that are normally regarded as environmentally friendly.

Agents of biotransformation

A great challenge for a specific biotransformation of a certain substrate is to find the appropriate microorganism, so that, traditionally, one of the most widely used techniques is screening with different microbial strains. Nowadays, biotransformation of compounds is performed by employing following systems:

1. By using microbes (fungi and bacteria)

2. By using plant and animal cell cultures

3. By using animals

Use of fungi in biotransformation

Fungi are playing a prominent role in the catalysis of organic compounds and in the production of commercially and industrially important compounds, because of their ability to catalyze novel reactions. Fungi are commonly used in industry for production of fermented beverages, foods, physiologically active substances, solvents, organic acids, polysaccharides, antibiotics etc⁴¹. Of the zygomycota, Mucor and Rhizopus are commonly used in industry. Rhizopus strains are important in citric acid production. Mucor strains make a significant number of important lipases, and catalyze the hydroxylation of a wide range of chemical compounds. In a survey of fungi with reference to their ability to hydroxylate the polycyclic aromatic hydro carbons naphthalene, 55 species of fungi were found to posses the same activity⁴².

Microbial enzymes and their reactions

In these biotransformation processes, several reactions that are difficult to achieve by chemical means have been accomplished such as:

Microbial enzymes (produced by micro organisms) show a diverse range of reactions including the insertion of oxygen into C-H and C-C bonds, introduction of hydroxyl groups into remote positions of terpenes; selective cleavage of the side chains of tetracyclic terpenoids to produce C19 steroids; regioselective glycosidic transfer reactions; selective ring cleavage through a Baeyer-Villiger-type oxidation to render seco-triterpenoids, and carbon skeleton rearrangements involving a methyl group migration, addition of oxygen to alkene, hydrolysis or formation of amides, transferring of acyl or sugar units from one substrate to another, epoxide, ester and nitriles hydrogenation, hydration and elimination of small units, racemization, epimerization and isomerization reactions and formation of C-C, C-O, C-N, and C-S bonds⁴³. Reduction of carboxyl groups and hydroxylation at non active sites are among the most valuable reactions catalyzed by the micro organisms.

Reduction/hydroxylation reactions

The enzymatic reduction takes place with a regio and stereo chemical homogeneity that are difficult to achieve chemically. These enzymatic methods possess an enantioselectivity which in many cases can be predicted on the basis of empirical rules. The reduction of a carbonyl, particularly to generate a new chiral center, is one of the most widely used biotransformations.

When a compound is incubated with microorganisms, a hydroxyl group may be inserted at a site that is remote from any other functionalities. This ability of micro organisms to hydroxylate compounds at chemically inactive sites is a valuable synthetic tool. The intact organism is normally used for these transformations because many of the enzyme systems that are involved are membrane bound and are consequently difficult to obtain in a stable form. The value of microbiological hydroxylation lies in the fact that the sites of hydroxylation are often different from those at which normal chemical reactions occur.

Monooxygenase are enzymes responsible for the microbiological hydroxylation and transfer of one oxygen atom to the organic substrate⁴⁴. The catalytic cycle of the enzymes has been deduced largely from a study of camphor hydroxylating enzymes isolated from Pseudomonas putida⁴⁵.

Use of plant cell cultures in biotransformations

It has been reported that the formation and the accumulation of some secondary metabolites does not normally occur in the cultured cells of higher plants^46–49, and it is found to be difficult to harness this potential to organic synthesis or industrial processes. To overcome these problems, the biotransformation of exogenous substrates mainly by the micro organism has extensively been investigated. In addition, plant cultured cells are also used to transform substrate.

Many studies have recently been focussed on the ability of the plant cultured cells to transform xenobiotic compounds^50–54. Plant cultured cells have the potential to be used as an excellent tool for the organic synthesis. The advantages of using the plant cultured cells as biocatalysts are: (i) The plant cultured cells can be grown in the laboratory. The cultured material is homogenous, and experiments can be performed and reproduced in all seasons. (ii) The plant cultured cells can accumulate high amounts of transformed products. (iii) The plant cultured cells can also be grown to an almost unlimited quantities of material in bioreactor.

Use of plant bacteria in biotransformations

Bacterial transformation involves changes in the structure of organic compounds through degradation pathways due to which many biologically inactive or compounds with low activities can be converted into more potent bioactive compounds^55,56.

Microbial enzymatic transformation of terpenes

The antitumour efficacy of several triterpenoids are currently being evaluated in phase I clinical trials⁸. Several reviews on microbial transformation of terpenoids have been published in recent years^57–60.

Microbial enzymatic transformation of some sesquiterpenes

The biotransformation of terpenoids is an important field of toxicology and xenobiochemistry⁶¹. These biotransformations have been used as in vitro models to mimic and predict the mammalian metabolism of biologically active triterpenoids. Several sesquiterpenoids have displayed analeptic, anthelmentic, anti-inflammatory, antitumor, hypotensive and sedative activities.

Terpenoidal substrates showed a variety of stereoselective reactions mainly oxidation, reduction, C-C bond formation, esterification etc. with various fungal cultures. Hydroxylation was found to be the most common reaction in sesquiterpenes pyrethrosin (5), plectranthone (6)⁶², costunolide (8)^38,63 and partheniol (7) (Scheme 1) afforded mainly their hydroxylated derivatives with various fungal strains. Published data related to the microbial enzyme transformation of terpenes showed higher susceptibility of this class of natural products towards the microbial enzymatic system⁶⁴.

Scheme 1.  Natural sesquiterpenes.

Ambrein oxidation and enzymatic transformation of ambrox

Ambrein (9) is the major constituent of the ambergris and possesses an amber-like odour. During drifting in the sea, ambrein is oxidatively decomposed by the action of sea water, air and sunlight to give rise to several odourous compounds (Scheme 2)⁶⁵ Microbial oxidation of ambrox has been carried out to obtain interesting metabolites, which were otherwise difficult to synthesize by using chemical methods.

Scheme 2.  Air degradation of ambrein 9.

Fermentation of ambrox with Fusarium lini afforded many new mono, di- and tri-hydroxylated metabolites such as 1α-hydroxy ambrox (12), 1α,11α-dihydroxyambrox (13), 1α,6α-dihydroxy ambrox (14), 1α,6α-11α−trihydroxy ambrox (15). Enantio selective α-hydroxylation occurred at C-1, C-6 and C-11 (Scheme 3).

Scheme 3.  Metabolism of compound 10 by Fusarium lini.

Incubation of ambrox with Rhizopus stolonifer, resulted in the formation of four metabolites, 3-oxoambrox (16), 3β-hydroxyambrox (17), 3β,6β-dihydroxyambrox (18) and its other cleaved product, tetranor-12,8-diol-labdane (21)⁶⁶ (Scheme 4). Fermentation of ambrox with Curvularia lunata yielded metabolites 3-oxoambrox and 3-oxoambrox (16), 3β-hydroxyambrox (20).

Scheme 4.  Metabolism of compound 10 by Rhizopus stolonifer.

Metabolites 3-oxoambrox, 3β-hydroxyambrox (20) and tetranor-12,8-diol-labdane (21) have been earlier reported as metabolites of ambrox by Cephalosporium aphidicola (wild type), Aspergillus niger (IFO 4049), C. lunata and Aspergillus cellulose (IFO4040). Incubation of ambrox with Cunninghamella elegans, resulted in the formation of two metabolites 3-oxoambrox (16) and 3β-hydroxyambrox (17) (Schemes 5 and 6)^66–68. The oxygenated metabolites of ambrox did not show any effective odour, while its ether cleaved product tetranor-12,8-diol-labdane (21) exhibited strong sweet odour, which is totally different from amber-like odour.

Scheme 5.  Metabolism of compound 10 by Curvularia lunata.

Scheme 6.  Metabolism of compound 10 by Cunninghamela elegans.

Biotransformation of (−)-ambrox with cell suspension cultures of Actinidia deliciosa (Kiwifruit) yielded the regio and stereospecific oxygenated products 3-oxoambrox,3β-hydroxyambrox,1α-hydroxyambrox,3β,6β-dihydroxyambrox,1α,6β-dihydroxyambrox, and 1α,3β-dihydroxyambrox. Metabolites 1α,6β-dihydroxyambrox and 1α,3β-dihydroxyambrox were found to be new compounds⁶⁸.

Microbial oxidations of sclareolide by C. elegans

Sclareolide exhibits good phytotoxic and cytotoxic activities against human cancer cell⁶⁹. Sclareolide is structurally different from ambrox only by the presence of a keto group at C-12. The effect of the presence of a lactone moiety on the metabolism was studied by incubating sclareolide (17) with C. elegans. This yielded a new set of metabolites, 3-oxosclareolide (22), 3β-hydroxysclareolide (23), 2α-hydroxysclareolide (24), 2α,3β-dihydroxyepisclareolide (25), 1α,3β-dihydroxyepisclareolide (26) and 3β-hydroxyepisclareolide (27). This indicated that the lactone functionality directs the enantioselective hydroxylations at C-2 and C-1, and epimerization at C-8 positions (Scheme 7).

Scheme 7.  Metabolism of sclareolide (17) by Cunninghamela elegans.

Metabolites 3-oxosclareolide (22), 3β-hydroxysclareolide (23) and 1α,3β-dihydroxyepisclareolide (26) have already been reported as fermented products of compound sclareolide by various fungi and have shown in vitro anti-cancer activities against human lungs, colon, skin and breast⁷⁰. All metabolites 22–27 showed significant phytotoxic activity against Lemna acquinoctialis Welv. except 2α,3β-dihydroxyepisclareolide (25).

Microbial enzymatic transformation of isolongifolen-4-one (30)

Longifolen is a tricyclic olefin and undergo a variety of rearrangements. Isolongifolen, isolongifolol and isolongifolen-4-one are the commonly rearranged products of 28. The microbial enzymatic transformation of (+)-isolongifolen-4-one by a number of fungi by means of a standard two-stage fermentation technique afforded (7R)-12-hydroxyisolongifolen-4-one, (7S)-13-hydroxyisolongifolen-4-one, (11R)-11 hydroxyisolongifolen-4-one, (10R)-10-hydroxyisolongifolen-4-one, and (9R)-9-hydroxyisolongifolen-4-one. The metabolites (7S)-13-hydroxyisolongifolen-4-one and (9R)-9-hydroxyisolongifolen-4-one showed potent tyrosinase inhibitory activity with an IC₅₀ = 51 µM⁷¹.

Derivatization of isolongifolen-4-one was carried out in order to find some derivatives with increased potency against tyrosinase. The transformation of isolongifolen-4-one with A. niger (ATCC 10549) was carried out and three new metabolites namely (7R)-12-hydroxyisolongifolin-4-one (32), (7S)-12-hydroxyisolongifolin-4-one (33), (11S) -9-hydroxyisolongifolin-4-one (34) were obtained (Scheme 8). Biotransformation of isolongifolen-4-one with C. aphidicola (IMI 68689) afforded compound (9S)-9-hydroxyisolongifolin-4-one. The metabolites (7R)-12-hydroxyisolongifolin-4-one, (7S)-12-hydroxyisolongifolin-4-one and (9S)-9-hydroxyisolongifolin-4-one showed enzyme inhibitory activity⁷¹ (Scheme 9). The transformation of isolongifolen-4-one with F. lini (NRRL 68751) afforded (9S)-9-hydroxyisolongifolin-4-one (35) and (10R)-9-hydroxyisolongifolin-4-one (36) (Scheme 10). Metabolites (7R)-12-hydroxyisolongifolin-4-one (32) and (7S)-12-hydroxyisolongifolin-4-one (33) were also obtained by the transformation of isolongifolen-4-one (30) with R. stolonifer (ATCC 10404) (Scheme 11).

Scheme 8.  Metabolism of compound 30 by Aspergillus niger.

Scheme 9.  Metabolism of compound 30 by Cephalosporium aphidicola.

Scheme 10.  Metabolism of compound 30 by Fusarium lini.

Scheme 11.  Metabolism of compound 30 by Rhizopus stolonifer.

The major metabolites (7R)-12-hydroxyisolongifolin-4-one (32), (7S)-12-hydroxyisolongifolin-4-one (33) and (9S)-9-hydroxyisolongifolin-4-one (35) were obtained in good yields.

Microbial enzymatic transformation of isolongifolol

A total of 20–30 natural products are known which contain the bridged tricyclic sesquiterpenes skeleton isolated from plants and fungi and possess interesting biological activities. Very few reports on the effect of fungal enzymatic system towards natural products possessing bridged tricyclic sesquiterpenes skeleton are found in literature^71–73. The incubation of bridged tricyclic sesquiterpene isolongifolol (31) with F. lini afforded its polar oxygenated metabolites named as, 10 oxo isolongifolol (37), 10α-hydroxy isolongifolol (38), 9α−hydroxy isolongifolol (39)⁷³ (Scheme 12). Incubation of compound isolongifolol with R. stolonifer afforded metabolites isolongifolol (38) and 9α−hydroxy isolongifolol (39).

Scheme 12.  Metabolism of compound 31 by Fusarium lini.

Biotransformation of caryophyllene oxide

Caryophyllene oxide reported to have strong anti-inflammatory activity^71,72. It also exhibits potent antimutagenic property⁷⁴. Caryophyllene oxide (40) was incubated with the cell suspension culture of Catharanthus roseus. It was observed that C. roseus cell culture can convert caryophyllene oxide into several metabolites 41–44 (Scheme 13)^73,75.

Scheme 13.  Metabolism of compound 40 by cell suspension culture.

Compound 41 was previously reported as a biotransformed product of compound 40 by Botrytis cinerea⁷⁶, while compound 42 was reported as a synthetic derivative of 5α-hydroxycaryophyll-8(13)-ene-3,4-epoxide⁷⁷. Metabolites 43 and 44 were found to be new metabolites (Scheme 13).

Fungal transformation of caryophyllene oxide (40) by a number of fungi afforded 15-hydroxymethyl caryophyllene oxide (41), 4β,5α-dihydroxy caryophyll-8 (13)-ene (42), clovane-5α,9β-diol (45), 14-hydroxy methyl caryophyllene oxide (46), 2β-hydroxy caryophyllane oxide-8-one (47), 8α-hydroxymethyl caryophyllane oxide (48), caryolane-5α,6β,13β-triol (49), 3β,8α-dihydroxy methyl caryophyllan oxide (50) and clovane-5α,9β-12-triol (51)^71,73,74,77 (Schemes 14–16).

Scheme 14.  Metabolism of (−) caryophyllene oxide 40 with Cephalosporium aphidicola.

Scheme 15.  Metabolism of (−) caryophyllene oxide 40 with Macrophomina phaseolina and Rhizopus stolonifer.

Scheme 16.  Metabolism of (−) caryophyllene oxide 40 with Aspergillus niger, Fusarium lini and Gibbrella fujikuroi.

The compound 14-hydroxymethyl caryophyllene oxide (46) showed a potent inhibitory activity against the butyryl cholinesterase enzyme⁷⁵, with an IC₅₀ = 10.9 µM. This was more potent than the parent caryophyllene oxide (IC₅₀ = 208.4 µM). Compound 8α-hydroxy methyl caryophyllan oxide (48) and caryolane-5α,6β,13β-triol caryolane-5α,6β,13β-triol (49) also showed inhibitory activity against enzyme butyryl cholinesterase.

The compounds clovane 5α,9β-diol, 14-hydroxymethyl caryophyllene oxide and caryolane-5α,6β,13β-triol caryolane-5α,6β,13β-triol showed an enhancing activity of α−glucosidase enzyme, whereas compounds 8α-hydroxymethyl caryophyllene oxide (48) and 3β,8α-dihydroxy methyl caryophyllan oxide (50) showed an average inhibitory activity against the enzyme, when compared with substrate caryophyllene oxide (40) (Scheme 16)^75,78,79.

Microbial enzymatic transformation of an anti-inflammatory sesquiterpene (−) caryophyllene oxide by a number of bacteria, afforded a known metabolite 4β,5α-dihydroxycaryophyll-8 (13)-ene (42) which was earlier reported as a synthetic derivative of 5α-hydroxycaryophyll-8(13)-ene-3,4-epoxide⁷⁶ (Scheme 15).

Microbial enzymatic transformation of triterpenes

Microbial transformation of triterpenoids has provided new derivatives that are potentially useful for pharmacological studies. Biotransformation of pentacyclic triterpenes, such as ursane^80–82 oleanane^83–86 have been reported to have anti-cancer activity⁸⁷.

Pentacyclic transformed triterpenes of the lupane type^88–93, such as lupeol, betulin and betulinic acid, have been reported to have interesting bioactivities including antiviral, in particular, against the human immunodeficiency virus^93–95, herpes simplex virus⁹⁶ and Epstein–Barr virus⁹⁷, anti-inflammatory⁹⁸ and antitumour against human melanoma and other types of human malignancies^99,100.

Examples of transformation of triterpenes include the microbial enzymatic transformation of two lupene-type triterpenes, betulin (53) and betulonic acid (54) by the fungus Chaetomium longirostre and microbial enzymatic transformation of a mixture of argentatin A (55) and incanilin (56) by Gibberella suabinetti and Septomyxa affinis^60,101,102 (Scheme 17).

Scheme 17.  Lupane type triterpenes.

Microbial transformation of 18β−glycyrrhetinic acid

18β glycyrrhetinic acid (57) has been used for the treatment of pulmonary diseases and inflammatory disorders. 18β glycyrrhetinic acid is well known for the anti-inflammatory activity^103,104. It was subjected to fermentation with C. elegans,¹⁰⁵ which afforded a hydroxylated metabolite, 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid (52) (Scheme 18). 3β,7β-Dihydroxy-11-oxo-olean-12-en-30-oic acid was earlier reported as a metabolite of glycyrrhetinic acid with C. lunata.

Scheme 18.  The incubation of 57 with Fusarium lini afforded a oxidative metabolite 3,11-dioxo-olean-12-en-30-oic acid (58) while with Cunninghamela elegans afforded a metabolite 3β, 7β-dihydroxy-11-oxo-olean-12-en-30-oic acid (52).

C. lunata, a well-known fungus used for 11-hydroxylation of steroids⁸⁴ also performed triterpenoid hydroxylations. The major product of the microbial transformation of glycyrrhetinic acid, also known as 18-glycyrrhetinic acid, by C. lunata ATCC 13432 was identified as 7-hydroxyglycyrrhetinic acid¹⁰⁶.

Epimeres of glycyrrhetinic acid, such as liquiritic acid, were partially converted by the same strain as well as by the fungi Cunninghamella sp. ATCC 3229 and Mucor griseo cyanus ATCC 1207-a to their 7-hydroxy, 15-hydroxy, and/or 7-15-dihydroxy derivatives. The fungal strains Mucor polymorphosporus AS 3.3443 and Mucor spinosus AS 3.3450 also produced 7-hydroxy glycyrrhetinic acid as a major metabolite from glycyrrhetinic acid.

The incubation of 18β-glycyrrhetinic acid (57) with F. lini afforded a oxidative metabolite 3,11-dioxo-olean-12-en-30-oic acid (58) from Maytenus undata¹⁰⁷, which was earlier reported as a semisynthetic derivative of 18β-glycyrrhetic acid¹⁰⁸ (Scheme 18). 3,11-Dioxo-olean-12-en-30-oic acid was previously reported as a microbial metabolite of glycyrrhetinic acid¹⁰⁹.

In mammalian cells, lipoxygenases catalyze the biosynthesis of a variety of bioregulatory compounds, such as hydroxyeicosatetraenoic acids, leukotrienes, lipoxins and hepoxylines¹¹⁰. It has been found that these lipoxygenase products play a major role in a variety of disorders, such as bronchial asthma, inflammation and tumour angiogenesis^89,111, raising the exciting possibility of using nonsteroidal anti-inflammatory drugs and Lox inhibitors as anti-angiogenesis agents for cancer treatment.

Compounds 18β-glycyrrhetinic acid and metabolites 52–58 were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid 58 showed a strong lipoxygenase inhibitory activity against the lipoxygenase, (IC₅₀ =144.2 µM) more than the parent 18β-glycyrrhetinic acid (57) (IC₅₀ = 225.1), whereas 3β, 7β-dihydroxy-11-oxo-olean-12-en-30-oic acid (52) showed only a low inhibitory activity against the enzyme with an IC₅₀ value of 300 µM.

In another biotransformation, betulonic acid was produced from betulinic acid using two Bacillus megaterium strains^90,112,113. Arthrobotrys converted betulonic acid into 3-oxo-7β-hydroxylup-20(29)-en-28-oic acid, 3-oxo-7β,15α-dihydroxylup-20(29)-en-28-oic acid and 3-oxo-7β,30-dihydroxy-lup-20(29)-en-28-oic acid. Colletotrichum converted betulinic acid into 3-oxo-15α-hydroxylup-20(29)-en-28-oic acid whereas, betulonic acid was converted into the same product and 3-oxo-7β,15α-dihydroxy-lup-20(29)-en-28-oic acid. Chaetophoma converted betulonic acid (4) into 3-oxo-25-hydroxy-lup-20(29)-en-28-oic acid and both Chaetophoma and Dematium converted betulinic acid into betulonic acid¹¹.

Biotransformation of cycloartenol, 24-methylenecycloartenol and cycloartenone

Most of these biotransformations have been performed onto tetracyclic triterpenoids. Biotransformation of three cycloartane-type triterpenes, cycloartenol (58a), 24-methylene cycloartenol (58b) and cycloartenone (58c), by the fungus Glomerella fusarioides was studied. Cycloartenol was converted into cycloartenone (58d), cycloart-25-ene-3β,24-diol (58e) and cycloartane-3β,24,25-triol (58f). 24-Methylene cycloartanol (58b) was metabolized to cycloeucalenol (58g) and two new compounds, 24-methylcycloartane-3β, 24,24-triol (58h) and 24-methoxy-24-methylcycloartane-3β,24-diol (58i). Cycloartenone was converted into two new metabolites, 4α,4β,14α-trimethyl-9β,19-cyclopregnane-3,20-dione (58j) and 25-hydroxy-24-methoxy cycloartan-3-one (58o), and four known compounds, viz., cycloartane-3,24-dione (58k), 24-hydroxycycloart-25-en-3-one (58l), (23E)-25-hydroxycycloart-23-en-3-one (58m) and 24,25-dihydroxycycloartan-3-one (58n) (Scheme 19)¹¹⁴.

Scheme 19.  Structures of cycloartenol (58a), 24-methylene cycloartenol (58b), cycloartenol (58c) and metabolites (58d–58o).

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb1, R. stolonifer and C. lunata produced four key metabolites, 3-O-[β-D-glucopyranosyl-(1,2)-β-D-glucopyranosyl]-20-O-[β-D-glucopyranosyl]-3β,12β,20(S) trihydroxydammar-24-ene, 3-O-[β-D-glucopyranosyl-(1,2)-β-Dglucopyranosyl]-20-O-[β-D-glucopyranosyl]-3β,12β,20(S)-trihydroxydammar-24-ol, 3-O-[β-D-glucopyranosyl-(1,2)-β-D-glucopyranosyl]-3β,12β,20(S)-trihydroxydammar-24-ene, and 3-O-β-D-glucopyranosyl-3β,12β,20(S)-trihydroxydammar-24-ene¹¹⁵. Transformation of G-Rb1, G-Rb3 and G-Rc by F. sacchari was respectively experimented. Kinetic evolutions of G-Rb1, G-Rb3 and G-Rc and their metabolites during the cell incubation were monitored by HPLC analysis. High-performance liquid chromatography was used for monitoring the transformation kinetics of bioactive compounds during F. sacchari metabolism¹¹⁶. Cucurbitacin E 2-O-P-~-glucopyranoside was transformed by Cuwularia lunata NRRL 2178 into cucurbitacin E 21 and three new metabolites. The novel metabolites were identified as (24R)-hydroxy-23,24-dihydrocucurbitacinE and (24S)-hydroxy-23,24-dihydrocucurbitacinE 14 and 51, and the 3-acetyloxy-3-methylbutyl ester of (23–27)-penta-norcucurbitacin I 22-oic acid¹¹⁷. More than seventy strains of aerobic bacteria showing glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar. Of these microorganisms, twelve strains could convert the major ginsenoside, Rb1, to the pharmaceutically active minor ginsenoside Rd. Three strains, Burkholderia pyrrocinia GP16, B. megaterium GP27 and Sphingomonas echinoides GP₅₀, were phylogenetically studied, and observed to be most potent at converting ginsenoside Rb1 almost completely within 48 h¹¹⁸.

Enzymatic transformation of β-amyrin acetate by Rhodobacter sphaeroides

The microbial transformation of β-amyrin acetate occurred in the culture of Rhodobacter sphaeroides producing three metabolites, β-amyrin,(3β)-olean-12-ene-3,23-diol and erythrodiol. Initially, it was found that β-amyrin acetate could be deacetylated and hydroxylated by microbial methods¹¹⁹.

Enzymatic transformation of oleanolic acid by F. lini

The fermentation of oleanolic acid (2) with F. lini afforded two oxidative metabolites, 2α,3β-dihydroxyolean-12-en-28-oic acid and 2α,3β,11β-trihydroxyolean-12-en-28-oic acid. 2α,3β,11β-trihydroxyolean-12-en-28-oic acid was found to be a new compound. These metabolites exhibited a potent inhibition of α-glucosidase enzyme⁸⁰. In another biotransformation, oleanolic acid was produced by the microbe Nocardia sp.NRRL. 5646 to selectively furnish their corresponding 28-methyl esters⁸⁰.

Microbial enzymatic transformation of diterpenes

Methyl ent-17-hydroxy-16β-kauran-19-oate was fed to a 2-day-old culture of the fungus R. stolonifer, fermenting at room temperature (25°C). Two novel compounds methyl ent-9α,17-dihydroxy-16β-kauran-19-oate and methyl ent-7α,17-dihydroxy-16β-kauran-19-oate were isolated from this experiment.

Many compounds of therapeutic and industrial interest were obtained by microbial transformation. Examples are the biotransformation of aphidicolin (59)¹²⁰ (Scheme 20) and methyl ent-15-oxokaur-16-en-19-oate (60)¹²¹ by various micro organisms ¹²².

Scheme 20.  Compounds undergo transformation by various micro organisms.

Sclareol (61), a labdane diterpene ditertiary alcohol, was first isolated from Salvia sclarea L. (Labiatae) in 1931¹²³ and also from Astrogalus brachystochys¹²⁴. Sclareol have a potent antibacterial activity. Commercially, sclareol, is used as a fixative and in perfumery, as a flavoring agent in the tobacco industry, and as a eynthon for the preparation of a series of ambra odourants in perfumery^125–127.

The metabolism of sclareol by R. stolonifer (ATCC 10404) yielded four metabolites 3β-hydroxy sclareol (62) 18α-hydroxy sclareol (63), 6α,18α-dihydroxy sclareol (64), 11S,18α-dihydroxy sclareol (65) (Scheme 21). Incubation of sclareol with R. stolonifer afforded in high yield a mixture of triols with 18-hydroxy-sclareol as the main component. The fermentation of sclareol with F. lini yielded two metabolites, 66–67 (Scheme 22).

Scheme 21.  Metabolism of 61 by Rhizopus stolonifer.

Scheme 22.  Metabolism of 61 by Fusarium lini.

Sclareol is reported to have an antibacterial activity against different pathogenic strains. The metabolites 62–67 produced from sclareol were screened for antibacterial activity against three pathogenic strains. 1β-Hydroxy sclareol (66) was found to be more active than sclareol, whereas compound 6α,18α-dihydroxy sclareol (64) and 11S,18α-dihydroxy sclareol (67) were found to be as active as sclareol against Bacillus subtilis¹²⁸.

Incubation of the diterpene 2β-hydroxy-ent-13-epi-manoyl oxide with Gibberella fujikuroi afforded in good yield 2β,6β-dihydroxy-ent-13-epi-manoyl oxide, 2β,12β-dihydroxy-ent-13-epi-manoyl oxide and 2β,20-dihydroxy-ent-13-epi-manoyl oxide, confirming that although ent-13-epi-manoyl oxide is a final metabolite of a biosynthetic branch in this fungus, more polar derivatives of this compound can be transformed by this micro-organism¹²⁹.

Microbial enzymatic transformation of monoterpene

Hydroxylation is found to be the most common reaction in monoterpene, such as in 1,4-cineole (68)¹³⁰, geraniol (70) and nerol (71)¹³¹ afforded mainly hydroxylated derivatives with various microbes. Scientists have investigated the microbial transformation of a monoterpene (1R,3R,4S)-(−)-menthol (72), by using various fungal strains.

In search of potent antibacterial compounds for commercial products as dental root canal sealers, antiseptics, food preservatives and feed supplements^132,133, a monoterpene (1R, 3R, 4S)-(−)-menthol (72), has been investigated¹³⁴.

Microbial enzymatic transformation of (−) menthol with Macrophomina phaseolina yielded the metabolites 73–77 (Scheme 23). Microbial enzymatic transformation of (1R, 3R,4S) (−) menthol with R. stolonifer yielded the metabolite (8R)-9-hydroxy menthol (75) (Scheme 24). Microbial enzymatic transformation of (1R, 3R, 4S)–(−), menthol (72) with C. elegans, while incubation of (−) menthol (64) with C. elegans yielded the metabolite 76 (Scheme 25).

Scheme 23.  Metabolism of (−) menthol (72) with Macrophomina phaseolina.

Scheme 24.  Metabolism of (−) menthol (72) with Rhizopus stolonifer.

Scheme 25.  Metabolism of (−) menthol (72) with Cunninghamela elegans.

Compound (1R,3R,4S)-(−)-menthol is known to possess antibacterial activity against food born pathogens such as Salmonella typhimurium, Staphylococcus aureus and Vibrio parahoemolyticus¹³⁵. (1R, 3R, 4S)-(−)-menthol was incubated with three fungal cultures to obtain five known metabolites 8-hydroxy menthol, 6α-hydroxy menthol, (8R)-9-hydroxy menthol, 1α-hydroxy menthol and 8R-9-hydroxy menthol (73–77) (Scheme 23). Metabolite 8-hydroxy menthol (73) has been reported as transformation product of (1R, 3R, 4S)-(−)-menthol by various Aspergillus species¹³⁶. Evaluation of (1R, 3R, 4S)-(−)-menthol and 73–77 for anti-bacterial activity against the gram positive bacteria had potent activities.

6α-Hydroxy menthol (74) reported earlier from the microbial enzymatic transformation of l-menthol by Rhizoctonia solani AG-11`-1A and 1B¹³⁷. (8R)-9-Hydroxy menthol, which was reported earlier as a synthetic derivative of (−)-(1R, 3R, 4S, 8R)-9-Phenyl thio menthol¹³⁸.

1α-Hydroxy menthol (76) reported earlier from the microbial transformation of l-menthol (64) by Rhizoctonia solani AG-1-1A and 1B¹³⁷. 8R,9-dihydroxy menthol (77) which was reported as synthetic derivative of (−)-isopulegol¹³⁹. Metabolites (73–77) are the strong candidates as menthol against Escherichia coli. Metabolites 1α-hydroxy menthol and 8R-9-hydroxy menthol 76–77 are the strong candidates as menthol against Pseudomonas aeruginosa. Metabolite 1α-hydroxy menthol is the strong candidate as menthol against Salmonella typhi. Compound 78 showed significant inhibitory potential against the BuCh inhibition with an IC₅₀ value of 91.9 µM, whereas compound 78 showed only low activity against AchE with an IC₅₀ value of 219.0 µM¹⁴⁰. This class of compounds also possess antibiotic and cytotoxic properties and can be good candidates for chemotherapy.

Microbial enzymatic transformation of sesterterpene leucosceptrine

The microbial transformation of a novel sesterterpene, leucosceptrine (78), the first member of class leucosesterterpenes, by R. stolonifer afforded two hydroxylated metabolites, 1α-hydroxyleucosceptrine (79), and 8α-hydroxyleucosceptrine (80)¹⁴³ (Scheme 26).

Scheme 26.  Metabolism of leucosceptrine (78) with Rhizopus stolonifer.

Conclusion

Enzyme plays a pivotal role in modifying the naturally occurring substance. Attempts are being made in our lab to enhance the activity of existing bioactive compounds and drug design. The hydrolytic and reductive capabilities of microorganisms have been known and are currently used in preparative and industrial reactions. Various classes of bioactive organic compounds have been subjected to enzymatic transformation in an effort to obtain more active and less toxic substances, or to elucidate their metabolic pathways.

Many drugs produced by chemical synthesis were racemates. However, their beneficial effects often reside in only one enantiomer. This need has given an immense stimulus to the use of enzymes in chiral synthesis. In recent years the synthesis of chiral molecules by enzymatic methods has led to the widespread use of the hydrolytic properties of enzymes such as esterases and lipases. Consequently, predictive models of these transformations were also developed.

In some cases, it is extremely difficult to provide sufficient amounts of active substances from isolation method and fungi due to their limited levels of biosynthesis. The limited quantity might be due to the rare occurrence of the organisms. Although fermentation of microorganisms is a possible way, it does not apply to metabolites produced strictly by macroorganisms. Use of biotransformation and biotechnological methods may help to obtain potent candidates for the treatment of diseases.

In sesquiterpenes, pyrethrosin, plectranthone and costunolide diverse microorganisms have oxidized hydroxyl group afforded mainly their hydroxylated derivatives with various fungal strains. In ambrox biotransformation with F. lini afforded many new mono, di- and tri-hydroxylated metabolites, 1α-hydroxy ambrox, 1α,11α-dihydroxyambrox, 1α,6α-dihydroxy ambrox, 1α,6α-11α−trihydroxy ambrox due to enantio selective α-hydroxylation at C-1, C-6 and C-11. In monoterpenes, the metabolite 1β-hydroxy sclareol produced from sclareol was screened for antibacterial activity, was found to be more active than sclareol, whereas compound 6α,18α-dihydroxy sclareol and 11S,18α-dihydroxy sclareol were found to be as active as sclareol against B. subtilis. In 3β-hydroxylated tetracyclic or pentacyclic triterpenes, diverse microorganisms have oxidized this hydroxyl group. Many triterpenes have been converted to 3,4-seco-type compounds, through a Baeyer-Villiger-type oxidation of these derivatives, followed by hydrolysis of the resulting seven-membered ring lactones.

Mycobacterium sp. (NRRL B-3805) has produced a series of complex chemical changes in tetracyclic terpenoids, such as cycloartenol, lanosterol and 24-methylenecycloartanol, including the selective cleavage of the side chain of these triterpenoids, to produce C19 steroids. Different microorganisms have been used to investigate the biotransformation pathways of ginsenosides, and also to convert major ginsenosides into more active minor saponins. These microorganisms have produced regioselective deglycosylation reactions to produce specific ginsenosides using an appropriate combination of ginsenoside substrates and specific microorganism.

Among the metabolites observed, approximately 80% were more potent than the parent compound and planned research can produce more potent terpenoids by using enzymatic transformation methods. Specifically, highly oxygenated terpenes are potential candidates responsible for antioxidant, antibiotic and anti-cancer properties can also be obtained by these biotransformed products. It is highly valuable to construct the optimal transformation system through process-optimization strategy. For the preparation of terpenoids, the reaction parameters have to be optimized with regard to high yields and biocatalysts stability, signifying low production costs. Further studies need to focus on the enzymatic evidences for terpenoid production.

Declaration of interest

The authors report no conflicts of interest.