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Biotransformations by endophytic fungi isolated from traditional Ecuadorian

medicinal plants: Connecting ethnomedicine with biotechnology

Laura Scalvenzi

Dirección de Investigación, Universidad Estatal Amazónica

Paso lateral, km 2½ vía Napo, Puyo, Pastaza

lscalvenzi@uea.edu.ec

Abstract

Ecuador, a small country with diverse ecosystems in the Amazon, Andes and Pacific

coastal regions is considered one of the 17 "megadiverse” countries, and the native

ethnic groups and rural communities have a strong ethnomedical tradition in the use

of native plants in healing. Traditional ethnobotanical knowledge can be used to

guide biotechnological research on medicinal plants, even when the new application

is an innovation only distantly related to the traditional use. Based on

ethnomedicalknowledge of indigenous communities, the following plants from the

Amazon and Andes regions were chosen for investigation: Piper aduncum

(Piperaceae), Maytenus macrocarpa (Celastraceae), Schinus molle (Anacardiaceae),

Tecoma stans (Bignoniaceae) and Myrcianthes hallii (Myrtaceae). The research was

focused on (i) assesing the presence of endophytic fungi in the selected plants, (ii)

isolating and subculturing in vitro pure endophytic strains, (iii) assessing the

biotransformation capacity of the isolated endophytes on pure compounds

(intermediates of pharmaceutical synthesis). The following compounds were chosen

as substrate models for biotransformations: (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one,

acetophenone, 1-indanone, 2-furyl methyl ketone, 2-methylcyclopentanone, 2-

methylcyclohexanone, 2-methoxycyclohexanone. A total of 364 fungal strains were

isolated in vitro; among these, five strains performed biotransformations on

acetophenone to (S)-1-phenylethanol, with important yields (78-97%) and

enantiomeric excess (78-100%). Three strains also yielded phenols, probably by

enzymatic reactions (Baeyer-Villiger oxidations). Fifteen fungal strains yielded the

lactones (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3-

oxabicyclo[3.3.0]oct-6-en-2-one from (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one,

probably as result of monooxygenase activation.

Resumen

Ecuador, un país pequeño con diversos ecosistemas en las regiones de la Amazonia,

los Andes y la costa del Pacífico, es considerado como uno de los 17países

"megadiversos", y los grupos étnicos nativos y las comunidades rurales tienen una

Revista Amazónica: Ciencia y Tecnología 1(3): 248-270. 2012.

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fuerte tradición etnomedicinal en el uso de plantas nativas en la curación. El

conocimiento etnobotánico tradicional puedes ser usado para guiar la investigación

biotecnológica en plantas medicinales, aún cuando la aplicación nueva e innovadora

no está relacionada estrechamente con el uso tradicional de las plantas. En base al

conocimiento etnomedicinal de las comunidades indígenas, las siguientes plantas de

la Amazoníay de los Andes del Ecuador fueron elegidas para la investigación: Piper

aduncum (Piperaceae), Maytenus macrocarpa (Celastraceae), Schinus molle

(Anacardiaceae), Tecoma stans (Bignoniaceae) y Myrcianthes hallii (Myrtaceae). La

investigación se enfocó en (i) determinar la presencia de hongos endofitos en las

plantas seleccionadas, (ii) aislar y cultivar in vitro las cepas de endofitos, (iii) evaluar

la capacidad de los endofitos aislados de biotransformar compuestos considerados

intermedios de la sintesis de medicamentos. Los siguientes compuestos fueron

investigados: (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, acetophenone, 1-indanone, 2-

furyl methyl ketone, 2-methylcyclopentanone, 2-methylcyclohexanone, 2-

methoxycyclohexanone. 364 cepas funginas han sido aisladas. Entre ellas, cinco

cepas han biotransformado el acetophenone a (S)-1-phenylethanol, con importantes

rendimientos (78-97%) y excesos enantiomericos (78-100%). Tres cepas han

producido también fenoles, probablemente debido a reacciones enzimáticas que

catalizan las oxidaciones de Baeyer-Villiger. Quince cepas funginas han producico

los lactones (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one y (-)-(1R,5S)-3-

oxabicyclo[3.3.0]oct-6-en-2-one a partir de (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one,

probablemente como resultado de la activación de enzimas monooxigenasas.

Key words: ethnomedicine, Amazonian plants, Andean plants, endophyte,

endophytic fungi, biotechnology, biotransformation

Introducción

Ecuador, a small country with

exceptionally diverse forest ecosystems

in the Amazon, Andes and Pacific

coastal regions, is considered one of the

17 "megadiverse” countries (Mittermeier

et al., 1997; Rai et al., 2003). It is well

known that South America is a

promising region for the study of the

health potential of plants as sources of

new pharmaceutical treatments. The

presence of a strong ethnomedical

tradition leads the research toward an in-

depth study of Ecuadorian biodiversity,

both under a chemical and biological

point of view. The Neotropical region,

including South America, contains a

large percentage of the world’s flora. At

the same time, 80% of humankind lives

in “emerging countries”, basing their

health needs on plant related traditional

remedies (WHO, 2006).

The indigenous people of

Ecuador, including Kichwa-speading

communities in the Andes and Shuar and

Achuar communities in the Amazon,

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with their strong ethnomedical culture,

constitute the background subject of this

research.Several studies had been

developed to determine the scientific

basis of ethnomedical uses of traditional

plants. In particular, the study of plant

compounds and their biological activity,

contributes to the development of

phytochemical fingerprinting of

traditional plants used by natives for

ethnopharmaceutical purposes. This can

be considered as a protection tool

towards misappropriations of

ethnomedical plants and the related

knowledge. Moreover the study of new

potential uses, far removed from the

ethnomedical tradition, is also very

interesting for science. In this sense

biotechnological applications of ethno

medicinal plants are extremely

innovative. In particular

biotransformations are a relatively new

branch of biotechnology.

Biotechnology and biotransformations

Biotechnology consists in the use

of live organisms, their derivatives or

their biomolecularprocesses to make

goods or provide services.

Biotechnology is applied in several

fields as agriculture, chemical industry,

medicine production, health, food

industry, environment and mining

industry. In particular biotransformations

are a relatively new branch of

biotechnology. From a chemical point of

view, "biotransformation" is the

conversion of a chemical compound

referred to as the "substrate", generally

not used as a nutrient by the

microorganisms, to another compound

referred to as the "product", with

different applications, through the

enzymatic activity of biological catalysts

(Bastoset al., 2007). Biotransformation

isa differentprocess from biosynthesis

and biodegradation. Biosynthesis is an

ex novo synthesis of complex products,

catalyzed by enzymes from simple

compounds such as carbon dioxide,

ammonia or glucose. Biodegradation is a

catabolic process, encompassing the

conversion of complex compounds into

different, simpler compounds.

Biotransformations are increasing among

biotechnological science and one of its

most appreciated features is catalyzing

regiospecific and stereospecific reactions

underchemical (pH) and physical

(temperature, pressure) conditions close

to ambient the ambient condition.

Moreover, biotransformations allow for

the production of new products as well

as improve the production of already

known molecules (Giri et al., 2001). A

huge number of studies were performed

about biotransformations due to

microorganisms as (i) sugar fermentation

by Saccharomyces cerevisiae cells, (ii)

conversion mechanism of alcohol to

citric acid by Bacterium xylinum, (iii)

conversion of lactose to lactic acid by

Lactobacillus bulgaricus and (iv) the

sucrose conversion to citric acid by

Aspergillus niger, used as flavour and

preservative in foods and beverages.

Biotransformations by endophytic fungi

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Biotransformations, bioconversions,

biodegradations and fermentations were

perceived as technologies able toreplace

traditional organic chemistry, due to the

enthusiasm enhanced by their potential

applications. Then, scientists understood

that biotransformations could play above

all support and synergy roles for organic

chemistry, rather than its substitution. In

fact, biotransformations were, and still

are, used to facilitate specific steps of

semi-synthesis and synthesis of chemical

reactions, difficult to perform through

traditional methods (complete synthesis)

(Csuk et al., 1991). A huge number of

microorganisms are used in

biotechnological applications, including

endophytic ones, which are those who

live inside living plants without causing

disease.

Endophytes and biotechnological

applications

Endophytes are bacteria or fungi

living in cells of higher plant tissues,

mainly located between the cell wall and

membrane. Generally, clear symptoms

are not induced. The most interested

plant tissues are epidermis and close

parenchyma. The physical and

physiological relationship between host

and endophyte remains really poor

studied. Some authors observed that, the

mutualistic relationship plant-endophyte

seems to consist in the constant

physiological oscillation between

parasitic and pathogenic condition. In

other words, it is not already clear, what

are the conditions inducing the fungus to

become an ecological enrichment for the

plant or a vector of plant pathology.

However, when the metabolic expression

of the plant host and the endophyte is

determined by a real symbiosis, a greater

resistance to biotic and abiotic stress has

been also observed (Strobel, 2003). In

some cases, a strong mutualism

relationship between plant and

endophyte has been observed in the

specie-specific expression of the

symbiosis; i.e. the metabolic expression

of the endophyte is strictly related to

taxonomical characters of plant and

endophyte. As metabolic expression of

this aspect, it could be stressed that some

endophytes isolated in vitro produce the

same metabolite as the plant host,

proving the fact that symbiosis could

determine also a selective pressure to

develop new metabolic pathways for the

endophyte.

As an example, Taxomyces

andeanae, a species-specific endophyte

isolated from Taxus brevifolia Nutt.,

produces in vitro the alkaloid taxol, a

secondary metabolite typical of the plant

host. The pharmaceutical and economic

importance of taxol is well known; it

used in the treatment of breast cancer.

The biotechnological perspective meets

the possibility to lower costs of the anti-

cancer drug production saving the

environment – in fact, pharmaceutical

taxol needs semi-synthetic steps – and

enhancing eco-friendly production

strategy limiting solvent use

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(Suryanarayanan et al., 2009; Tan et al.,

2001). There are a lot of studies on

biotechnology applications of fungal

endophytes that improve biotechnologies

in terms of increasing product yields and

lower costs. The laccase enzymes,

isolated from endophytes belonging to

the fungal genus Monotospora sp.

isolated from the weedy grass Cynodon

dactylon (L.) Pers., represent a

successful example for the paper

industry. Laccases remove lignans from

cellulose in a particular selective way,

leaving cellulose fibers with a high

purity. This evidence represents a

relevant biotechnological perspective for

paper industry, bioremediations, bio-

fuels production and pharmaceutical

industry (i.e. excipients as

microcrystalline cellulose) (Wang et al.,

2005). Therefore, studying plants with

ethnomedical importance offers the

possibility to obtain pharmaceutically

important chemicals through

biotechnological processes. Recent

studies have shown that 50% of active

substances isolated from endophytic

fungi were previously unknown, while

for the soil microflora the same index is

considerably lower (38%) (Strobel,

2003).

The aims of the research

The present study was focused on

the extension of the scientific knowledge

related to a group of medicinal plants

from Andean and Amazonian Ecuador,

focusing on potential health applications,

integrated to biotechnological

application of endophytic fungi isolated

from these plants. The research was

performed both in Ecuador (Salesian

Polytechnic University, Quito), and in

Italy (University of Ferrara,

pharmaceutical biology labs). Based on

ethnomedical knowledge of indigenous

communities, the following plants from

the Amazon and Andes regions were

selected for this research: a) Amazonian

plants: Piper aduncum L. (Piperaceae;

common name in Ecuador “matico”);

Maytenus macrocarpa (Ruiz & Pav.)

Briq. (Celastraceae, common name

“chuchuguazo”), b) Andean plants:

Schinus molle L. (Anacardiaceae,

common name “falsopepe” or “molle”);

Tecoma stans (L.) Juss. ex Kunth

(Bignoniaceae, common name “tepla”);

Myrcianthes hallii (O. Berg) McVaugh

(Myrtaceae, common name “arrayán”).

The general outline of the

research is shown in the diagram below

(Figure 1).

Biotransformations by endophytic fungi

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Figure 1. General outline of the research.

The research was focused on

biotechnology, and especially on (i)

checking the presence of endophytic

fungi in the selected plants, (ii) isolating

and subculturing pure endophytic strains,

(iii) checking the biotransformation

capacity of the isolated endophytes on

pure compounds. The target of the

research was to identify and isolate

endophytic fungi from Andean and

Amazonian plant species of

ethnopharmaceutical interest, estimating

their biotransformation properties. Then,

quality and quantity of biotransformation

metabolites have been considered,

focusing on oxidation products

considered as the result of reaction

catalyzed by monooxygenaseenzimes.In

particular, Baeyer-Villiger reactions

have been considered, catalyzed by

flavoenzymes that catalyze oxidation

and enantioselective reactions,

converting linear and cyclic ketones into

esters and lactones respectively. These

kinds of reactions are very important in

bioremediation, in the pure chemical and

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pharmaceutical compounds synthesis

(Urlacher et al., 2006).

Materials and Methods

The research areas were the

Amazonian and Andean regions of

Ecuador, in particular the provinces of

Morona Santiago and Pichincha, as

indicated in the map (Figure 2).

The cities of Macas and Quito

were chosen for collecting vegetal plant

material. These areas have the following

environmental conditions: a) Macas,

equatorial climate at 1200 m.a.s.l., mean

temperature of 22°C, relative humidity

80%, annual precipitation 3000 mm; b)

Quito, Andean climate at 2500 m.a.s.l.,

mean temperature of 15°C, relative

humidity of 70% , annual precipitation

500 mm.

Sampling and taxonomic identification

of plants

The choice of the plant species

was made starting from the medicinal

properties attributed to plants by

Amazonian and Andean indigenous

communities; the ethnomedical

knowledge of Natives was collected by

direct interview and bibliographic

documents (Kloucek et al., 2006). This

approach gave rise to the choice of

species for which knowledge and

traditional uses are still mainly based on

oral traditions.

Figure 2. Map of Ecuador showing Morona Santiago and Pichincha provinces.

Biotransformations by endophytic fungi

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Figure 3. Images of Pichincha and Morona Santiago, the research areas.

Amazonian plant samples were

collected in March and April 2007 at

Wapu reserve and Sevilla Don Bosco

(Morona Santiago province; 2°22'`S,

78°08'W, Figure 2) and Andean plants

were collected in Quito (Pichincha

province; 0°15'S, 78°35'W). Three

different areas have been identified

(figure 3); samples of plant species were

taken from these areas, in order to

guarantee scientific significance of the

acquiring data. The identification of

plant sources was made with the help of

expert Natives, while taxonomic

identifications were carried out under the

supervision of Marco Cerna, M.Sc., an

expert in tropical botany at the Salesian

Polytechnic University (UPS) and the

National Herbarium of Ecuador (QCNE)

in Quito.

Phytochemical and biological

knowledge of selected plants

Piper aduncum, known in

Ecuador as “Matico” (Figura 4), belongs

to the Piperaceae family, characterized

by tropical plants, usually shrubs and

vines. The family includes four main

genera and more than 2000 species.

From an economic standpoint, the

species are important as they provide

black pepper (Piper nigrum L.), cubeba

(P. cubeba L. f.) and kawa (P.

methysticum G. Forst.) from which the

natives of the Pacific islands get an

alcoholic drink with sedative properties

(Schultes, 1995). Piper aduncum is a

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branched shrub that can reach 5 m in

height. It widespread as a native plant

throughout the American tropics,

including the Carribbean, Mexico, and

Central and South America and often

acts as a weed colonizing marginal areas

of urban centres (Pennington, 2004).

Piper aduncum contains as functionally

compounds terpenes (mono-, sesqui-, di-

) and alkaloids. Sevral species of Piper

are used in traditional medicine for their

antiseptic, insecticidal and antibiotic

properties. An infusion made with leaves

and roots is used to treat diarrhea,

nausea, genital and urinary infections

and also to control the bleeding in

haemorrhage. The essential oil is known

to have insecticidal properties,

molluscicides and antibacterial activity

(Guerrini et al., 2009).

Figure 4. Habit and infructescence of Piper

aduncum

Maytenus macrocarpa belongs to

the Celastraceae (Figura 5), which

includes about 50 genera and 800

species of plants with different habits:

trees, shrubs and lianas. Maytenus

comprises about 200 species in the

American and Old World tropics

(Schultes, 1995; Pennington et al.,

2004). Maytenus macrocarpa is a tree up

to 25 m tall, well branched, with reddish

bark, leaves entire, alternate, leathery,

elliptical, light green, with very small

axillary flowers (Pennington et al.,

2004).

The genus Maytenus presents a

complex and relevant, but scarcely

investigated, phytochemistry. It is rich in

particular compounds including the

macrocyclic alkaloids, which are closely

similar to fungal substances, known as

ansa-macrolide and generally

characterized by strong antibiotic

properties, named chuchuhuanine

(Shirota et al., 2004) and laevisine

(Piacente et al., 1999).

The compounds currently known

to be biologically active are alkaloids,

saponins, tannins, anthraquinones and

glycosides. Extracts from M.

macrocarpa have antibacterial activity

towards Escherichia coli and antifungal

activity towards Trichophyton rubrum

(Villacres et al., 1995)

In traditional Amazonian

medicine, M. macrocarpa is used for

production of a bark decoction with anti-

inflammatory, antirheumatic and anti-

diarrheal properties (Schultes, 1995).

Recent investigations assign to the genus

analgesic, anti rheumatic, tonic and

antianemic activities (Rios et al., 2007).

Biotransformations by endophytic fungi

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Figure 5. Leaves and bark of Maytenus

macrocarpa

Schinus molle (Anacardiaceae) is

an evergreen tree native to the inter-

Andean valleys of Ecuador and Peru

(Figure 6), and widely grown as an

ornamental street tree in tropical and

warm temperate regions such as Mexico,

southern California and Australia. It

reaches heights between 3 and 15 m; it is

characterized by rather short trunk and

fibrous dark brown bark with deep

fissures. The branches are slender and

pendent. The leaves are compound,

narrow and lance-shaped, smooth and

deep green with a characteristic smell

similar to that of pepper, if rubbed. The

hermaphrodite flowers are small,

grouped in a terminal panicle. The fruit

is a drupe similar to a pink common

peppercorn in size (Barceloux, 2008).

Extract of S. molle shows antibacterial

properties towards Klebsiella

pneumoniae, Pseudomonas aeruginosa,

Escherichia coli, Acinobacter

calcoacetica and antifungal activity

towards Aspergillus ochraceus,

Aspergillus parasiticus, Fusarium

culmorum, and Alternaria alternatea

(Gundidza, 1993).

The essential oil obtained by

steam distillation of fresh leaves of S.

molle is reported to have a significant

antifungal activity against the most

common fungi detectable in food

spoiling. The toxicity of the essential oil

persists even at temperature above 80°C

and over 90 days of storage, but

decreased significantly when autoclaved.

Its chemical composition includes 50

different compounds (Dikshit, 1986).

Figure 6. Habit of Schinus molle and its

seeds.

Tecoma stans (Bignoniaceae) is

an evergreen shrub native to the

mountains of Central and South America

(Figura 7), and attains 5-7 m in height.

The bark has a color ranging from pale

brown to gray, roughened with

increasing age. The leaves are compound

and imparipinnate with 2-5 pairs of

leaves; the leaflets are lanceolate, about

10 cm long and with an entire margin.

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The flowers are clustered at the distal

part of the branches, trumpet shaped

with yellow corolla. The fruits are

narrow and slightly flattened, about 20

cm long; they are green when immature,

and pale brown at maturity, growing on

the plant for several months (Pennington

et al., 2004). Extract of T. stans has

antibacterial activity against P.

aeruginosa and other Gram+ and Gram-

bacteria (Ramesh et al., 2009), as well as

antifungal activity towards Rhizoctonia

solani, F. oxysporum, Penicillium

expansum, A. parasiticus, Pythium

ultimum among others (Meela et al.,

2008). An infusion of leaves is known to

have diuretic properties and it is also

used to treat diabetes, intestinal and

stomach problems (Orwa et al., 2009).

Figure 7.Habit of Tecoma stans, flowers

and bark.

Myrcianthes hallii (Myrtaceae),

“Arrayán”, is native to the Andean

forests of Ecuador and Peru (Figure 8).

The genus Myrcianthes comprises about

30 species of trees extending from

Mexico to Chile. The inflorescence is

axillary, ramiflorous and sometimes

clustered at the shoot apex. Flowers have

4-5 petals and are arranged in a panicle

or sometimes solitary; the stamens are

numerous. The fruit is a berry crowned

by the persistent calyx (Pennington et

al., 2004). There are no reports regarding

the antibacterial activity of M. hallii, but

the aromatic leaves are used to flavor a

traditional beverage in Ecuador known

as “colada morada”, the fruits are edible

and infusions of the leaves have several

traditional medicinal uses in the Andes

of Ecuador (Jaramillo, 2012).

Figure 8. Flowers of Myrcianthes hallii.

Detection, isolation and identification

of endophytic fungi

All of the plant samples were

obtained from adult plants in the

flowering stage. In order to have samples

representative of the entire tree, samples

were taken at different levels of the plant

for each species: upper, middle and at

the base of the stem. The plant samples

included leaves, petioles, young twigs

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and pieces of bark. The plant samples for

endophyte isolation were immediately

wrapped in a moistened paper and then

immediately transported to the

laboratory and stored at 4 °C. Within 48

hours all the collected material was

subjected to the isolation of endophytic

fungi.

The plant material was

previously washed with running water

and then dried. The plant samples were

sanitized according to the protocol

reported by Andreotti (2004). This

operation eliminates the microorganisms

on the external surface of the plant

samples, without damaging the possible

endophytes inside the plant parenchyma.

The protocol consists in washing the

plant material in 70% ethanol (1 min),

then, dipping it in 5% sodium

hypochlorite solution (5 min) followed

by a final rinse in sterile distilled water

(10 min). The entire operation is

performed under a laminar flow hood

using sterile equipment. The samples

were cut into fragments of about 1cm

2

for leaves and bark, and about 2 cm

2

for

branches and stems. The sample

fragments were placed on the agar

surface, previously autoclaved and with

addition of 200mg/l of the antibiotic

chloramphenicol. Four different types of

culture media were used in order to

isolate as many strains of endophytes as

possible: Malt Agar (DIFCO), Malt

Extract Agar (DIFCO) and Soy Peptone

(DIFCO), Mycosel Agar (Becton

Dickinson). In practice, a different

composition in culture medium generally

contributes to easily noticeable

differences in fungal morphology

(Andreotti, 2004, Moreno 2010). A total

of 64 x 3 Petri dishes were prepared for

each species: 4 plates for each kind of

medium, 4 plates for each sample

(leaves, bark, stems, branches); each

experiment was carried out in triplicate.

After several days of incubation at room

temperature, fungal hyphae were visible

from the edge of the samples. Hyphal

samples were removed and transferred to

PDA dishes with the aim to obtain pure

culture of each fungus. The strains with

similar macroscopic characteristics – i.e.

color of the mycelium and of the

medium, the shape of mycelia margins –

were grouped and coded. The strains that

gave the best results in terms of

biotransformation activity have been

sent to the Fungal Biodiversity Centre of

the Central Bureau Voor

Schimmelcultures (CBS) in Utrecht, the

Netherlands to be taxonomically

identified. The taxonomic identifications

of the most promising strains for

biotransformations were not completed

as this article went to press. However,

suggestions about their classification

were made on the basis of our previous

experience (Andreotti, 2004; Moreno

2010).

Biotransformation activity of fungal

strains

The isolated endophytes were

tested in vitro for biotransformation

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capacities on various chemicals with the

specific aim to evaluate biocatalytic

reactions with regio- and stereo-

selective results. The chemicals

employed to assess biotransformations

were chosen for their importance as

molecular patterns similar to compounds

of pharmaceutical interest (Masood et

al., 2010; Iwaki et al., 2006).

Seven different substrates were

tested (Table 1): 2-furylmethylketone

(Fluka), acetophenone (Aldrich), cis-

bicyclo-[3,2,0]-hept-2-en-6-one (Fluka),

1-indanone (Fluka), 2-methyl-

cyclohexanone (Aldrich), 2-methoxy-

cyclohexanone (Aldrich), acetylfuran

(Fluka), 2-methyl-cyclopentanone

(Aldrich).

The endophytes were inoculated

in PDB liquid medium (Potato Dextrose

Broth, Liofilchem srl, Italy), sampling a

portion of mycelium,

previouslysuspended in tubes containing

2 ml of sterile water. The fungi samples

were then transferred to bioreactors (20

ml sterile flasks) to assess their

biotransformation capacities. Sterile

flasks (20 ml) were prepared with a

quantity of liquid medium corresponding

to a 1:5 ratio with respect to the

bioreactor volume (Andreotti, 2004;

Moreno, 2010). After inoculation, the

flasks were incubated at 27°C and

maintained under constant shaking

(120rpm). After 7 days, the fungi

reached an adequate biomass to perform

biotransformations, showing typical

mycelia with a globular shape. At this

step the compounds to assess for

biotransformations were added to the

bioreactors. To the previously prepared

culture broth, ketone solutions were

added, obtained by dissolving 0,1 mg of

ketone compounds in 1 ml DMSO

(dimethylsulfoxide).

An amount of 0.2 ml of ketone

solution was added to 20 ml of culture

broth, for each group of endophyte. One

ml of culture broth was sampled every 1,

3, 7, 10 days after inoculation to monitor

biotransformations.

Each biotransformation sample

was extracted immediately after

inoculation by adding ethyl acetate (1ml)

and anhydrous sodium sulfate (Na

2

SO

4

).

The vigorous shaking of the mixture

allowed the dissolving of possible

bioreaction products from the broth

solution to the ethyl acetate solvent,

related in polarity to the expected

alcoholic products of the substrate

reduction.

Figure 9. Flasks with liquid cultures of

endophytic fungi after 7 days of culturing. Note

the typical globular shape.

Biotransformations by endophytic fungi

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Table 1. Ketones subjected to biotranformation and pharmaceutical selection criteria

considered.

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The organic extract was initially

analyzed by silica gel TLC, using

hexane-ethyl acetate 5:1 as eluent for

acetophenone, indanone and acetylfuran;

while a mixture of petroleum ether and

diethyl ether 7:3 for the bicycloeptenone

was employed. The organic extracts of

the other substrates were directly

analyzed by GC. The products on TLC

were assessed and detected by UV light

or by spraying phosphomolybdic

solution. Once the presence of

biotransformed products was verified,

the analyses were carried out by gas

chromatography (GC 6000 Vega Series

2-Carlo Erba). The chromatographic

analyses were processed using a

capillary column (MEGADEX OV 1701

containing dimethyl-n-pentyl-β-

cyclodextrin; 25 m x 0,25 mm). Helium

(80 kPa) was used as carrier gas; air (100

kPa) and hydrogen (50 kPa) were used

for flame ionization detector. Injector

and detector temperatures were 250 °C

and 220 °C respectively.

Results and Discussion

Endophytic fungi

A total of 364 fungal strains were

isolated from aerial parts of adult plants

of Piper aduncum, Maytenus

macrocarpa, Schinus molle, Tecoma

stans, and Myrcianthes hallii,

Amazonian and Andean plants known

for their ethnomedical relevance. Each

strain was coded with reference to its

macroscopic aspect (color, colony

border, texture mycelia, exudates,

changes in medium color during

culturing).

Then groups were established

according to the similar features of the

strains and coded with an

alphanumerical code.

After one month of incubation,

80% of the plant samples on the plates

showed the presence of emerging

endophytes (Figure 10). An average of 2

fungal mycelia – easily noticeable by the

different macroscopic morphology -

could be detected for each positive plate.

All the isolated strains were

employed to test biotransformation on

pure chemicals chosen in light of their

chemical structure similar to

pharmaceutical drug intermediates. The

taxonomic identification of the

endophytes was carried out only for the

strains that were most efficient in term of

biotransformation capabilities.

Table 2. Plant sources, number of isolated

fungal strains and code assigned to strain

group.

Plant Source

No. of

isolated

fungal

strains

Code

Piper aduncum

116

from

EC01

to

EC65

Maytenus

macrocarpa

127

Schinus molle

28

from

FE1

to

FE110

Tecoma stans

32

Myrcianthes

hallii

61

TOTAL

364

Biotransformations by endophytic fungi

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Figure 10. Emerging fungal hyphae from plant tissues on agar medium.

Figure 11. Selection of fungal endophytes isolated from plants with traditional

ethnobotanical uses in Ecuador.

These strains were sent to the “Fungal

Biodiversity Centre” at the Centraal

Bureau voor Schimmelcultures (CBS) in

Utrecht, the Netherlands for

identification; this work is still in

progress as this article goes to press.

However, a preliminary identification

based on morphological aspects was

carried out by the research group. In

particular the preliminary determinations

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indicate that EC19 and EC37 are in the

genus Fusarium, while EC46, EC49,

EC59 and EC61 are Penicillium.

Fungal strains cultivated in liquid

culture medium PDB (Potato Dextrose

Broth) showed a different macroscopic

morphology if compared with ones

grown in solid culture medium PDA

(Potato Dextrose Agar). In fact, fungal

mycelia acquired a globular shape when

stirred in liquid medium. Thus, fungi

showed different size, morphology and

color compared to ones grown in solid

medium, as shown in the figures below.

Evaluation of biotransformation activity

Table 3 lists the fungal strains which

performed the most relevant

biotransformations, with descriptions of

the macromorphology of the fungal

colonies.

Endophytes isolated from P.

aduncum and M. macrocarpa showed

the most relevant results in terms of

biotransformation capabilities. In Table

4 and Table 5 are summarized the most

significant results obtained in the

biotransformation of pharmaceutical

ketones: the first table is referred to

alcohols (reduction products), the second

to the Baeyer-Villiger oxidation

products.

The strains EC17, EC19, EC37,

EC38, EC46, EC49, EC50, EC60, EC61,

FE40 and FE86 gave the best results in

terms of kind of products, yield and

enantiomeric excess (ee).

Figure 12. Fungal mycelia in liquid PDB culture medium.

Figure 13. Detail of globular shaped mycelium grown in stirred PDBliquid medium.

Biotransformations by endophytic fungi

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Table 3. Vegetal source, plant part used for endophyte isolation, culture medium and

macroscopic morphology of the fungal colonies that performed the most relevant

biotransformations.

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Table 4. Fungal strains, biotransformation products, yields, enantiomeric excesses (ee) of

pharmaceutical ketones biotransformation

Biotransformations by endophytic fungi

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Table 5. Fungal strains, biotransformation products, yields, enantiomeric excesses (ee) of

Baeyer-Villiger oxidation products.

Among all the strains, 5 of them

performed biotransformations on

acetophenone to (S)-1-phenylethanol,

with important yields (78-97%) and

enantiomeric excess (78-100%). Three

strains gave also phenols, probably by

enzymatic reactions (Baeyer-Villiger

oxidations). 15 fungal strains gave the

lactones (-)-(1S,5R)-2-

oxabicyclo[3.3.0]oct-6-en-3-one and (-)-

(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-

one from (+/-)-cis-bicyclo[3.2.0]hept-2-

en-6-one, probably as result of

monooxygenase activation.

Concerning the Baeyer-Villiger

oxidation, on cis-bicyclo[3.2.0]hept-2-n-

6-one, EC17, EC33, EC49 andEC61

showed the best results with total yield

and enantiomericexcess (Table 5). The

low production of alcohols indicate that

fungal strains preferably catalyze

oxidation reactions,in particular the

Bayer-Villiger reaction, leading to the

production of the two lactones (-)-

(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-

one and (-)-(1R,5S)-3-

oxabicyclo[3.3.0]oct-6-en-2-one.

Final Note: Ethical Implications

The scientific publications

derived from this kind of research profile

– from ethnomedicine to laboratory -

could be one of the starting points for a

public policy of protection for

indigenous peoples and rural

communities as well as natural habitats

in the Amazon and Andes region, against

bio-piracy and misappropriation of

natural sources and related knowledge

by third parties. On the other hand, if

traditional ethnomedical knowledge

provides science with opportunities to

find new sources for solutions to human

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health needs, new chemicals for new

drugs to treat old and new diseases, the

research protocols must address the

ethical implications of these values. The

present research is one example of an

attempt to use this approach based on

traditional ethnobotanical knowledge.

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