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Słupskie Prace Biologiczne

Nr 13 ss. 295-316 2016

ISSN 1734-0926 Przyjęto: 7.11.2016

© Instytut Biologii i Ochrony Środowiska Akademii Pomorskiej w Słupsku Zaakceptowano: 16.01.2017

ANTI-PSEUDOMONAS FLUORESCENS EFFICACY

OF ETHANOLIC EXTRACTS DERIVED FROM THE LEAVES

OF VARIOUS FICUS SPECIES (MORACEAE)

Halyna Tkachenko1

Lyudmyla Buyun2

Elżbieta Terech-Majewska3

Zbigniew Osadowski1

Vitaliy Honcharenko5

Andriy Prokopiv4, 5

1 Pomeranian University in Słupsk Institute of Biology and Environmental Protection Department of Zoology and Animal Physiology Arciszewski Str. 22B, 76-200 Słupsk, Poland e-mail: tkachenko@apsl.edu.pl 2 M.M. Gryshko National Botanical Garden

National Academy of Science of Ukraine, Kyiv, Ukraine 3 Department of Epizootiology, University of Warmia and Mazury in Olsztyn, Poland 4 Botanical Garden of Ivan Franko Lviv National University, Lviv, Ukraine 5 Ivan Franko Lviv National University, Lviv, Ukraine

ABSTRACT

Various studies have been carried out in discovering the different medicinal

properties of plants, although these studies are not enough to encompass the whole

plant biodiversity and the traditional use of medicinal plants. In this concern, the

plants of the Ficus genus have attracted considerable attention from pharmacologists

due to a wide range of biological properties, including antimicrobial activity. There-

fore, the main purpose of this investigation was to evaluate the susceptibility of

Pseudomonas fluorescens in vitro to the ethanolic extracts obtained from the leaves

of plants of various Ficus species, cultivated under glasshouse conditions. This in-

vestigation is in line with our previous works which have revealed a great potential

of Ficus species as plants with potent antimicrobial properties. In preparation for

this study, ethnobotanical literature on the traditional medical uses of various Ficus

species in various tropical regions of the world was surveyed. The antimicrobial ac-

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tivity of plant extracts, derived from the leaves of various Ficus species, was evalu-

ated using agar disc diffusion method. In our study, most ethanolic extracts obtained

from Ficus spp., proved effective against the bacterial strain of Gram-negative

P. fluorescens, with 8-22 mm zones of inhibition being observed. Current investiga-

tion has shown that among various species of Ficus the most effective against P. flu-

orescens were the ethanolic extracts of the leaves of ten Ficus species: F. hispida,

F. binnendijkii, F. pumila, F. rubiginosa, F. erecta, F. erecta var. sieboldii, F. sur,

F. benjamina, F. craterostoma, F. lyrata, F. palmeri (the species are listed in order of

effectiveness against pathogen tested). Thus, the extract with the greatest antibacte-

rial activity was that of F. hispida leaf (inhibition zone was 22.0 mm). Consequently,

the results of the present study confirmed the importance of the studied plants of Fi-cus spp. as a source of bioactive compounds for the treatment of P. fluorescens relat-

ed infectious diseases, in aquaculture, particularly. Finally, these findings justify the

traditional uses of these plants for therapeutic purposes.

Key words: Moraceae, Ficus spp., antimicrobial properties, fish pathogen, disc dif-

fusion technique

INTRODUCTION

Members of the genus Pseudomonas inhabit a wide variety of environments,

which is reflected in their versatile metabolic capacity and broad potential for adap-

tation to fluctuating environmental conditions (Silby et al. 2011). Pseudomonas fluo-rescens, a Gram-negative bacterium, is a member of the fluorescent pseudomonad

group. It is an aquaculture pathogen with a broad host range, which can survive and

replicate in moist reservoirs (Wong et al. 2011). P. fluorescens has generally been

regarded to be of low virulence and an infrequent cause of human infection, usually

in immunocompromised patients (Wong et al. 2011). The bacteria in the P. fluo-

rescens species complex are motile rods that are primarily aerobic, unable to ferment

glucose, and chemoorganotrophic and grow at pH values, ranging from 4 to 8

(Scales et al. 2014). Isolates of P. fluorescens derived from non-mammalian samples

have a permissive growth range from 4 to 32°C, while isolates from humans and

other mammals have an elevated upper range extending to 37°C (Scales et al. 2014).

Optimal growth generally occurs at lower temperatures than those for P. aeruginosa,

which can make identification difficult at the standard microbiology laboratory in-

cubation temperature of 37°C. It can grow at temperatures as low as 4°C, tempera-

tures at which blood products, distilled water, and disinfectants provide the ideal en-

vironment for proliferation (Pier and Ramphal 2004).

Pseudomonads exist throughout the aquatic environment and are associated with

both healthy and diseased fish (Daly and Aoki 2011). It is generally believed that

these bacteria can be opportunistic pathogens or secondary infections. For example,

when carp (Cyprinus carpio) are infected with Aeromonas salmonicida ssp. nova,

the causative agent of carp erythrodermatitis, both pseudomonads and motile aer-

omonads are isolated readily from the internal viscera (Evenberg et al. 1988). P. flu-orescens causes white nodules in the spleen and abscesses in the swim-bladder of ti-

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lapia (Sarotherodon niloticus) (Miyashita 1984). Natural mortalities were highest

when water temperatures were 15-20°C. Intramuscular injections of the bacteria caused

mortalities and pathology similar to those observed in naturally infected fish. Granulo-

mas were found in the spleen, liver and kidney (Miyashita et al. 1984). The same bacte-

rium has been reported to cause mortalities in 2-week-old tilapia fry (Duremdez and Lio-

po 1985) and causes disease in a number of other fish species, including goldfish

(Carassius auratus) (Bullock 1965), tench (Tinea tinea) (Ahne et al. 1982) and rainbow

trout (Oncorynchus mykiss) (Li and Fleming 1967; Li and Traxler 1971). P. (Alter-

omonas) putrefaciens was identified as the causative agent of a disease outbreak in cul-

tured rabbit fish (Siganus rivulatus) in the Red Sea (Saeed et al. 1987). Moribund fish

were discoloured, had haemorrhagic necrosis on the body and mouth and exhibited ex-

ophthalmia and frayed fins. The authors were able to reinfect and kill fish when bacteria

were injected intraperitoneally into healthy rabbit fish (Saeed et al. 1987, 1990). P. chlo-

roraphis has been reported to cause high mortalities in Amago trout (O. rhodurus) in

a hatchery in Japan (Hatai et al. 1975), where fish exhibited large amounts of ascitic flu-

id and haemorrhaging in various parts of the body. P. pseudoalcaligenes was isolated

from skin lesions on rainbow trout infected with Yersinia ruckeri type I (Austin and

Stobie 1992). Experimental injection of 105 cells/fish of P. pseudoalcaligenes, either in-

traperitoneally or intramuscularly, resulted in total mortalities within 7 days. Intramuscu-

lar injection resulted in some muscle liquefaction around the injection site. P. putida and

P. luteola have also been implicated in disease of rainbow trout in Turkey and Japan (Al-

tinok et al. 2006).

The impact of the intensive use of antimicrobial agents worldwide for prophylac-

tic and therapeutic purposes has been associated with the increase of bacterial re-

sistance in the exposed microbial environment. In the last three decades, pathogenic

resistant bacteria caused major health problems throughout the world in human and

veterinary medicines although the pharmacological industries produced quantities of

antibiotics (Nascimento et al. 2000). Pathogenic bacteria often develop drug re-

sistance if exposed to antibacterial drugs long term and this makes infections more

difficult to treat (Sugita et al. 2002). Many infectious diseases have been identified

to be treated with herbal products throughout the history of mankind (Wadud et al.

2007). Natural products provide enormous opportunities for the development of new

drugs, especially antimicrobials, which can have therapeutic potential to treat infec-

tious diseases (Mukherjee and Wahile 2006). There is a continuous need to discover

new antimicrobial compounds with suitable chemical structures and novel mode of

actions against pathogens. An increasing amount of evidence suggests that antimi-

crobial compounds of plant origin have an enormous therapeutic potential to treat

many infectious diseases (Mukherjee and Wahile 2006). Furthermore, numerous

plant materials are widely used in aquaculture for preventing diseases by controlling

the pathogenic microbes and enhancing the immunity (Baskaralingam Vaseeharan

and Rajagopalan Thaya 2014). In particular, leaf extracts of Tamarindus indicus,

Terminalia chebula, Citrus aurantifolia, Eugenia caryophyllata and Spondias pinna-

ta were found to inhibit the growth of all of the P. fluorescens strains, isolated from

bacterial hemorrhagic septocaemia infected carp and catfish (Foysal et al. 2011).

Medicinal plants are natural sources of compound that can be used against many dis-

eases today (Kubmarawa et al. 2007). Natural plant products have been known for cen-

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turies for their biological activities and have been widely evaluated against various bio-

logical targets such as bactericidal, virucidal, fungicidal, antiparasitic, insecticidal, anti-

cancer agents, cholesterol lowering agents, cosmetics and other pharmaceutical applica-

tions. These antimicrobial activities have been resulted after screening of a wide range of

plant species (Prabuseenivasan et al. 2006, Parasuraman et al. 2016). In recent times,

there has been renewed interest on plants as sources of antimicrobial agents due to their

use historically and the fact that a good portion of the world’s population, particularly in

developing countries, rely on plants for the treatment of infectious and non-infectious

diseases (Wadud et al. 2007, Ayoola et al. 2008). Thus, considering the undesirable side

effects of synthetic antibiotics, there is an urgent need to develop alternative treatments

that are less dangerous to humans and animals, and that have less impact on the envi-

ronment (Wei et al. 2012).

Various studies have been done which utilized plants in investigating possible an-

timicrobial remedies and in discovering the different medicinal properties of plants,

although these studies are not enough to encompass the whole plant biodiversity and

the traditional use of medicinal plants (Bekheet et al. 2011). Therefore, it is reasona-

ble to assume that natural plant products can be a valuable source to explore their

antibacterial properties against multidrug resistant pathogens, in aquaculture as well.

To test this suggestion, the genus Ficus L. (Moraceae), one of the most species-rich

and ecologically important plant genera in lowland tropical rainforests, was chosen

for evaluation of antimicrobial activity, in particular, of leaf extracts. In preparation

for this study, ethnobotanical literature was screened on the traditional medical uses

of various species of this large genus (Ali and Chaudhary 2011, Majumder and Pari-

dhavi 2013).

The genus Ficus is known for its horticultural value in most countries and as tradi-

tional medicinal of China and India. From a survey of the literature on this subject, it

was noticed that several medicinal properties (e.g., anti-diabetic, antipyretic, anti-

adipogenic, immune regulatory, cure to skin diseases, anti-tumour, antimutagenic) and

numerous phytochemicals (e.g., benzyl derivates, phenanthrene derivates, alkaloids, fla-

vonoids, pigments, sesquiterpenoids) have been recorded and reviewed in many Ficus species (Peraza-Sánchez et al. 2002, Rakesh et al. 2010, Ali and Chaudhary 2011, Bi-

darigh et al. 2011, Solomon-Wisdom et al. 2011, Shahriar et al. 2013, Afifi et al. 2014,

Woon et al. 2014, Abubakar et al. 2015, Yap et al. 2015).

Our preliminary investigations of biological activity of extracts derived from var-

ious Ficus species revealed that they have promising control effects on both Gram-

positive and Gram-negative pathogens (Tkachenko et al. 2016a-g). Being encour-

aged by the novelty in the use of crude extracts obtained from Ficus leaves, several

of these species were also used for monitoring the in vitro control efficacy against

fish pathogenic bacteria (Tkachenko et al. 2016a-g). Indeed, higher plant extracts

with an array of phytocompounds have never been overcome by any pathogenic bac-

teria. Continuing this line of work, several important Ficus species were chosen to

evaluate their antimicrobial efficacy against Pseudomonas fluorescens, which is

documented here. With this background, an attempt was made to study the in vitro

antimicrobial activity of the ethanolic extracts of various species of Ficus against the

most common fish pathogen Pseudomonas fluorescens. So the present study was

conducted to investigate in vitro antimicrobial activity of crude ethanolic extracts

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obtained from leaves of various Ficus species against fish pathogen, Pseudomonas fluorescens, isolated locally from internal organs of rainbow trout (Oncorhynchus

mykiss Walbaum) with clinical features of furunculosis.

MATERIALS AND METHODS

Collection of Plant Material. The leaves of F. aspera G. Forst, F. benghalensis L.,

F. benjamina L., F. benjamina ‘Reginald’, F. binnendijkii Miq., F. binnendijkii ‘Amstel

Gold’, F. binnendijkii ‘Amstel King’, F. carica L., F. craterostoma Warb. ex Mildbr. &

Burret, F. cyathistipula Warb., F. deltoidea Jack, F. drupacea Thunb., F. drupacea ‘Black Velvet’, F. elastica Roxb., F. elastica ‘Variegata’, F. erecta Thunb., F. erecta

var. sieboldii (Miq.) King, F. hispida L.f., F. luschnathiana (Miq.) Miq., F. lyrata Warb.,

F. macrophylla Desf. ex Pers., F. mucuso Welw. ex Ficalho, F. natalensis Hochst. subsp. natalensis, F. natalensis Hochst. subsp. leprieurii (Miq.) C.C. Berg, F. palmeri

S. Watson, F. platypoda (Miq.) A. Cunn. ex Miq., F. pumila L., F. religiosa L., F. ru-

biginosa Desf. ex Vent., F. sagittata Vahl, F. septica Burm. f., F. sur Forssk., F. sy-comorus L., F. vasta Forssk., F. villosa Blume were collected in M.M. Gryshko Na-

tional Botanical Garden (Kyiv, Ukraine) and Botanical Garden of Ivan Franko Lviv

National University (Lviv, Ukraine) (Photo 1). The whole collection of tropical and

subtropical plants both at M.M. Gryshko National Botanical Garden and Botanical

Garden of Ivan Franko Lviv National University (including Ficus spp. plants) has the

status of a National Heritage Collection of Ukraine. The species author abbreviations

were followed by Brummitt and Powell (Authors… 1992).

Photo 1. Exhibition area designed in domed glasshouse at M.M. Gryshko National Botanical

Garden of National Academy of Sciences of Ukraine (Kyiv, Ukraine) with emergent “cano-

py” trees of various Ficus species

Source: photo by V. Vakhrushkin

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Preparation of Plant Extracts. The sampled leaves of Ficus spp. were brought into

the laboratory for antimicrobial studies. Freshly crushed leaves were washed, weighted,

and homogenized in 96% ethanol (in proportion 1:10) at room temperature. The extract

was then filtered and was investigated for their antimicrobial activity.

Method of Culturing Pathological Sample. Pseudomonas fluorescens (strain E

1/7/15) isolated locally from internal organs of rainbow trout (Oncorhynchus mykiss

Walbaum) with clinical features of furunculosis (kidneys were gray, liver was pale and

fragile, normal spleen without exudate in the body cavity) was used as test organism.

Fish infection had a mixed character (Aeromonas hydrophila complex, P. orysihabitans). The increased mortality among trout has been occurred. Fish having 4-9.6 gram of body

mass with clinical signs of disease were euthanized with Propiscin (Inland Fisheries In-

stitute, Poland), anesthetic designed for fish, in immersion using 2 ml per liter of water.

Samples of internal organs (kidneys, spleen, liver) were taken and homogenized before

preincubation in TSB broth (Tripticase Soya Broth, Oxoid) for 24 hrs.

Identification Method of the Bacteria. After preincubation, bacterial culture

was transferred to different cultivation media: TSA (Tripticase Soya Agar, Oxoid),

BHIA (Brain Heart Infusion Agar, Oxoid) supplemented with 5% of sheep blood,

Kinga B agar (Oxoid) and selectiv agar GSP Lab-agar TM Base (Glutamate starch

penicillin agar, Biocorp) (Austin and Austin 1999). After 48 hrs of incubation at

27°C, characteristic confluent and translucent colonies were selected for further ex-

amination. P. fluorescens colonies on the GSP agar were blue. Bacterial species were

identified with the use of the oxidase, catalase test and API E test kit (Biomerieux,

France). The results of the test were interpreted in accordance with the manufactur-

er’s protocol, after 24 hrs of incubation at 27°C.

Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion

Method. Strain tested was plated on TSA medium (Tryptone Soya Agar) and incu-

bated for 24 hrs at 25°C. Then the suspension of microorganisms were suspended in

sterile PBS and the turbidity adjusted equivalent to that of a 0.5 McFarland standard.

Antimicrobial activity of extracts was evaluated by using agar well diffusion method

(Bauer et al. 1966). Muller-Hinton agar plates were inoculated with 200 µl of stand-

ardized inoculum (108 CFU/ml) of bacterium and spread with sterile swabs.

Sterile filter paper discs impregnated by extract were applied over each of the culture

plates, 15 min after bacteria suspension was placed. The antimicrobial susceptibility test-

ing was done on Muller-Hinton agar by disc diffusion method (Kirby-Bauer disk diffu-

sion susceptibility test protocol). The P. fluorescens isolates were individually tested

against 4 antibiotics. The tested antibiotics were as follows: oxytetracycline (30 µg), en-

rofloksacin (5 µg), gentamicin (10 µg); sulphamethoxazole/trimethoprim (25 µg).

A negative control disc impregnated by sterile ethanol was used in each experiment. The

sensitivity of strain was also studied to the commercial preparation with extracts of garlic

(in dilution 1 : 10, 1 : 100 and 1 : 1000). After culturing bacteria on Mueller-Hinton agar,

the disks were placed on the same plates and incubated for 24 hrs at 25°C. The diameters

of the inhibition zones were measured in millimeters, and compared with those of the

control and standard susceptibility disks. Activity was evidenced by the presence of

a zone of inhibition surrounding the well. Each test was repeated six times.

Statistical analysis. All statistical calculation were performed on separate data

from each species with STATISTICA 8.0 (StatSoft, Poland) (Zar 1999). The follow-

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ing zone diameter criteria were used to assign susceptibility or resistance of bacteria

to the phytochemicals tested: Susceptible (S) ≥ 15 mm, Intermediate (I) = 11-14

mm, and Resistant (R) ≤ 10 mm (Okoth et al. 2013).

RESULTS

The results of screening study of antimicrobial activity of ethanolic extracts ob-

tained from Ficus spp. leaves are presented in Figs 1-8. Ethanolic extracts of Ficus

species were tested against P. fluorescens for antibacterial properties by the agar-

well diffusion method and the values of zones of inhibition were recorded (Figs 1

and 2). A comparison of susceptibility categories, i.e. susceptible, intermediate, and

resistant, for the disk diffusion method is shown in Figs 1 and 2.

Our results showed that the P. fluorescens revealed resistance (diameters of inhibi-

tion zone were within 8-10 mm) concerning to ethanolic extracts obtained from

F. aspera, F. benghalensis, F. binnendijkii ‘Amstel Gold’, F. binnendijkii ‘Amstel

King’, F. carica, F. deltoidea, F. drupacea, F. drupacea ‘Black Velvet’, F. elastica,

F. elastica ‘Variegata’, F. luschnathiana, F. macrophylla, F. natalensis subsp. leprieurii, F. natalensis subsp. natalensis, F. sagittata, F. septica, and F. villosa (Fig. 1).

Fig. 1. Resistance of P. fluorescens against ethanolic extracts obtained from Ficus spp. leaves

(n = 6)

Source: own research

Ethanolic extracts obtained from F. benjamina, F. binnendijkii, F. craterostoma,

F. cyathistipula, F. erecta, F. erecta var. sieboldii, F. hispida, F. lyrata, F. mucuso,

F. luschnathiana

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F. palmeri, F. platypoda, F. pumila, F. rubiginosa, F. sur, F. sycomorus revealed an-

tibacterial efficacy against P. fluorescens (Fig. 2). The ethanolic extracts of F. hispi-

da and F. binnendijkii had a zone of inhibition size of 20 mm and 16 mm against

P. fluorescens (Fig. 2).

Fig. 2. Intermediate susceptibility of P. fluorescens against ethanolic extracts obtained from

Ficus spp. leaves (n = 6)

Source: own research

Thus, the most effective 15 plants at least causing a zone of inhibition 11-20 mm

were F. benjamina, F. binnendijkii, F. craterostoma, F. cyathistipula, F. erecta, F.

erecta var. sieboldii, F. hispida, F. lyrata, F. mucuso, F. palmeri, F. platypoda, F. pu-

mila, F. rubiginosa, F. sur, and F. sycomorus. The unique plant that controlled path-

ogen was F. hispida. Moderate control capacity was exhibited by the following

eighteen plants: F. aspera, F. benghalensis, F. binnendijkii ‘Amstel Gold’, F. binnen-

dijkii ‘Amstel King’, F. carica, F. deltoidea, F. drupacea, F. drupacea ‘Black Vel-

vet’, F. elastica, F. elastica ‘Variegata’, F. luschnathiana, F. macrophylla, F. na-

talensis subsp. leprieurii, F. natalensis subsp. natalensis, F. sagittata, F. septica, and

F. villosa. The remaining four plants F. religiosa, F. benjamina ‘Reginald’, F. vasta

exhibited the least control over microorganism tested. Detailed data regarding the

zones of inhibition by the ethanolic plant extracts were recorded (Figs 3-8).

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A B

Fig. 3. Antimicrobial activity of ethanolic extracts obtained from F. mucuso (3), F. bengha-

lensis (4), F. benjamina (5) (A), as well as F. sycomorus (9) and F. aspera (12) against

P. fluorescens (B) measured as inhibition zone diameter

Source: own research

A B

Fig. 4. Antimicrobial activity of ethanolic extracts obtained from F. cyathistipula (14), and

F. lyrata (15), F. binnendijkii (18) (A), as well as F. sur (21) and F. pumila (22) against P.

fluorescens (B) measured as inhibition zone diameter

Source: own research

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A B

Fig. 5. Antimicrobial activity of ethanolic extracts obtained from F. craterostoma (27) (A),

as well as F. natalensis subsp. leprieurii (34) and F. binnendijkii ‘Amstel Gold’ (35) against

P. fluorescens (B) measured as inhibition zone diameter

Source: own research

A B

Fig. 6. Antimicrobial activity of ethanolic extracts obtained from F. erecta var. sieboldii

(37), F. rubiginosa (40) and F. erecta (41) (A), as well as F. palmeri (50), F. hispida (51),

and F. lyrata (44) against P. fluorescens (B) measured as inhibition zone diameter

Source: own research

305

A B

Fig. 7. Antimicrobial activity of ethanolic extracts obtained from F. palmeri (50), F. hispida

(51), and F. pumila (53) (A), as well as F. benjamina (56) against P. fluorescens (B) meas-

ured as inhibition zone diameter

Source: own research

A B

Fig. 8. Antimicrobial activity of the commercial preparation with extracts of garlic (in dilu-

tion 1:10, 1:100 and 1:1000) (A), as well as selected antibiotics against P. fluorescens (B)

measured as inhibition zone diameter

Source: own research

DISCUSSION

This work demonstrated that ethanolic extracts of 10 plants, F. hispida, F. bin-

nendijkii, F. pumila, F. rubiginosa, F. erecta, F. erecta var. sieboldii, F. sur, F. ben-

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jamina, F. craterostoma, F. lyrata, F. palmeri in order of effectivity as stated, were

the most effective against P. fluorescens (Figs 1-2).

Our observations are in well agreement with the reports by several workers who

have investigated antimicrobial properties of Ficus species previously (Olusesan et

al. 2010, Solomon-Wisdom et al. 2011, Parmar Namita and Rawat Mukesh 2012,

Salem et al. 2013). For example, F. hispida was traditionally used for the treatment

of dysentery, diarrhea, ulcers, biliousness, psoriasis, anemia, piles and jaundice due

to the presence of various important pharmacological activities like antioxidant, car-

dioprotective, hepatoprotective, anticancer, anti-inflammatory, etc. All parts of this

plant are found to be acrid, astringent, bitter, coolant, while the fruit juices along

with honey act as a good antihemorrhagic agent. Additionally, the fruit is known to

be active as aphrodisiac, tonic, lactagogue and emetic (Ali and Chaudhary 2011).

Secondary plant products can have a variety of functions in plants. It is likely

that their ecological function may have some bearing on potential medicinal effects.

For example, secondary products involved in plant defense through cytotoxicity to-

wards microbial pathogens could prove useful as antimicrobial agents, if not too tox-

ic. The role of secondary products as defense chemicals would not only ensure effec-

tiveness against a wide range of pathogens but would also decrease the chances of

these organisms developing resistance or adaptive responses (Singh and Watal

2010).

Phytochemical profiling studies on plants of various Ficus species discovered

that they contain secondary metabolites such as saponins, flavonoids, tannis, poly-

phenols, triterpenoids, and proanthrocyanins (Ghosh et al. 2004, Salem et al. 2013).

Preliminary phytochemical screening of F. hispida has shown the presence of alka-

loids, carbohydrates, proteins and amino acids, sterols, phenols, flavonoids, gums and

mucilage, glycosides, saponins, and terpenes (Ghosh et al. 2004). The bark of F. his-

pida contains lupeol acetate, β-amyrine acetate, β-sitosterol (Acharya and Kumar 1984),

while in the dried bark powder, acetates of n-triacontanol, β-amyrin and gluanol

were extracted (Acharya and Kumar 1984). A new norisoprenoid, ficustriol, and the

known phenanthroindolizidine alkaloid O-methyltylophorinidine, were isolated from

a CHCl3 extract of the leaves and twigs of F. hispida. O-Methyltylophorinidine

showed potent cytotoxic activity when tested against a small panel of human cancer

cells, while ficustriol was inactive (Peraza-Sánchez et al. 2002). In the research car-

ried out by Venkatachalam and co-workers (1982), two substantial phenanthroindoliz-

idine alkaloids, 6-O-methyltylophorinidine and 2-demethoxytylophorine, and a novel

biphenylhexahydroindolizine hispidine from stem and leaves of F. hispida were iso-

lated. Hispidacine, an 8,4'-oxyneolignan featuring incorporation of an unusual 2-hydro-

xyethylamine moiety at C-7, and hispiloscine, a phenanthroindolizidine alkaloid,

were isolated from the stem-bark and leaves of the Malaysian F. hispida. Hispida-

cine induced a moderate vasorelaxant activity in rat isolated aorta, while hispiloscine

showed appreciable antiproliferative activities against MDA-MB-231, MCF-7, A549,

HCT-116 and MRC-5 cell lines (Yap et al. 2015). The potential synergism of anti-

proliferative effects induced by individual alkaloid extracts of F. fistulosa, F. hispida

and F. schwarzii combined with δ- and γ-tocotrienols against human brain glioblasto-

ma (U87MG), lung adenocarcinoma (A549) and colorectal adenocarcinoma (HT-29)

cells was explored by Abubakar and co-workers (2015). Cell viability and morpho-

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logical results demonstrated that extracts containing a mixture of alkaloids from the

leaves and bark of F. schwarzii inhibited the proliferation of HT-29 cells, whereas the

alkaloid extracts of F. fistulosa inhibited the proliferation of both U87MG and HT-29

cells and showed synergism in combined treatments with either δ- or γ-tocotrienol re-

sulting in 2.2-34.7 fold of reduction in IC50 values of tocotrienols (Abubakar et al.

2015). Two new pyrrolidine alkaloids, ficushispimines A (1) and B (2), a new ω-

(dimethylamino)caprophenone alkaloid, ficushispimine C (3), and a new indolizidine al-

kaloid, ficushispidine (4), together with the known alkaloid 5 and 11 known isoprenylat-

ed flavonoids 6-16, were isolated from the twigs of F. hispida by Shi and co-workers

(2016). Isoderrone (8), 3'-(3-methylbut-2-en-1-yl)biochanin A (11), myrsininone A (12),

ficusin A (13), and 4',5,7-trihydroxy-6-[(1R*,6R*)-3-methyl-6-(1-methylethenyl)cyclo-

hex-2-en-1-yl]isoflavone (14) showed inhibitory effects on α-glucosidase in vitro (Shi et

al. 2016).

Together with fifteen known secondary metabolites including cellobiosylsterol (3),

β-sitosterol (4), stigmasterol (5), β-sitosterol 3-O-β-D-glucopyranoside (6), lupeol ace-

tate (7), ursolic acid (8), procatechuic acid (9), 2-methyl-5,7-dihydroxychromone 8-C-β-

D-glucoside (10), apigenin (11), (-)-epicatechin (12), (+)-catechin (13), N-benzoyl-L-

phenylalanilol (14), α-acetylamino-phenylpropyl α-benzoylamino-phenylpropionate (15),

asperphenamate (16) and bejaminamide (17), structures of compounds of two new sphin-

golipids mucusamide (1) and mucusoside (2) have been isolated from methanol soluble

part of the stem bark of F. mucuso by Bankeu and co-workers (2010). Analysis of F. ben-

jamina indicated four phenolic compounds (chlorogenic, p-coumaric, ferulic and syrin-

gic acids) in roots, three (chlorogenic p-coumaric and ferulic acids) in stem and only one

(caffeic acid) in leaves (Imran et al. 2014). Flavonoids have also been reported to have

antimicrobial activities (Bylka et al. 2004). The presence of flavonoids in plant extracts

could be responsible for their antimicrobial activity. This justifies the ethnomedicinal use

of the plants. Flavonoid compounds have been reported to display strong antimicrobial

activity (Özçelik et al. 2008).

Flavonoids, hydroxyl groups on their β-rings are more active against microorgan-

isms and have also been found that the more hydroxylation, the greater the antimi-

crobial activity (Sato et al. 1996). Levels of total phenolics, total flavonol and total

flavonoid compounds in F. bengalensis aerial roots in 70 mg/g of extract, 3 mg/g

quercetin equivalent and 5 mg quercetin equivalent/gm extract have also been re-

ported (Sharma et al. 2009).

Ficus deltoidea var. deltoidea and var. angustifolia is traditionally used in herbal

remedies to treat hypertension, diabetes, headache, and fever, and to reduce the risk

of cancer (Woon et al. 2014). Results of study of Imran and co-workers (2014) indi-

cate that F. benjamina extracts can be used as a potential antimicrobial agent to in-

hibit the growth of various dangerous microbes. The extracts and fractions of stem,

root and leaves exhibited considerable antimicrobial activity against four bacterial

and two fungal strains (Aspergillus niger ATCC 10595, Candida albicans ATCC

32612, Pseudomonas aerugonisa locally isolated, Escherichia coli ATCC 25922,

Bacillus subtilis JS 2004, Bacillus cereus locally isolated). The butanol fraction from

leaves again showed higher antimicrobial potential as is evident from its values

19.50 mm and 19.75 mm against B. cereus and C. albicans, respectively. The meth-

anol, n-hexane, chloroform and ethyl acetate fractions of leaves showed moderate

308

activity with considerable value shown by chloroform fraction (12 mm). However,

n-butanol fraction of leaves showed strong activity against B. cereus. Comparatively,

n-hexane and ethyl acetate fractions denoted lower antimicrobial potential (Imran et

al. 2014).

The crude extract of F. hispida and its different partitionates when subjected to an-

timicrobial screening at 400 μg/disc, the crude ethanol extract and its methanol soluble

fraction had been reported to reveal antimicrobial activity against microorganisms

(Bacillus cereus, B. megaterium, B. subtilis, Staphylococcus aureus, Sarcina lutea,

Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphi, S. typhi, Shigella boydii, S. dysenteriae, Vibrio mimicus, V. parahemolyticus, Candida albicans, Asper-

gillus niger, Sacharomyces cerevacae) having the zone of inhibition ranging from 9 to

12 mm (Shahriar et al. 2013). Sanowar and co-workers (2014) suggests that active an-

timicrobial agents present in the extract of F. hispida fruits may have potential for the

treatment of bacterial infection. They have investigated the methanolic extract of

F. hispida fruits and its fractions (chloroform, ethyl acetate and aqueous fractions) for

their in vitro antimicrobial activities. The extract and its fractions exhibited reasonable

antibacterial activities against three Gram-positive (Bacillus cereus, Staphylococcus aureus, Agrobacterium species) and three Gram-negative (Escherichia coli, Shigella

dysenteriae, Shigella sonnei) pathogenic bacteria. Ethyl acetate fraction (EAF) showed

highest zone of inhibition (20.5 and 28 mm in diameter) against E. coli at a concentra-

tion of 200 and 400 μg/disc, respectively. Besides, they revealed that the activity of

crude methanolic extract was higher (20 mm in diameter) than EAF against Gram-

positive bacteria. The chloroform fraction did not show any activity against both

Gram-positive and Gram-negative bacteria (Sanowar Hossain et al. 2014).

In study of Sayed and co-workers (2015), the inhibitory effect of F. sycomorus

aqueous extract on methicillin-resistant Staphylococcus aureus growth was noted.

The effect of F. sycomorus alcoholic extract was bacteriostatic while it was bacteri-

cidal for the aqueous extracts of F. sycomorus leaves. The leaves of F. sycomorus

were reported to contain calcium, phosphorous, iron, magnesium and zinc. Addi-

tionally, Sandabe and co-workers (2006) reported, that the stem bark extract contains

tannins, saponins, reducing sugars, flavones, aglycones, anthraquinone glycosides,

flavonoid glycosides and condensed tannins.

Chemical investigation of leaf extracts from F. cyathistipula led to the isolation of

α-amyrin palmitate (1), lupeol acetate (2), taraxerol (3), β-sitosterol (4), protocatechuic

acid (5) and 3-O-caffeoyl quinic acid (6) that were identified by El-Sakhawy and co-

workers (2016). Presence of flavonoid glycosides, phenolic acids, isoflavones, couma-

rins and fatty acids was also revealed. Fractions obtained by successive partition of etha-

nolic extract of F. cyathistipula possessed antioxidant and antimicrobial activities (El-

Sakhawy et al. 2016). Essien and coauthors (2016) proved that F. mucoso essential oil

could also be used to induce significant antimicrobial activity against the Gram-positive

bacteria, Bacillus cereus (ATCC 14579) and Staphylococcus aureus (ATCC 29213) and

the Gram-negative bacteria, Pseudomonas aeruginosa (ATCC 27853) and Escherichia

coli (ATCC 254922).

F. lyrata has been reported to have numerous bioactive compounds such as arab-

inose, β-amyrins, β-carotenes, glycosides, β-setosterols and xanthotoxol (Jeong and

Lachance 2001, Vaya and Mahmood 2006). The secondary metabolite profiles of

309

F. lyrata leaves and fruits (flavonoids, phenolic acids and fatty acids) were assessed

by Farag and co-workers (2014). A total of 72 metabolites were evaluated. Seven-

teen flavonoids were characterised and tentatively identified with the main constitu-

ents being catechins/procyanidins, O- and C-linked flavonoid glycosides. The major

procyanidins were dimers and trimers comprising (epi)catechin and (epi)afzelechin

units, whereas the predominant flavones were C-glycosides of luteolin and apigenin.

Aside from these major flavonoid classes, a group of benzoic acids, caffeoylquinic

acids, fatty acid and sphingolipids were also annotated (Farag et al. 2014). Bidarigh

and co-workers (2011) also studied the antimicrobial activity of ethyl acetate latex

extract of F. lyrata and Nystatin on 65 clinical isolates of Candida albicans from

Vulvovaginal candidiasis and standard strain of C. albicans (ATCC 5027). Inhibitory

effect of the crude extract was analyzed by using the disk diffusion technique (Bauer

et al. 1966). F. lyrata extract has inhibitory effect on clinical isolates and type strain

of C. albicans in lower concentrations than Nystatin drug with the diameters of inhi-

bition zones ranging from 22 to 26 mm and 30 to 32 mm for clinical isolates and

standard strains of C. albicans, respectively. The diameter of inhibition zones for

Nystatin was between 16 to 20 mm and 21 to 24 mm for standard strain and clinical

isolates of C. albicans, respectively. Based on the data analysis (Macrobroth dilution

method), the best Minimal Inhibitory Concentration (MIC) of F. lyrata ethyl acetate

latex extract on clinical isolates and type strain of C. albicans were 25 mg/ml and

2.5 mg/ml, respectively. In another study, the chemical analysis of latex showed that

extract contains alkaloids, flavonoids, phenols, tannins, terpenoid (Bidarigh et al.

2011). Ethyl acetate extract of F. lyrata latex possesses compounds with antibacteri-

al and anticandidal properties which can be used as antimicrobial agents in new

drugs for therapy of infectious diseases (Bidarigh et al. 2011). Bidarigh and co-

workers (2011), on the other hand, stated that the methanolic extract had no effect

against bacteria except for Proteus mirabilis while the ethyl acetate extract had inhi-

bition effect on the multiplication of five bacteria species (Enterococcus faecalis,

Citrobacter freundii, Pseudomonas aeruginosa, Escherichia coli and Proteus mira-

bilis).

The biological screening results obtained by Solomon-Wisdom and co-workers

(2011) are indicative of the potential of F. sur as antimicrobial substance. The anti-

microbial activity and chemical constituents of the leaves and stem bark extract of

F. sur were investigated. The extracts at crude level were shown in vitro to inhibit

Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida pseudotropi-calis of the six test organisms at 2 mg/ml. Pseudomonas aeruginosa and Salmonella

typhimorium were not inhibited at the same concentration. The stem bark extract had

a wide spectrum of activity against some micro-organisms at minimum inhibitory

concentration of 0.5 mg/ml. The leaf extract also had activity on the microorganisms

but at 1.0 mg/ml. Saponins, saponin glycosides, tannins, phenols and volatile oils

were the important phytochemical components found in the plant parts which may

be responsible for the biological properties of this plant (Solomon-Wisdom et al.

2011).

Six compounds were isolated for the first time from the leaves of F. platypoda and

were identified as 3-methoxycarpachromene [1], pheophorbide-a methyl ester [2], phe-

ophorbide-b methyl ester [3], (6S, 9R)-3-oxo-6-hydroxy-α-ionol [4], β-sitosterol-3-O-β-

310

D-glucopyranoside [5] and rutin [6] (Afifi et al. 2014). The different crude extracts (pe-

troleum ether, ethyl acetate and n-butanol) showed selective antimicrobial activities

against two Gram-positive bacteria, seven Gram-negative bacteria and two fungi. More-

over, compound 2 showed potent antimalarial activity against both chloroquine-sensitive

and chloroquine-resistant strains of Plasmodium falciparum with IC50 values in the range

of 3.9 μg/ml without showing any cytotoxicity to mammalian cells but showing weak

antileishmanial activity with IC50 value of 40 μg/ml. The petroleum ether and ethyl ace-

tate extracts showed remarkable cytotoxic activities against two human tumor cell li-

nes: Human colon carcinoma (HCT-116) and Human breast cancer (MCF-7) cell lines,

whereas week activities were detected with the n-butanol extract (Afifi et al. 2014).

Our present study has also revealed a great potential of Ficus species as plants

with potent antimicrobial properties. In our study, most ethanolic extracts obtained

from Ficus spp., proved effective against the bacterial strain of Gram-negative

P. fluorescens, with 8-22 mm zones of inhibition being observed. Current investiga-

tion has shown that among various species of Ficus the most effective against P. flu-

orescens were the ethanolic extracts of the leaves of ten Ficus species: F. hispida,

F. binnendijkii, F. pumila, F. rubiginosa, F. erecta, F. erecta var. sieboldii, F. sur, F. benjamina, F. craterostoma, F. lyrata, F. palmeri (the species are listed in the or-

der of effectiveness against pathogen tested). Thus, the extract with the greatest anti-

bacterial activity was that of F. hispida leaf (inhibition zone was 22.0 mm). Conse-

quently, the results of the present study confirm the importance of the studied plants

of Ficus spp. as a source of bioactive compounds for the treatment of P. fluorescens

related infectious diseases, in aquaculture, particularly.

It should be noted, that apart from limited research being carried out on various

Ficus species so far, several species from this pantropical genus are categorized as

vulnerable (Kumara et al. 2013). Therefore, maintenance of living plants collection

of Ficus species at Botanical Gardens in temperate climate zone can be considered

as valuable tool for both ex situ biodiversity conservation and accumulation the trop-

ical plants with promising antimicrobial potential.

CONCLUSIONS

Our results indicated that ethanolic leaf extracts of Ficus species tested offer

a promising alternative to the use of antibiotics in controlling Pseudomonas fluo-rescens growth. In fact, most ethanolic extracts obtained from Ficus spp., proved ef-

fective against the bacterial strain of Gram-negative P. fluorescens, with 8-20 mm

zones of inhibition being observed. It should be noted that P. fluorescens demon-

strated the highest susceptibility to F. hispida leaf extracts. Current investigation has

shown that among various species of Ficus the most effective against P. fluorescens were the ethanolic extracts of the leaves of ten Ficus species: F. hispida,

F. binnendijkii, F. pumila, F. rubiginosa, F. erecta, F. erecta var. sieboldii, F. sur,

F. benjamina, F. craterostoma, F. lyrata, F. palmeri (the species are listed in the or-

der of effectiveness against pathogen tested).

Therefore, the preliminary screening assay indicated that the leaves of some Fi-cus spp. with antibacterial properties may offer alternative therapeutic agents against

311

bacterial infections in aquaculture industry. On the base of the present results, it is

proposed that these products can be used in aquaculture as therapeutic and prophy-

lactic agents against fish pathogens, with antimicrobial properties.

Nevertheless, more extensive studies should be conducted prior to the develop-

ment of novel antimicrobial pharmaceuticals from Ficus spp. extracts. The potential

antimicrobial compounds comprising in the extracts of various Ficus species tested

should be isolated, purified, and further examined. Finally, the mechanisms of action

of these potential active compounds should also be assessed.

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SUMMARY

For a long period of time, plants have been a valuable source of natural products

both for maintaining human health and for use in veterinary. The medicinal proper-

ties of plants have been investigated worldwide. In recent times, there has been re-

newed interest on plants as sources of antimicrobial agents because pathogenic re-

sistant bacteria caused major health problems throughout the world in human and

veterinary medicines despite a large number of antibiotics, produced by the pharma-

cological industry. Extensive studies have been undertaken which used plants in in-

vestigating different medicinal properties of plants and in discovering possible anti-

microbial agents, although these studies are not enough to encompass the whole

plant biodiversity and the traditional use of medicinal plants. In preparation for this

study, ethnobotanical literature was surveyed on the traditional medical uses of vari-

ous Ficus species in various tropical regions of the world. Antibacterial activity of

ethanolic extracts obtained from the leaves of plants of various Ficus species, culti-

vated under glasshouse conditions, was assayed against the most common fish path-

ogen Pseudomonas fluorescens. The main purpose of this investigation was to eval-

uate the susceptibility of P. fluorescens in vitro to these extracts. The antimicrobial

activity of plant extracts, derived from the leaves of various Ficus species, was eval-

uated using agar disc diffusion method. In our study, most ethanolic extracts ob-

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tained from Ficus spp., proved effective against the bacterial strain of Gram-

negative P. fluorescens, with 8-22 mm zones of inhibition being observed. Current

investigation has shown that among various species of Ficus the most effective

against P. fluorescens were the ethanolic extracts of the leaves of ten Ficus species:

F. hispida, F. binnendijkii, F. pumila, F. rubiginosa, F. erecta, F. erecta var. sieboldii,

F. sur, F. benjamina, F. craterostoma, F. lyrata, F. palmeri (the species are listed in

the order of effectiveness against pathogen tested). Thus, the extract with the great-

est antibacterial activity was that of F. hispida leaf (inhibition zone 22.0 mm). There-

fore, the preliminary screening assay indicated that the leaves of some Ficus spp.

with antibacterial properties may offer alternative therapeutic agents against bacteri-

al infections in aquaculture industry. These products can be used in aquaculture as

therapeutic and prophylactic agents against fish pathogens, with antimicrobial and/or

immunostimulant properties.