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Słupskie Prace Biologiczne

Nr 13 ss. 277-294 2016

ISSN 1734-0926 Przyjęto: 7.11.2016

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

IN VITRO MICROBIOLOGICAL INVESTIGATION

OF ETHANOLIC EXTRACTS OBTAINED

FROM LEAVES OF VARIOUS BEGONIA SPECIES

AGAINST ESCHERICHIA COLI

Halyna Tkachenko1

Lyudmyla Buyun2

Zbigniew Osadowski1

Yana Belayeva2

1 Pomeranian University in Słupsk

Institute of Biology and Environmental Protection Department of Zoology and Animal Physiology Arciszewski St. 22B, 76-200 Słupsk, Poland e-mail: tkachenko@apsl.edu.pl 2 M.M. Gryshko National Botanical Garden National Academy of Science of Ukraine Timiryazevska St. 1, Kiev 01014, Ukraine

ABSTRACT

Many plants of the family Begoniaceae are used in the treatment of different diseas-

es. Traditionally leaves of Begonia are applied to cure venereal diseases and give cooling

effects to the body, to increase the body vigor and the body vigor and weight, for de-

creasing of fleeting pain in the limbs and joints, for blood purification, to reduce the

body temperature, for treating anemia, for the treatment of respiratory infections, diar-

rhea, blood cancer and skin diseases, peptic ulcer, conjunctivitis, colic and dyspepsia,

dysentery and mouth ulcer. Moreover, the leaves of Begonia species are used for the

treatment of cancer; beside they possess the anti-HIV activity. Some of the plants of the

genus Begonia were previously reported for their antimicrobial activities. The present

work was designed to assess the anti-Escherichia coli activity of the ethanolic extracts

from the leaves of Begonia species plants, cultivated under glasshouse conditions at

M.M. Gryshko National Botanical Garden (NBG), National Academy of Science of

Ukraine. The leaves of B. solimutata L.B. Sm. & Wassh., B. goegoensis N.E.Br., B. fo-

liosa Kunth, Begonia × bunchii L.H. Bailey (syn. Begonia × erythrophylla Hérincq),

B. thiemei C.DC., B. peltata Otto & Dietr., B. heracleifolia Cham. & Schltdl., B. dregei Otto & Dietr., B. mexicana G. Karst. ex Fotsch were sampled for study. Antimicrobial

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activity was determined using the agar disk diffusion assay. The present study has shown

that ethanolic extracts obtained from leaves of Begonia species have moderate activity

against E. coli. The diameters of inhibition zone for B. solimutata were 14 mm, 11.5 mm

for B. goegoensis, 13 mm for B. foliosa, 13.5 mm for Begonia × bunchii, 15 mm for B. thiemei, 19 mm for B. peltata, 12 mm for B. heracleifolia, 11.5 mm for B. dregei, and

16 mm for B. mexicana. The highest antimicrobial effect was recorded for B. peltata, B. mexicana, and B. thiemei. The most antimicrobially effective plant against E. coli was

B. peltata, being highly active with the ethanolic extract (diameter of inhibition zone was

19 mm). The highly active antimicrobial effects noted against E. coli are worthy of high-

lighting. The obtained results highlighted the interesting antimicrobial potency of various

Begonia species and provided scientific basis for the traditional use of these plants in the

treatment of microbial infections.

Key words: Begonia, Escherichia coli, leaves, ethanolic extract, antimicrobial ac-

tivity, agar disk diffusion assay

INTRODUCTION

Plants have been crucial in sustaining human health and well-being of mankind

and are the primary source of therapeutics (Wadud et al. 2007). The main advantages

and benefits associated with the use of medicinal plants are their cost-effectiveness and

global availability as well as their safety compared to other medicinal products and the

lack of major side-effects. Each part of the plant including seeds, root, stem, leaves,

and fruit, potentially contains bioactive components (Yamani et al. 2016).

Plant preparations and medications continue to be used in the treatment of numer-

ous disorders (Mohanty and Cock 2010). The revival of interest in herbal medications

is due to a lower incidence of adverse reactions to plant preparations compared to syn-

thetic pharmaceuticals, as well as an alarming increase in the incidence of new infec-

tions diseases and the development of resistance to the antibiotics in current clinical

use (Kamazeri et al. 2012). For many plant treatments, the antimicrobial activity has

been proven. On the other hand, for many plant-based antiseptics, the mechanisms of

action are not clear. Many traditionally used antiseptic agents have yet to be subjected

to thorough scientific investigation (Mohanty and Cock 2010).

Studies of medicinal plants are important sources of knowledge for development of

less harmful and more effective antimicrobial agents for treatment of various infec-

tions. There has been an increased interest in looking at antimicrobial properties of ex-

tracts from various plants. Plant based antimicrobial agents have enormous therapeutic

potentials as they can serve the purpose with lesser side effects that are often associat-

ed with synthetic compounds. Antimicrobial properties of medicinal plants are being

increasingly reported from different parts of the world. In an effort to expand the spec-

trum of antibacterial agents from natural resources, genus Begonia belonging to Be-

goniaceae family has been selected, because among plant-based antimicrobials, the

antimicrobial activity of Begonia species has been well studied (Ramesh et al. 2002,

Maridass 2009, Dan-Ping et al. 2012, Jeeva et al. 2012, Indrakumar et al. 2014, Amu-

tha and Sreedevikumari 2016, Nisha Shrestha et al. 2016).

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Begonia is one of the most species-rich angiosperm genera with approximately

1500 species currently recognized (Frodin 2004). Begonia is now considered to be

one of the five largest genera of vascular plants (Hoover et al. 2004), and the genus

is increasingly used as a model for understanding the evolution of species-rich gene-

ra (Dewitte et al. 2011).

Most species of Begonia are monoecious herbs with cymose inflorescences

(Ågren and Schemske 1991). They present staminate and pistillate flowers in differ-

ent phases of anthesis, with staminate flowers usually open before pistillate flowers.

Typically, pistillate flowers open and become receptive only after staminate flowers

have senesced (Wyatt and Sazima 2011). It was shown that gender specialization in

Begonia facilitates the unusual pollination strategy of the genus, wherein male flow-

ers supply a pollen as reward, whereas pistillate flowers are non-rewarding intersex-

ual mimics of staminate flowers and are pollinated by deceit (Ågren and Schemske

1991).

The genus has a near pantropical distribution being absent only from the Austral-

ian tropical forest (Photo 1).

Photo 1. Begonia sp. plants, growing in natural habitats, Vietnam, Lam Dong Province

Source: photo by L. Buyun

There are centers of diversity in both the Neotropics and mainland Asia but the

genus is relatively species poor in Africa. The African species, despite being rela-

tively small in number, show the greatest morphological diversity (Neale et al.

2006). The recent phylogenetic studies of Begoniaceae have indicated that the most

basal Begonia species are African, from which both Asian and American Begonia

species are derived (Forrest and Hollingsworth 2003, Harrison et al. 2016). Species

radiation in the South America, South-East Asia and Africa, has generated many ex-

amples of parallel evolution, for example in leaf form, plant architecture, inflo-

rescence arrangement and drought tolerance, reproductive systems, including di-

chogamy (Neale et al. 2006, Twyford et al. 2014).

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Although Begonia species are typical herbs for wet rainforest, the genus also ex-

hibits substantial diversity in ecology, with ranges from dry desert scrub through to

wet rainforest, and at altitudes from sea level to over 3,000 meters (Tebbitt 2005).

Therefore, the large numbers of species, pantropical distribution and a solid horticul-

tural background make Begonia an excellent system for the study of plant evolution

in tropical environments (Neale et al. 2006). In particular, Begonia is an ideal study

system to investigate the evolution of floral sex ratios and the relationship with in-

florescence architecture (Twyford et al. 2014).

Many begonias are popular ornamental plants. Species have been in cultivation for at

least 600 years (Neale et al. 2006). Begonias are usually easily recognized by their more

or less asymmetrical leaves and dry three-winged fruit (in most species). However, there

is a huge amount of morphological diversity contained within the genus, making it an

excellent group for studying the evolution of a large variety of traits.

Its leaves, flowers, and roots are used in diverse ailments in traditional and folk-

lore remedies. Traditionally leaves of Begonia floccifera Bedd. are used to cure ve-

nereal diseases and give cooling effects to the body among the tribal inhabitants of

Tirunelveli Hills of Western Ghats (India) (Ayyanar and Ignacimuthu 2008). In In-

dia, Begonia floccifera is used to increase the body vigor, the body weight and to al-

leviate fleeting pain in the limbs and joints. The juice of the fresh leaves is given to

the young babies for proper development of teeth and bone. It also arrests the gum

and teeth diseases. The juice of the leaves mixed with honey is taken in as a tonic

and they believe that is having the rejuvenation capacity (Ariharan et al. 2012).

The juice of Begonia malabarica Lamk is used along with honey for blood puri-

fication. It is given in for fever to reduce the body temperature and it is taken as

a general health tonic. The leaf juice mixed with ginger is taken for treating anemia

(Ariharan et al. 2012). The leaves are used for the treatment of respiratory infec-

tions, diarrhea, blood cancer and skin diseases (Nisha et al. 2009).

The juice of B. picta is taken to relieve headache and also consumed in treatment

of peptic ulcer. The paste is applied to stop bleeding from cuts and wound and is ap-

plied externally on ringworm and scabies. The root juice is used as eyes wash to

treat conjunctivitis. The whole plant is feed to sterile animals to help them conceive.

Whole plant is used as appetizer and juice of leaves is given to relieve the fever.

Plant decoction is used in colic and dyspepsia. Paste of young shoot also has taken

for respiratory tract infections. B. picta also called patherchattha in India is used in

dysentery and mouth ulcer. Juice of B. picta was used as slight venom. Other species

of Begonia, i.e. B. nepalensis is used as anthelmintic in Nepal (Shrestha et al. 2016).

Moreover, the leaves of Begonia species are used for the treatment of cancer and

possess the anti-HIV activity (Wu et al. 2004). These researchers have isolated and char-

acterized three compounds: begonanline (1) nantoamide (2) and methyl (S)-glycerate (3)

as well as forty-four known compounds from the rhizomes of B. nantoensis. Among

them, cucurbitacin B (4), dihydrocucurbitacin B (5), cucurbitacin E (6), dihydrocucur-

bitacin E (7), cucurbitacin I (8), and (-)-auranamide (9) showed cytotoxicity against four

human cancer cell lines. 3β,22α-Dihydroxyolean-12-en-29-oic acid (10), indole-3-carbo-

xylic acid (11), 5,7-dihydroxychromone (12), and (-)-catechin (13) demonstrated signifi-

cant activity against HIV replication in H9 lymphocyte cells (Wu et al. 2004). Some of

the plants of the genus Begonia were previously reported for their antimicrobial activi-

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ties (Ramesh et al. 2002, Maridass 2009, Dan-Ping et al. 2012, Jeeva et al. 2012, In-

drkumar et al. 2014, Amutha and Sreedevikumari 2016, Shrestha et al. 2016).

The antimicrobial activities of volatile compounds of intact plants of 24 Begonia

species have been assessed against several pathogenic microorganisms (i.e. Staphylo-coccus epidermidis, Escherichia coli, and Candida albicans). As a result, 14 Begonia

species, possessing well expressed phytoncidal activity have been recommended to

use as indoor plants, based on their ability to reduce microbial air pollution indoor by

a factor of 1,5-3, in particular, by decreasing the Staphylococcus aureus load (Karpova

et al. 2009, Tsybulya et al. 2010).

Since poor hospital indoor air quality may lead to hospital-acquired infections, sick

hospital syndrome and various occupational hazards (Verde et al. 2015), use of plants,

Begonia in particular, may be considered as one of air-control measures, which are cru-

cial for reducing dissemination of airborne biological particles in hospitals.

Considering the points highlighted above and based on previous results obtained

in our laboratory, in the present study, we propose to evaluate the antimicrobial ef-

fects of nine ethanolic extracts obtained from leaves of Begonia species against

Escherichia coli strain.

MATERIALS AND METHODS

Collection of Plant Material. The leaves of Begonia plants, cultivated under

glasshouse conditions, were sampled at M.M. Gryshko National Botanical Garden

(NBG), National Academy of Science of Ukraine. The leaves of Begonia solimutata

L.B. Sm. & Wassh., Begonia goegoensis N.E.Br., Begonia foliosa Kunth, Begonia ×

bunchii L.H. Bailey (syn. Begonia × erythrophylla Hérincq), Begonia thiemei C.DC.

(syn. Begonia macdougallii Ziesenh.), Begonia peltata Otto & Dietr. (syn. Begonia kellermanii C.DC.), Begonia heracleifolia Cham. & Schltdl., Begonia dregei Otto

& Dietr., Begonia mexicana G. Karst. ex Fotsch were sampled for study (Photo 2).

The antimicrobial screening of Begonia leaf extracts has been carried out.

Preparation of Plant Extracts. Freshly leaves were washed, weighted, crushed,

and homogenized in 96% ethanol (in proportion 1 : 19) at room temperature. Then

the extracts were filtered and investigated for their antimicrobial activity.

Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion Meth-

od. Antimicrobial activity was determined using the agar disk diffusion assay (Bauer

et al. 1966). Culture of Escherichia coli (ATCC 25922) was suspended in sterile so-

lution of 0.9% normal saline and the turbidity adjusted equivalent to that of a 0.5

McFarland standard. Culture was inoculated onto Mueller-Hinton (MH) agar plates.

Sterile filter paper discs impregnated with extracts were applied over each of the cul-

ture plates. Isolates of bacteria were then incubated at 37°C for 24 h. The plates were

then observed for the zone of inhibition produced by the antibacterial activity of var-

ious ethanolic extracts obtained from leaves of Begonia spp. The presence of inhibi-

tion zones around each of paper discs after the period of incubation was regarded as

the presence of antimicrobial action while the absence of any measurable zone of in-

hibition was interpreted as absence of antimicrobial action. A negative control discs

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Begonia × bunchii L.H. Bailey (syn. Begon-

ia × erythrophylla Hérincq)

Begonia dregei Otto & Dietr.

Begonia foliosa Kunth

Begonia goegoensis N.E.Br.

Begonia heracleifolia Cham. & Schltdl. Begonia mexicana G. Karst. ex Fotsch

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Begonia peltata Otto & Dietr.

Begonia solimutata L.B. Sm. & Wassh.

Begonia thiemei C.DC.

Photo 2. Overview of the growth habits of Begonia species studied (plants are cultivated at

NBG’s glasshouses)

Source: photo by L. Buyun

impregnated with sterile ethanol were used in each experiment. The antimicrobial

activities of the extracts tested were evaluated at the end of the inoculated period by

measuring the inhibition zone diameter around each paper disc in millimeters. The

plates were observed and photographs were taken. For each extract six replicate tri-

als were conducted. Zone diameters were determined and averaged.

Statistical analysis. All statistical calculation were performed on separate data

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

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

Antimicrobial activity of various ethanolic extracts obtained from leaves of Be-

gonia species against E. coli measured as inhibition zone diameter is presented in

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285

Figs 1 and 2. The present study has shown that ethanolic extracts obtained from

leaves of Begonia species have moderate activity against E. coli. The diameter of in-

hibition zone for B. solimutata was 14 mm, 11.5 mm for Begonia goegoensis, 13 mm

for B. foliosa, 13.5 mm for Begonia × bunchii, 15 mm for B. thiemei, 19 mm for B. peltata, 12 mm for B. heracleifolia, 11.5 mm for B. dregei, and 16 mm for B. mexi-

cana (Figs 1 and 2).

Detailed data regarding the zones of inhibition by the various plant extracts were

recorded and presented in Fig. 2.

A B

C D

E F

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G H

I

Fig. 2. Antimicrobial activity of various ethanolic extracts obtained from leaves of B. soli-

mutata (A), B. goegoensis (B), B. foliosa (C), Begonia × bunchii (D), B. thiemei (E),

B. peltata (F), B. heracleifolia (G), B. dregei (H), B. mexicana (I) against Escherichia coli

measured as inhibition zone diameter

Source: own research

DISCUSSION

The effects of the plant extracts on the growth of standard strains of E. coli are

shown in Figs 1 and 2. Ethanolic extracts of the plant materials decrease the growth of

the standard strain of E. coli. The effects varied significantly according to the Begonia species. It was observed in the present study that ethanolic extracts inhibited the

growth of bacteria tested moderately. The highest antimicrobial effect was recorded for

B. peltata, B. mexicana, and B. thiemei (Figs 1 and 2). It should be noted that the most

antimicrobially effective plant against E. coli was B. peltata, being highly active with

the ethanolic extract (diameter of inhibition zone was 19 mm). The highly active anti-

microbial effects noted against E. coli are worthy of highlighting.

The antimicrobial activity of Begonia species has been well studied (Ramesh et al.

2002, Maridass 2009, Dan-Ping et al. 2012, Jeeva et al. 2012, Indrkumar et al. 2014,

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Amutha and Sreedevikumari 2016, Shrestha et al. 2016). For example, Jeeva and co-

workers (2012) have investigated the antibacterial activity and phytochemical proper-

ties of Begonia floccifera Bedd. methanolic flower extracts against the selected patho-

gens (Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococ-cus aureus, Bacillus cereus, Salmonella typhi, Serratia marcescens, Proteus mirabilis,

Enterococcus faecalis and Streptococcus pyogenes) by the disc diffusion method. The

results of the phytochemical screening revealed that phenol, tannins, xanthoproteins,

steroids, phytosterols, triterpenoids, sapogenins, coumarins, and carbohydrates are

comprised in the methanolic extracts of B. floccifera. The antibacterial activity has

been observed in the methanolic extracts of B. floccifera against the tested bacteria

with varied activity. The maximum diameter of inhibition zone was 28 mm for B. ce-reus, 25 mm for S. aureus, 15 mm for E. coli, 13 mm for P. mirabilis, 7 mm for K.

pneumonia. The other pathogens viz., P. aeruginosa, S. typhi, S. marcescens, Entero-

bacter spp., E. faecalis and S. pyogenes showed the minimal susceptibility to inhibi-

tion by extracts tested. Thus, methanolic flower extracts of B. floccifera can be used to

treat nausea, vomiting, diarrhea, urinary tract infections, nosocomial infections, pneu-

monia, septicemias etc. (Jeeva et al. 2012).

The study undertaken by Indrkumar and co-workers (2014), highlighted the anti-

microbial and in vitro antioxidant potential of extracts of Begonia dipetala. Antimicrobi-

al activity against Staphylococcus aureus, Salmonella typhi, E. coli and fungi Epi-

dermophyton floccosum, Microsporum gypseum and Trichophyton mentagrophytes,

free radical scavenging activity, Superoxide anion scavenging activity, Nitric oxide

scavenging activity and Ferric reducing antioxidant power assay were carried out on

different concentration of the extracts. The petroleum ether, chloroform and ethanol

extracts of B. dipetala were tested for growth inhibiting activity against three bacte-

rial strains (S. aureus, S. typhi, E. coli) in three varying concentration. The results of

Indrkumar and co-workers (2014) showed that the plant possess appreciable amount

of antibacterial activity against the strains tested. The ethanolic extract of the plant

was found to be more active than the rest of the extracts. The zone of inhibition was

much comparable to that of the standard. The activity was found to be more on the

S. aureus than the Gram-negative organisms tested. The chloroform extract of the

plant was found to inhibit the organism only at the higher concentration (50 μl). The

petroleum ether extract was found to be active only against S. aureus and E. coli at

the concentration of 50 μl. The petroleum ether, chloroform and ethanol extracts of

B. dipetala were tested for activity against three fungal strains in three varying con-

centration. The ethanolic extracts of the plant was found to be active against all the

strains and was found to inhibit the growth of T. agrophytes more than the other two

strains. The chloroform and petroleum ether extract was found to exert only a mini-

mal activity on the fungal strains tested. The petroleum ether extract did not show

any inhibition on M. gypseum at 20 μl and 35 μl. Moreover, it is concluded that eth-

anolic extract of B. dipetala exhibits high antioxidant and free radical scavenging

activities. It also chelates iron and has reducing power. This plant extract is a signifi-

cant source of natural antioxidant, which might be helpful in preventing the progress

of various oxidative stresses (Indrkumar et al. 2014).

The inhibitory activity against bacteria and fungi of sterol extracts from B. sinensis

rhizome was investigated by Dan-Ping and co-workers (2012). The results showed that

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sterol extracts had strong inhibition on S. aureus, B. subtilis, E. coli, Salmonella spp.,

Shigella spp., Aspergillus niger and Penicillium digitatum. Among these microorgan-

isms, sterol extracts showed the strongest inhibition against S. aureus with inhibition di-

ameters larger than 9 mm and Minimal Inhibitory Concentration (MIC) of 0.5 mg/ml.

It was also found that the inhibition of microorganism by sterol extracts was positively

correlated with their concentration but negatively correlated with culture time (Dan-Ping

et al. 2012).

The aqueous and organic solvent (hexane, chloroform and methanol) extracts of

B. malabarica were tested against ten human pathogenic bacteria (Aeromonas hy-drophila, Chromobacterium violaceum, E. coli, Klebsiella pneumoniae, P. aerugino-

sa, Salmonella typhi, Vibrio cholerae and Vibrio parahaemolyticus, B. subtilis, and S. aureus) and four fungal strains (Aspergillus flavus, Aspergillus fumigatus, Asper-

gillus niger, and Candida albicans) by Ramesh and co-workers (2002). Regarding

antimicrobial activity the hexane extract of B. malabarica did not show activity

against the tested bacteria and fungi. All the extracts did not show activity against

the tested fungi. The chloroform extract showed activity against all the bacteria and

maximum activity was observed against C. violaceum and V. parahaemolyticus. The

methanol extract also showed activity against all the bacteria and the maximum ac-

tivity was observed against K. pneumoniae. All the three aqueous extracts (cold,

boiled and autoclaved) showed activity against the Gram-negative bacteria except

V. parahaemolyticus and no activity was observed against all the Gram-positive bac-

teria. The activity of the same extracts against V. parahaemolyticus suggests the use

of the plant in diarrhea. The activity of the chloroform and aqueous extracts against

C. violaceum supports the use of plant against skin lesions and pyameia (Ramesh et

al. 2002).

Nisha Shrestha and co-workers (2016) have revealed that the hexane and meth-

anolic extract of B. picta showed no indicative antibacterial activity against S. aure-us (ATCC 25923), Bacillus cereus (clinical isolated), E. coli (ATCC 25922), Salmo-

nella typhi (clinical isolated). Only the ethyl acetate extract shows the indicative

antimicrobial activity against S. typhi among the tested stamps of microorganisms.

Phytochemical present in different extract of B. picta were alkaloid, tannin, sapon-

nin, terpenoid, glycoside, carbohydrate, flavonoid, phenol, cardiac glycoside and an-

thraquinone glycoside. Phytochemical analysis and antioxidant activity determina-

tion revealed that the phytochemicals, including polyphenol and flavonoid may be

responsible for the antimicrobial and antioxidant activity (Shrestha et al. 2016).

The antibacterial activity of the leaves extract of B. alba-coccininea (Hook.),

B. cordifolia (Wight) Thwaites, B. dipetala R. Grah., B. fallax A.DC., and B. floccif-

era Bedd. against S. aureus, B. subtilis, E. coli and P. aeruginosa was determined by

Maridass (2009). All the extracts were active against the zone of inhibition ranges

from < 6 to 10 mm, 11 to 15 mm and > 16 mm above for resistant, intermediate and

sensitive bacteria. B. floccifera and B. fallax showed strong activity against S. aureus

and P. aeruginosa. Zones of inhibition ranging from 6 to 10 mm for the leaves ex-

tract of B. alba-coccinia, B. dipetala and B. floccifera against B. subtilis, while

B. cordifolia and B. fallax were more active against S. aureus. Leaves extract of

B. alba-coccinia, B. cordifolia and B. dipetala inhibited the growth of the Gram-

negative E. coli strain. This shows that the sensitivity of the bacteria to most of the

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active extracts could be considered as intermediate, with high sensitivity to two ex-

tract, B. floccifera and B. fallax (Maridass 2009).

The antibacterial efficiency of B. cordifolia leaf extracts using hexane, ethyl ace-

tate, acetone and aqueous solution as solvents and tested against pathogens like Vibrio harvayie and Pseudomonas aeroginosa using disc diffusion method were examined by

Amutha and Sreedevikumari (2016). The maximum zone of inhibition was observed

in methanolic extract of B. cordifolia against V. harvayie. The study also revealed that

hexane, ethyl acetate extract shows moderated and acetone and aqueous extract shows

minimum antimicrobial activity (Amutha and Sreedevikumari 2016). The presence of

antibacterial activity in a particular part of a particular species may be due to the pres-

ence of one or more bioactive compounds such as alkaloids, glycosides, flavonoids,

steroids, saponins etc. (Balandrin and Klocke 1988).

Variation in the chemical profile of extracts could influence their biological ac-

tivities. Therefore, it was important to know the chemical composition of extracts to

correlate with their antimicrobial activities. A study conducted by Kalpanadevi and

Mohan (2012) has shown that the extracts of B. malabarica and B. floccifera contain

higher quantities of phenolic compounds, which exhibit antioxidant and free radical

scavenging activity. In vitro assay systems confirm B. malabarica and B. floccifera

whole plants as natural antioxidants. The phenolics and flavonoids could be the rea-

son for its antioxidant activity. The preliminary phytochemical studies revealed the

presence of flavone, sterol, triterpene in hexane, chloroform and methanol extracts;

phenol in chloroform and methanol extracts of B. malabarica and quinone, saponin,

tannin and starch in methanol extract. All the extracts did not answer for alkaloid

(Ramesh et al. 2002). Preliminary phytochemical screening of B. floccifera and

B. malabarica conducted by Ariharan and co-workers (2012) showed the presence

of a number of bioactive constituents such as vitamin C. The antimicrobial activity

could be due to the presence of this phytoconstituent. The contents of flavonoids (in-

cluding glycosides of quercetin and kaempferol), anthocyanins and ascorbic acid in

overground part of plants of 7 species and cultivars of genus Begonia L. (B. bahiensis,

B. bowerae, B. carolineifolia, B. fischeri, B. heracleifolia, B. ‘Erythrophylla’, B. ‘Hel-

en Teupel’) were determined by Karpova and co-workers (2009). The contents of flavo-

noids were 24-650 mg% of dry weight, including glycosides of quercetin – 3-76 mg%.

Kaempferol glycosides was detected only in species of section Gireoudia (1.2-5.7 mg%).

The contents of anthocyanins were between 60 and 157 mg%, ascorbic acid – 5-43 mg%

of fresh weight. Studied plants of Begonia can be considered as the sources of biologi-

cally active compounds with antioxidant and antimicrobial activity (Karpova et al.

2009).

In 1998, Frei and co-workers have reported about six known cucurbitacins isolated

from the rhizomes of B. heracleifolia. The structures were established as cucurbitacin

B (1), cucurbitacin D (2), 23,24-dihydrocucurbitacin D (3), 23,24-dihydrocucurbitacin

F (4), 2-O-beta-glucopyranosyl-cucurbitacin B (5), and 2-O-beta-glucopyranosyl-cucur-

bitacin D (6). All isolated compounds showed no activity against several bacterial, fun-

gal and yest targets in an agar overlay method [Bacillus subtilis (ATCC 6633), Micro-coccus luteus (ATCC 9341), Escherichia coli (ATCC 25922), Bacillus cereus (ATCC

10720), Mycobacterium fortuitum, Staphylococcus aureus (ATCC 25923), Staphylococ-cus epidermidis (ATCC 12228), Pseudomonas aeruginosa (ATCC 27853), Candida al-

290

bicans (H29 ATCC 26790)]. The antibacterial activity of the crude extract must therefore

be due to other compounds. On the other hand, varyingly strong antiproliferative activity

towards tumor and immune cells was observed for three compounds (1-3), due to differ-

ent structural features (Frei et al. 1998). The main active constituents of luteolin, querce-

tin and β-sitosterol were isolated from the leaves of B. malabarica (Kiritkar and Basu

1975). Cordell and Young Geun Shin (1999) have reported that cucurbitacin D, hexa-

norcucurbitacin D, cucurbitacin B and dihydrocucurbitacin B were isolated from the

roots of B. parviflora L.

It is well documented that the presence of these chemicals is responsible for var-

ious medicinal properties of plants. There are many reports available to support the

role of phytochemicals and their activity against specific diseases.

CONCLUSIONS

The examined ethanolic extracts showed different antibacterial activities against

E. coli. The highest antimicrobial effect was recorded for B. peltata, B. mexicana, and B. thiemei. The most antimicrobially effective plant against E. coli was B. pel-

tata, being highly active with the ethanolic extract (diameter of inhibition zone was

19 mm). This study indicated that ethanolic extracts of Begonia species have anti-

bacterial activity and may have potential medical use. The results of this study pro-

vide informative data for the use of the crude ethanolic extract obtained from Begon-ia species against bacterial microbial infections caused by E. coli. Phytochemical

constituents in the plant samples are known to be biologically active compound and

they are responsible for different activities such as antioxidant, antimicrobial, anti-

fungal, and anticancer (Suresh and Nagarajan 2009). All secondary metabolite com-

ponents displayed antioxidant and antimicrobial properties through different biolog-

ical mechanisms (Hossain and Nagooru 2011). The identification of precise

molecular mechanism addressing how these extracts inhibit bacterial growth are

need to be explored. We believe that the present paper adds knowledge about anti-

microbial properties of plants in general and for Begonia species in particular.

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SUMMARY

Antimicrobial properties of medicinal plants are being increasingly reported from

different parts of the world. In an effort to expand the spectrum of antibacterial

agents from natural resources, genus Begonia belonging to Begoniaceae family has

been selected, because among plant-based antimicrobials, the antimicrobial activity

of Begonia species has been well studied. Moreover, the leaves of Begonia species

are used for the treatment of cancer; beside they possess the anti-HIV activity. Some

of the plants of the genus Begonia were previously reported for their antimicrobial

activities. The present work was designed to assess the anti-Escherichia coli activity

of the ethanolic extracts from the leaves of Begonia species plants, cultivated under

glasshouse conditions at M.M. Gryshko National Botanical Garden (NBG), National

Academy of Science of Ukraine. The leaves of B. solimutata L.B. Sm. & Wassh.,

B. goegoensis N.E.Br., B. foliosa Kunth, Begonia × bunchii L.H. Bailey (syn. Be-

gonia × erythrophylla Hérincq), B. thiemei C.DC., B. peltata Otto & Dietr., B. hera-cleifolia Cham. & Schltdl., B. dregei Otto & Dietr., B. mexicana G. Karst. ex Fotsch

293

were sampled for study. Antimicrobial activity was determined using the agar disk

diffusion assay. The present study has shown that ethanolic extracts obtained from

leaves of Begonia species had moderate activity against E. coli. The diameters of in-

hibition zone for B. solimutata were 14 mm, 11.5 mm for B. goegoensis, 13 mm for B. foliosa, 13.5 mm for Begonia × bunchii, 15 mm for B. thiemei, 19 mm for B. pel-

tata, 12 mm for B. heracleifolia, 11.5 mm for B. dregei, and 16 mm for B. mexicana. The highest antimicrobial effect was recorded for B. peltata, B. mexicana, and B.

thiemei. The most antimicrobially effective plant against E. coli was B. peltata, be-

ing highly active with the ethanolic extract (diameter of inhibition zone was 19 mm).

The highly active antimicrobial effects noted against E. coli are worthy of highlight-

ing. Phytochemical constituents in the plant samples are known to be biologically

active compound and they are responsible for different activities such as antioxidant,

antimicrobial, antifungal, and anticancer. All secondary metabolite components dis-

played antioxidant and antimicrobial properties through different biological mecha-

nisms. The obtained results highlighted the interesting antimicrobial potency of var-

ious Begonia species and provided scientific basis for the traditional use of these

plants in the treatment of microbial infections. This study indicated that ethanolic

extracts of Begonia species have antibacterial activity and may have potential medi-

cal use. The results of this study provide informative data for the use of the crude

ethanolic extract obtained from Begonia species against bacterial microbial infec-

tions caused by E. coli. The identification of precise molecular mechanism address-

ing how these extracts inhibit bacterial growth are need to be explored. We believe

that the present paper adds knowledge about antimicrobial properties of plants in

general and for Begonia species in particular.

294