The antibacterial activity of certain Sansevieria species against Escherichia coli

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Institute of biology and environmental protection, Pomeranian university in Slupsk

M.M. Gryshko National Botanical Garden, National Academy of Science of Ukraine

The antibacterial activity of certain Sansevieria species against Escherichia coli

Tkachenko H.,

candidate of biological sciences

Buyun L.,

doctor of biological sciences

Osadowski Z.,

doctor of biological sciences

Maryniuk M.,

engineer

Abstract

H. Tkachenko, L. Buyun, Z. Osadowski, M. Maryniuk

The present study was aimed to investigate in vitro antimicrobial activity of ethanolic extracts of seventeen species of Sansevieria genus [Sansevieria canaliculata Carriere, S. trifasciata Prain, S. cylindrica Bojer ex Hook., S. parva N.E. Br., S. fischeri (Baker) Marais, S. kirkii Baker, S. aethiopica Thunb., S. metallica Gerome & Labroy, S. caulescens N.E. Br.,

S. francisii Chahin, S. arborescens Cornu ex Gerome & Labroy, S. volkensii Gurke, S. forskaliana (Schult. & Schult.f.) Hep - per & J.R.I. Wood, S. gracilis N.E. Br., S. hyacinthoides (L.) Druce, S. roxburghiana Schult. & Schult.f., S. suffruticosa

N.E. Br.] against Escherichia coli (ATCC 25922). The crude extracts were screened for antimicrobial activity using agar diffusion method. All the extracts showed variable degree of diameters of zone inhibition against tested bacterium. Therefore, the results revealed the antimicrobial potential of these extracts. In fact, the test organism was susceptible to extracts of S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens,

S. metallica, S. aethiopica with diameters of inhibition zone from 12 to 24 mm. E. coli isolate was resistant only to S. hyacinthoides extract and the diameter of zone inhibition around the rest ranged from 8 to 10 mm. The ethanolic extracts obtained from leaves of S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica possess antibacterial potency against E. coli isolates and may be used as natural antiseptics and antimicrobial agents in medicine.

Key words: Sansevieria, leaves, extract, antimicrobial activity, paper disc diffusion method.

Анотація

Висвітлено дослідження in vitro антимікробної активності етанольних екстрактів сімнадцяти видів роду San - sevieria [Sansevieria canaliculata Carriere, S. trifasciata Prain, S. cylindrica Bojer ex Hook., S. parva NEBr., S. fischeri (Baker) Marais, S. kirkii Baker, S. aethiopica Thunb., S. metallica Gerome & Labroy, S. caulescens NEBr., S. francisii Chahin, S. arborescens Cornu ex Gerome & Labroy, S. volkensii Gurke, S. forskaliana (Schult. & Schult.f.) Hepper & JRIWood, S. gracilis NEBr., S. hyacinthoides (L.) Druce, S. roxburghiana Schult. & Schult.f., S. suffruticosa N.E. Br.] щодо Escherichia coli (ATCC 25922). Антимікробну активність визначали за допомогою диско-дифузійного методу. Всі досліджені екстракти виявили різний ступінь інгібування зони росту протестованої бактеріальної культури, що свідчить про протимікробний потенціал цих екстрактів. Тестовий мікроорганізм є сприйнятливим до екстрактів листя S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica з діаметром зони пригнічення в інтервалі 12-24 мм. Ізолят E. coli був резистентним лише до екстракту S. hyacinthoides, в той час як діаметр зони пригнічення навколо інших досліджуваних екстрактів становив 8-10 мм. Етанольні екстракти, отримані з листя S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica мають антимікробні властивості щодо ізолятів E. coli і можуть бути використані в медицині як природні антисептики і протимікробні препарати.

Ключові слова: Sansevieria, листя, екстракт, антибактеріальна активність, диско-дифузійний метод.

Аннотация

Антибактериальная активность некоторых видов сансевиерий относительно кишечной палочки

Г.М. Ткаченко, Л.И. Буюн, З. Осадовский, М. Маринюк

Освещено исследования in vitro антимикробной активности этанольных экстрактов семнадцати видов рода Sansevieria [Sansevieria canaliculata Carriere, S. trifasciata Prain, S. cylindrica Bojer ex Hook., S. parva N.E. Br., S. fischeri (Baker) Marais, S. kirkii Baker, S. aethiopica Thunb., S. metallica Gerome & Labroy, S. caulescens N.E. Br., S. francisii Chahin, S. arborescens Cornu ex Gerome & Labroy, S. volkensii Gurke, S. forskaliana (Schult. & Schult.f.) Hepper & J.R.I. Wood, S. gracilis N.E. Br., S. hya - cinthoides (L.) Druce, S. roxburghiana Schult. & Schult.f., S. suffruticosa N.E. Br.] в отношении Escherichia coli (ATCC 25922). Антимикробную активность определяли с помощью диско-диффузионного метода. Все исследованные экстракты показали различную степень ингибирования зоны роста протестированной бактериальной культуры, что свидетельствует о про - тивомикробном потенциале этих экстрактов. Тестовый микроорганизм является восприимчивым к экстрактам листьев S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica с диаметром зоны ингибирования в интервале 12-24 мм. Изолят E. coli был резистентным лишь к экстракту S. hyacinthoides, в то время как диаметр зоны ингибирования вокруг остальных исследуемых экстрактов составлял 8-10 мм. Этанольные экстракты, полученные из листьев S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica обладают антимикробными свойствами в отношении изолятов E. coli и могут быть использованы в качестве природных антисептиков и противомик - робных препаратов в медицине.

Ключевые слова: Sansevieria, листья, экстракт, антибактериальная активность, диско-диффузионный метод.

Main part

Introduction. It was estimated that 70-80% of people worldwide rely chiefly on traditional, largely herbal; medicine to meet their primary healthcare needs [10, 12]. The literature search on this issue has shown that Sansevieria Thunb., a genus with diverse ethnobotanical uses in its geographical distribution range, has occupy an important place among plant genera applied for treatment of a broad spectrum of diseases and disorders [16, 23, 24]. During the last years, many Sansevieria species were screened and plants with high bioactive compounds were identified [1, 9, 15].

Genus Sansevieria, belonging to Asparagaceae family [19], comprises ca. 70 species worldwide, distributed mainly in dry or arid areas of the Old World tropics and subtropics [23], with a distribution range from Africa to south east Asia and the islands of the Indian Ocean [3]. Representatives of this genus are usually xerophytic perennial rhizomatous plants that occur in dry tropical and subtropical parts of the world [23]. Africa is the center of diversity for Sansevieria [6]. Common English names for Sansevieria species are snake plant or bowstring-hemp, zebra lily, cow tongue, leopard lily, devil's tongue, good luck plant along with «mother-in-law's tongue» for the widely cultivated horticultural plant S. trifasciata [23, 24].

The medicinal use of rhizomes and leaves of S. aethiopica is widespread in southern Africa. In Zimbabwe, the leaves are heated and the sap is squeezed into the ear to treat ear-infections, while the rhizome is warmed and used for treating toothache [14]. Fresh or boiled rhizome are eaten to treat haemorrhoids, stomach-ache, ulcer, diarrhoea and internal parasites. In Namibia Bushmen apply the heated, pounded leaves to a stiff neck to give relief. Leaf sap is applied to wounds to accelerate healing and to maternal breast to stimulate milk production. Rhizomes and leaves contain ruscogenin and related sapogenins, which have anti-inflammatory and venotomic properties. Nevertheless, antibacterial tests have given negative results [5].

For example, S. roxburghiana Schult. & Schult.f. is used for coughs, rheumatism; as an expectorant, febrifuge, purgative, and tonic [11]. The study of Haldar and co-workers (2010) has demonstrated that the hydroalcoholic extract of S. roxburghiana rhizome exhibited remarkable antitumor activity against Ehrlich ascites carcinoma in Swiss mice that is plausibly attributable to its augmenting endogenous antioxidant mechanisms. In addition, diethyl ether, alcohol, and acetone extracts of S. roxburghiana rhizome showed antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus [15].

The assessment of antimicrobial activity of the methanolic leaf extract of S. liberica, conducted by Adelanwa and Habibu (2015), has revealed that Bacillus cereus and S. aureus were sensitive to the methanolic extract of S. liberica while Salmonella typhi and E. coli were resistant to the extract [1]. In addition, these authors carried out the phytochemical screening of S. liberica leaf extract, which demonstrated the presence of carbohydrate, triterpenes, flavonoids and cardiac glycosides; absence of anthraquinones and alkaloids in the plant. However tannins, saponins and steroids were found to be absent in S. liberica [1]. These results are consistent with those of Eze and co-workers (2011), which have assessed the antimicrobial activity of the leaf extract of S. liberica [9].

Phytochemical analysis of the crude extract and the fractions showed the presence of various bioactive substances such as alkaloids, saponins, flavonoids, terpenoids, steroids, glycosides, reducing sugars, tannins, resins, carbohydrates, proteins, acidic compounds, fats and oils [9].

Although Escherichia coli can be an innocuous resident of the gastrointestinal tract, it also has the pathogenic capacity to cause significant diarrheal and extraintestinal diseases [7]. E. coli is a Gramnegative, oxidase-negative, rod-shaped bacterium from the family Enterobacteriaceae. It is able to grow both aerobically and anaerobically, preferably at 37°C, and can either be nonmotile or motile, with peritrichous flagella. Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide [7].

In addition, the development of bacterial resistance to presently available antibiotics has necessitated the search for new antimicrobial agents. Another challenging factor for the renewed interest in plant antimicrobial agents in the past 20 years has been the threatening rate of plant species extinction [18].

Hence, an attempt has been made to evaluate antibacterial activity of seventeen species of Sansevieria genus against E. coli. So the present study was conducted to investigate in vitro antimicrobial activity of ethanolic extracts of seventeen species of Sansevieria genus [Sansevieria canaliculata Carriere, S. trifasciata Prain, S. cylindrica Bojer ex Hook., S. parva N.E. Br. (syn. S. dooneri N.E. Br.), S. fischeri (Baker) Marais, S. kirkii Baker, S. aethiopica Thunb., S. metallica Gerome & Labroy, S. caulescens N.E. Br., S. francisii Chahin, S. arborescens Cornu ex Gerome & Labroy, S. volkensii Gurke (syn. S. intermedia N.E. Br.), S. forskaliana (Schult. & Schult.f.) Hepper & J.R.I. Wood, S. gracilis N.E. Br., S. hyacinthoides (L.) Druce (syn. S. grandis Hook.f.), S. roxburghiana Schult. & Schult.f., S. suffruticosa N.E. Br.] against E. coli strain.

Materials and methods. Collection of Plant Materials. The leaves of Sansevieria plants, cultivated under glasshouse conditions, were sampled for antimicrobial potency assessment at M.M. Gryshko National Botanical Garden, National Academy of Science of Ukraine.

Preparation of Plant Extracts. Freshly leaves were washed, weighted, crushed, and homogenized in 96% ethanol (in proportion 1:19) at room temperature. The extracts were then filtered and investigated for their antimicrobial activity. All extracts were stored at 4°C until use.

Bacterial test strain and growth conditions. For this study, a strain of E. coli (ATCC 25922) was used. The cultivation medium was trypticase soy agar (Oxoid, UK), supplemented with 10% defibrinated sheep blood. Cultures were grown aerobically for 24 h at 37°C. The cultures were later diluted with sterile solution of 0.9% normal saline to approximate the density of 0.5 McFarland standard. The McFarland standard was prepared by inoculating colonies of the bacterial test strain in sterile saline and adjusting the cell density to the specified concentration.

Determination of antibacterial activity of plant extracts by the disk diffusion method. Antimicrobial activity was determined using the agar disk diffusion assay [4]. Culture of E. coli was inoculated onto Mueller-Hinton (MH) agar plates. Sterile filter paper discs impregnated with extracts were applied over each of the culture plates. Isolates of bacteria were then incubated at 37 ^oC for 24 h. The plates were then observed for the zone of inhibition produced by the antibacterial activity of various ethanolic extracts obtained from leaves of Sansevieria species. A negative control disc impregnated with sterile ethanol was used in each experiment. At the end of the period, the inhibition zones formed were measured in millimeters using the vernier. For each extract, six replicates were assayed. The plates were observed and photographs were taken. Zone diameters were determined and averaged.

Statistical analysis. All statistical calculation was performed on separate data from each species (Statistica 8.0, StatSoft, Poland). The following 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 [20].

Results. The results of antimicrobial activity of ethanolic extracts obtained from leaves of Sansevieria species are presented in Figs 1 and 2.

Mean diameters of inhibition zone

Fig. 2. Antibacterial activity of the ethanolic extract obtained from the leaves of S. parva (A), S. volkensii (B), S. arborescens (C), and S. kirkii (D) against E. coli strain

antimicrobial sansevieria escherichia coli

Extracts from the leaves of S. kirkii and S. arborescens were particularly active against tested organism (diameters of inhibition zones were 24 and 22.5 mm, respectively). It was followed by the activities of extracts from S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica leaves (diameters of inhibition zones were ranged between 13.5 and 12.0 mm). Finally, the ethanolic extracts of S. gracilis, S. suffruticosa, S. fischeri, and S. hyacinthoides showed less antimicrobial activities (diameters of inhibition zones were ranged from 8 to 11.5 mm) (Figs 1 and 2).

Discussion. The practice of traditional herbal medicine is widespread and natural products derived medicines are widely used for effective infectious disease treatment [22]. The antimicrobial investigation carried out in this study involved the determination of the sensitivity pattern of E. coli to the leaf extracts of seventeen species of Sansevieria genus. The results revealed the antimicrobial potential of these extracts. The test organism was susceptible to extracts of S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica with inhibition zone diameter between 12-24 mm. E. coli isolate was resistant only to S. hyacinthoides extract and the diameter of inhibition zone around the rest ranged from 8 to 10 mm.

In agreement with the results obtained from the present study, previous research papers documented the noticeable antimicrobial potency of the ethanolic extracts from S. fischeri, S. francisii, S. parva, S. kirkii, S. aethiopica, S. caulescens, and S. metallica against Staphylococcus aureus [8]. The microbial growth inhibition capacity was attributed to the presence of the rich variety of phytochemicals including carbohydrates, saponin, flavonoids, phenols, alkaloid, anthocyanin and cyanine, glycosides, proteins and phytosterols [8]. According to Deepa Philip and co-workers (2011), the methanolic and acetone extracts of leaves of S. roxburghiana diluted showed antibacterial activity against Grampositive bacteria such as Micrococcus luteus, Bacillus cereus, Enterococcus spp., S. aureus, Gramnegative bacteria such as Proteus vulgaris, Pseudomonas aeruginosa, P. fluorescense, Salmonella typhi, S. paratyphi, Klebsiella pneumoniae, Shigella sonnie, and E. coli, fungal strains Cryptococcus spp. and Candida albicans [Deepa Philip et al. 2011]. Ethyl acetate extracts of rhizomes also exhibited appreciable antimicrobial activity against most of the pathogens tested. The minimum inhibitory concentrations (MIC) of the various extracts by agar dilution method ranged from 1.0 to 8.0 mg per mL. The leaf extracts exhibited better antimicrobial activity than rhizomes [8].

In a study conducted by Poonam Sethi (2013), the ethanolic extract of rhizome of S. roxburghiana displayed remarkable antibacterial activity against the four pathogenic bacteria, S. typhi, P. fluo - rescens, P. aeruginosa and E. coli [21]. Maximum activity was seen in the case of P. fluorescens where the zone diameter was 32 mm (300 pg/ml). The MIC study revealed that the value for the S. typhi and E. coli as 80 and 60 pg/ml for P. fluorescens and P. aeruginosa [21]. Hanumanth Kumar and Pramoda Kumari (2015) reported about potential bioactive secondary metabolites and revealed the possible antimicrobial activities of leaf extracts of S. roxburghiana. Antimicrobial screening revealed significant antimicrobial activity against P. vulgaris, S. typhi, P. aeruginosa, K. pneumoniae, and E. coli [13]. Qualitative analysis, conducted by these authors, confirmed by the presence of various primary and secondary plant metabolites such as alkaloids, terpenoids, flavonoids, saponins, steroids, phenols, tannins, and quinine in selected parts of S. roxburghiana.

S. roxburghiana also exhibited good inhibition effect against S. aureus and P. aeruginosa whereas S. trifasciata manifested good antimicrobial effect against E. coli, S. aureus and P. aeruginosa. It is interesting to note that the combined effect of antibiotics and plant extract has enhanced the antimicrobial effect of the extracts obtained against pathogenic microorganisms. The percentage inhibition of combined effect was calculated and it was observed that the leaves of S. roxburghiana possess antimicrobial effect (50%) against S. aureus combined with norfloxacin whereas the leaf extract of S. trifasciata when combined with tetracycline it showed 36% of inhibition against S. aureus. The methanolic extract from the leaves of S. roxburghiana and S. trifasiata was effective against Gram-positive and Gram-negative pathogenic microorganisms. The 50 mg per mL of the methanolic extracts manifested effective antimicrobial effect against pathogens [17].

Also, the results obtained from the present research showed antimicrobial potential of eleven extracts obtained from leaves of Sansevieria genus against E. coli. So, these plants extracts can be used as antiseptics and antimicrobial agents in medicine. The antibacterial activity in Sansevieria genus may be due to presence of alkaloids, saponins, terpenoids, steroids, glycosides, tannins, acidic compounds, fats and oils in their composition. Also flavonoids have several therapeutic effects such as antioxidant and anti-inflammatory [2].

Conclusions. In conclusion, we would like to emphasize that taking into account rapid losses and degradation of natural habitats in the tropics and associated with them catastrophic declines of many species of angiosperms, including Sansevieria spp., maintenance of living plants collection ex situ and assessment of their medicinal properties are very important.

Thus, our findings demonstrate that the ethanolic extracts obtained from leaves of S. kirkii, S. arborescens, S. roxburghiana, S. francisii, S. forskaliana, S. cylindrica, S. trifasciata, S. canaliculata, S. caulescens, S. metallica, S. aethiopica possess antibacterial potency against E. coli strain. Considering the medicinal importance of the tested microorganism these extracts may be used as a natural antiseptics and antimicrobial agents in medicine.

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