International Journal of Modern Biology and Medicine, 2017, 8(1): 24-46 International Journal of Modern Biology and Medicine ISSN: 2165-0136 Florida, USA Journal homepage: www.ModernScientificPress.com/Journals/IJBioMed.aspx Article Phytochemical Screening and Antibacterial Activities of Bidens pilosa L. and Tridax procumbens L. on Skin Pathogens Oluwole O. Owoyemi* and Muftau K. Oladunmoye Department of Microbiology, Federal University of Technology, P. M. B. 704, Akure. Ondo state, Nigeria *Author to whom correspondence should be addressed; E-Mail: [email protected] Article history: Received 29 July 2017, Revised 10 November 2017, Accepted 30 November 2017, Published 6 December 2017. Abstract: The antibacterial activities of the plants used by Rattus norvegicus (brown rat) in the construction of its nest were studied. The quadrat sampling results of various brown rats’ nests sampled revealed the use of Bidens pilosa L. and Tridax procumbens L. leaves by R. norvegicus (Brown rat) to constructing its nest. Hence, these plants were harvested and extracted with solvents of distinct polarities and the extracts were qualitatively and quantitatively screened for phytochemicals which revealed the presence of tannins, alkaloids, flavonoids, saponins and cardiac glycosides in both plants. The antibacterial activities of the ethanolic, aqueous and chloroform extracts of these plants at different concentrations of 50 mg/mL, 100 mg/mL and 200 mg/mL were evaluated on clinical skin pathogens and their corresponding type cultures using agar well diffusion method. The ethanoic extract of T. procumbens at 200 mg/mL exhibited broad spectrum activities on all the test bacteria except B. subtilis. The zones of inhibition of T. procumbens extracts on the test bacteria at 200mg/mL ranged from 5.00±0.00 mm to 15.20±0.20 mm, the lowest inhibitory effect was observed on Escherichia coli, while the highest was observed on Staphylococcus aureus ATCC 43300. It was observed that the Gram positive organisms were more susceptible to the extracts compared to the Gram negative organisms. However, B. pilosa extracts had more antibacterial activities on Gram negative organisms compared to Gram positive organisms. The results of this study revealed the therapeutic potentials of these plants selected by the brown rat’s in controlling pathogenic microorganisms from its environment especially skin- Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 25 related infectious microorganisms such as S. aureus evaluated and can also serve as a basis for controlling the adverse effect of these pathogens in humans too. Keywords: Bidens pilosa, Tridax procumbens, Rattus norvegicus, Phytochemical, Antibacterial activities, Clinical, Type, Pathogens. 1. Introduction Human is surrounded by countless microorganisms. The disease producing microbes play a very important role in human life. Pathogenic microorganisms are always trying to develop resistance to the various antimicrobial agents used for their control. Therefore, the chemotherapy of infectious diseases has proved to be a continuous struggle. Scientists are always in search of new antimicrobial agents to control the ever increasing menace of the microbes (Bushra and Ganga 2003). Thus, it is paramount for microbiologists to conduct series of research even from the animal environments in order to seek for alternative therapy for the resistant strains of infectious microbes. Rat’s nest is a structure usually made up of plants material in which a rat lives and breeds its young ones. There are different types of rat nest; each nest is always specific to a particular rat. Rats use different types of leaves to constructing their nest depending on the specie and habitat of the rat (Reichman and Smith, 1990). Roof rats find harborage in plants such as Algerian illy, bougainvillea and dead fronds of palm tree. Wood rats sleeping nests were constructed of lichens, mosses, dried grasses, shredded bark and wood and varied considerable in mass [30-215 g] (Hemmes et al., 2002). Wood rats use bay leaf to control nest-borne ectoparasites (Hemmes et al., 2002). Bay leaf has a high monoterpenoid content, which is noted for its biocidal activity, which has been shown to be toxic when fed to laboratory mice (Hemmes et al., 2002). Rats are specific about the plants they use in constructing their nests, using their instinct; they carefully select plants that can confer anti-parasite and antibacterial activities which are used in protecting themselves against insect herbivores (Langenheim, 1994) and pathogenic microorganisms (Deans and Ritchie, 1987; Knobloch et al., 1989).Rat nest plays vital roles in the life span of a rat by providing good health benefits to it such as regulating the nest temperature, protection against predators, harborage and protection from infectious microbes. Rattus norvegicus which is also referred to as brown rat, emóigbó in yoruba land (Nigeria), Norway rat and street rat is one of the best known and most common rats. One of the largest muroids, it is a brown or grey rodent with a body up to 25 cm long, and a similar tail length; the male weighs on average 350 g and the female 250 g. Thought to have originated in northern China, this rodent has now spread to all continents except Antarctica, and is the dominant rat in Europe, Africa and much of North Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 26 America—making it by at least this particular definition the most successful mammal on the planet after humans (Fragaszy et. al., 2003). With rare exceptions, the brown rat lives wherever humans live, particularly in urban areas. Selective breeding of R. norvegicus has produced the laboratory rat, a model organism in biological research, as well as pet rats (Baker et al., 1979). Like mice, these rats are frequently subjects of medical, psychological and other biological experiments, and constitute an important model organism. This is because they grow quickly to sexual maturity and are easy to keep and to breed in captivity. When modern biologists refer to "rats", they almost always mean R. norvegicus. A 2007 study found brown rat’s to possess metacognition, a mental ability previously only found in humans and some primates (Science Daily, 2007), but further analysis suggested they may have been following simple operant conditioning principles (Smith et al., 2008). The quadrat sampling results of various brown rats’ nests sampled revealed the use of Bidens pilosa and Tridax procumbens leaves to constructing their nests. Previous studies on these plants show that they belong to a class of medicinal plants (Dimo et al., 2001; Ali et al., 2001). Medicinal plants are gifts of nature which are used to cure limitless number of diseases among human beings (Bushra and Ganga, 2003). The abundance of plants on the earth’s surfaces has led to an increasing interest in the investigation of different extracts obtained from traditional medicinal plants as potential sources of new antimicrobial agents (Bonjar and Farrokhi, 2004). Tridax procumbens L. belongs to the family of Asteraceae and commonly known as ‘Gaddichamanthi’ in Telugu, in Ayurvedic as Jayanthi, in Sidda/Tamil asVettukkaaya-thalai, in Folk as Akalakohadi and in English as Coat buttons/Mexican Daisy, because of the appearance of its flowers and is an ethno botanically important medicinal plant. The plant has been considered as a gregarious weed, distributed throughout the tropics and sub tropics. It has been extensively used in Indian traditional medicine as anticoagulant, antifungal and insect repellent; in bronchial catarrh, diarrhea and dysentery (Ali et al., 2001). T. procumbens is extensively used in the Indian Ayurvedic system of medicine for the treatment of diarrhea, as an insect repellent, hair tonic and wound healer, i.e. the leaf juice is used to check hemorrhage from cuts and bruises (Bhat et al., 2007). It is a well-known remedy for liver disorders and has been shown to possess antidiabetic activity (Bhagwat et al., 2008). B. pilosa is an erect, perennial herb widely distributed across temperate and tropical regions. B. pilosa is either glabrous or hairy, with green opposite leaves that are serrate, lobed, or dissected. It has white or yellow flowers, and long narrow ribbed black achene’s (seeds). It grows to an average height of 60 cm and a maximum of 150 cm in favorable environments (Alcaraz and Jimenez, 1988). B. pilosa, either as a whole plant or different parts, has been reported to be useful in the treatment of more than 40 disorders such as inflammation, immunological disorders, digestive disorders, infectious Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 27 diseases, cancers, metabolic syndrome, wounds, and many others (Pereira et al., 1999; Dimo et al., 2001). B. pilosa is usually ingested; however, it can also be utilized externally. For instance, fresh B. pilosa is used to treat snake bites and wounds (Dharmananda, 2013), and in Trinidad and Tobago the aqueous solution of the leaves of B. pilosa is used to bathe babies and children (Lans, 2007). This research investigated the phytochemical components in the two plants (B. pilosa and T. procumbens) selected by the brown rat in constructing its nest as well as the antibacterial activities of these plants on selected human skin pathogens. 2. Materials and Methods The identification of the plants used by R. norvegicus in constructing its nest was investigated. The physical observations of various newly-made nests of the brown’s rat and quadrat sampling of the plants in the specified area of the rat nest region were carried out. The species of plants adopted by the brown rat in constructing its nest were identified and authenticated by Prof. Y. A. Awodun in the Department of Crop, Soil and Pest Management, The Federal University of Technology, Akure (FUTA), Nigeria which were: Bidens pilosa L. and Tridax procumbens L. 2.1. Collection of Rattus Norvegicus Nest-leaves Composition, Preparation of the Extracts and Percentage Recovery of the Extracts The plant materials used by R. norvegicus in constructing its nests (B. pilosa and T. procumbens) were collected from the nest environment in the outskirt bush of Edo-lodge Street Oke-ijebu Akure, Ondo state. Having harvested the leaves, they were washed with tap water and air-dried at room temperature (25±2ºC) for two weeks. The dried leaves were pulverized by grinding machine (type N model) into smooth powder and subsequently sieved using 1.18mm sieve. The method of Green (2004) was utilized for the extraction of the plants using solvent to sample ratio of 10:1 (v/w). Two hundred grams portions of the pulverized leaves were separately weighed and soaked in 2500 mL ethanol, aqueous and chloroform at ambient temperature for 72 h under regular shaking condition. The extracts were filtered using Whatman filter paper. The filtrates were recovered by removal of the solvents using rotary evaporator under reduced temperature at 40ºC. The recovery rate of extracts was calculated using the formula below; WA % Recovery of extract = 𝑥 100 IW Where WA = Weight of extracts recovered after extraction, IW = Initial weight of extracts. 2.2. Qualitative Phytochemical Screening of B. pilosa and T. procumbens Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 28 The phytochemical analysis for qualitative detection of total flavonoids, tannins, alkaloids, saponins, phlobatannin, cardiac glycosides, steroids, terpenoids and anthraquinone were performed on the extracts as described by AOAC (2007). Determination of saponin Exactly 0.5000 g of the plant extract (B. pilosa or T. procumbens) was mixed with distilled water in a test tube and shook together, frothing which persist on warming was taken as preliminary evidence for the presence of saponins, absence of frothing on warming indicates negative result (AOAC, 2007). Determination of tannin Exactly 0.5000 g of the extract was stirred with 100 mL of sterile distilled water, filtered and ferric chloride reagent was added to the mixture, a blue-black green or blue green precipitate was taken as evidence for the presence of tannin while absence of a blue-black coloration indicates negative result (AOAC, 2007). Evaluation of phlobatannin Deposition of red precipitate when 0.5000 g of the extract was boiled with 1% aqueous HCl was taken as evidence for the presence of phlobatannin while the absence of red precipitate indicated negative result (AOAC, 2007). Determination of flavonoid A 0.5000 g of the extract was stirred with 20 mL of dilute ammonia solution, a yellow coloration was observed, and the disappearance of the yellow color after the addition of 1 mL concentration of H SO indicated the presence of flavonoid while the presence of yellow coloration after the addition of 2 4 1 mL concentration of H SO indicated negative result (AOAC, 2007). 2 4 Determination of terpenoid A 0.5000 g of the extract was mixed with 20 mL of chloroform and filtered. 3 mL of concentrated H SO was added to the filtrate to form a layer. A reddish brown colour at the interface was observed 2 4 which indicated the presence of terpenoids while the absence of a reddish brown coloration indicated negative result (AOAC, 2007). Determination of cardiac glycosides Exactly 1.000 g of the extract was dissolved in pyridine and few drops of 1% sodium nitroprusside with few drops of 20% NaOH were added. A deep red coloration which faded to a brownish yellow indicated the presence of cardiac glycosides while the presence of a deep red coloration which does not fade away to a brownish yellow indicated the absence of cardiac glycosides. Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 29 2.3. Quantitative Phytochemical Analysis of B. pilosa and T. procumbens The quantitative analysis of the two plant extracts were carried out according to the method described by AOAC (2007). 2.4. Collection of Test Microorganisms Both clinical and type microorganisms were used for the study. The clinical organisms which include: Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae were obtained from Don Bosco Catholic Hospital Akure, Ondo state, Nigeria and their corresponding type organisms were also obtained in Pathcare, Lagos State. These were: Staphylococcus aureus ATCC 43300, Streptococcus pyogenes ATCC 29212, Bacillus subtilis ATCC 21332, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218 and Klebsiella pneumonia ATCC 48891. 2.5. Standardization of Inoculums The inocula were prepared from the stock cultures which were maintained on nutrient agar slant at 4OC and sub cultured onto nutrient broth aseptically using sterile inoculating loop and incubated at 37OC for 18-24 hours. The setup were suspended in saline solution (0.85% NaCl) and adjusted with the aid of a spectrophotometer (Unico 1100RS) to match a turbidity of 0.5 McFarlands standard at wavelength of 540 nm according to the method described by CLSI (2010). 2.6. Antibacterial Assay of B. pilosa and T. procumbens Leaf Extracts on Bacterial Test Organisms Antibacterial activities of extracts were determined by agar well diffusion method as described by Esimore et al. (1998) with slight modification. After standardization using 0.5 Mcfarland standard of the inoculum, sterile Petri dishes were inoculated aseptically with 0.1 mL of the 18 hours old broth cultures of the bacterial test organisms each, while 15 ml of sterilized nutrient agar was poured aseptically in the inoculated plates. The plates were swirled carefully for even distribution and allowed to gel. With the aid of a sterile cork borer of 6 mm in diameter, wells were made on the solidified agar plate aseptically. A concentration of 50, 100 and 200 mg/mL of the extracts were prepared using 30% dimethylsulphoxide (DMSO) as the reconstituting solvent and sterilized using 0.2 µm sterile membrane pore filter paper. Using micropipette, each extract of 0.1 ml was then pipetted into the wells of appropriately labelled plates and holes. The plates were allowed to stand on the laboratory bench for 15 minutes to allow proper in flow of the solution into the medium before incubating the plates at 37ºC for 24 hours. The control was prepared by using 0.1 ml of reconstituting solvent (30% DMSO) and incubated alongside with the extracts plates. After incubation, the zones of inhibition (diameter) formed in the Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 30 medium were measured in millimeter to determine antibacterial effectiveness of the extracts on the test organisms. The experiment was carried out in triplicates. 2.7. Determination of Minimum Inhibitory Concentrations (MIC) of B. pilosa and T. procumbens Extracts on Test Organisms The tube dilution susceptibility test was used to determine the MIC values of the plant extracts on the test organisms using the method of CLSI (2006). A series of Mueller-Hinton broth tubes containing varying two fold concentrations of the various plant extracts in the range of 300 mg/mL to 12.5 mg/mL were prepared and incubated with a previously standardized density of the test organisms (0.5 mL). The lowest concentration of the plant extract resulting in no growth following visual inspection after 18-24h of incubation for bacteria was recorded as the MIC. 3. Results and Discussion 3.1. Percentage Recovery of Plant Extracts The percentage recovery of each extract after the extraction processes was evaluated and shown in table 1. Table 1: Result of the percentage recovery of plant extracts Solvent Original weight/input Extracted (%)recovery= (g) weight/output(g) 𝐖𝐀 𝒙 𝟏𝟎𝟎 𝐈𝐖 A. Ethanol 250 16.00 6.40% 1. Ethanol 250 18.70 7.48% B. Aqueous 250 11.20 4.48% 2. Aqueous 250 10.20 4.08% C. Chloroform 250 9.70 3.88% 3. Chloroform 250 14.50 5.80% Key: 1, 2, 3 = Bidens pilosa extracts; A, B, C = Tridax procumbens extracts. WA = Weight of extracts recovered after extraction, IW = Initial weight of extracts. The ethanolic extract of B. pilosa had the highest percentage recovery of 7.48%, followed by chloroform extract with 5.80% and the least was observed on aqueous extract with 4.08% extracting value. Moreover, the ethanolic extract of T. procumbens had the highest percentage recovery of 6.40%, followed by aqueous extract with 4.48% extraction value and the least was recovered from chloroform extract with 3.88% (Table 1). Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 31 3.2. Qualitative Phytochemical Screening of B. pilosa and T. procumbens The ethanoic, aqueous and chloroform leaf extracts obtained from B. pilosa and T. procumbens were qualitatively screened for phytochemicals. As in table 2, the result of the phytochemical screening revealed the presence of tannins, alkaloids, flavonoids, saponins and cardiac glycosides in both plants [B. pilosa and T. procumbens]. Table 2: Qualitative Phytochemical screening of B. pilosa and T. procumbens Phytochemical Extracts BEE BAE BCE TEE TAE TCE Saponin + + + + + + Tannin + + + + + + Phlobatannin - - - - - - Flavonoid + + + + + + Steroid + + - + + - Terpenoid + - - - - - Cardiac glycoside + + - + + - Alkaloid + + + + + + Anthraquinone - - - - - - Key: + = present, – = absent, BEE= Ethanolic extract of Bidens pilosa, BAE = Aqueous extract of Bidens pilosa, BCE = Chloroform extract of Bidens pilosa, TEE = Ethanolic extract of T. procumbens, TAE = Aqueous extract of T. procumbens, TCE = Chloroform extract of T. procumbens. 3.3. Quantitative Phytochemical Analysis 3.3.1. Quantitative phytochemical analysis of B. pilosa The ethanolic, aqueous and chloroform leaf extracts obtained from B. pilosa were screened quantitatively for phytochemicals. The results presented in fig. 1. Tannin content had the highest value in ethanolic extract containing 23.50±0.45 mg/g, followed by cardiac glycoside content of 20.52±0.58 mg/g. The phytochemical contents present in aqueous extract ranges from 7.13±0.01 to 16.18±0.02 mg/g with steroids being the lowest and cardiac glycosides being the highest. The chloroform extract of B. pilosa extract components consist saponin, tannin, flavonoid and alkaloid with saponin content being the highest (15.63±0.38 mg/g) and flavonoid content being the lowest (8.41±0.30 mg/g). The tannin content recorded was 12.63±0.00mg/g and alkaloid content was 12.97±0.01mg/g (Fig. 1). Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 32 Fig 1: Quantitative phytochemical components of B. pilosa 3.3.2. Quantitative phytochemical analysis of T. procumbens The ethanoic, aqueous and chloroform leaf extracts obtained from T. procumbens were quantitatively screened for phytochemicals. The quantitative phytochemical analysis of T. procumbens ethanoic extract displayed high flavonoid content compared to all other phytochemicals present containing 25.54±0.25 mg/g followed by alkaloid content of 6.13±0.01 mg/g. The lowest value was observed in aqueous extract of T. procumbens for tannin content of 2.83±0.01 mg/g, followed by steroids content of 3.09±0.13 mg/g (Fig. 2). Fig 2: Quantitative phytochemical components of T. procumbens 3.4. Comparative Antibacterial Activities of Bidens pilosa Extracts at 50 mg/ml on Test Organisms Copyright © 2017 by Modern Scientific Press Company, Florida, USA Int. J. Modern Biol. Med.2017, 8(1): 24-46 33 The antibacterial activities of crude ethanoic, aqueous and chloroform leaf extracts of Bidens pilosa at 50 mg/ml concentration on clinical and type test microorganisms after 24 hours of incubation are presented in Table 3. Table 3: Comparative antibacterial activities of Bidens pilosa at 50mg/ml on test microorganisms (Zones of inhibition, mm) Organisms Source Ethanol Aqueous Chloroform Control *E. coli 4.30±0.10b 0.00±0.00a 0.00±0.00a 0.00±0.00a E. coli ATCC35218 3.13±0.20c 3.03±0.05c 2.13±0.15b 0.00±0.00a *K. pneumoniae 2.36±0.15b 0.00±0.00a 0.00±0.00a 0.00±0.00a K. pneumoniae ATCC48891 3.13±0.15c 0.00±0.00a 2.26±0.25b 0.00±0.00a *P. aeruginosa 4.40±0.30c 2.36±0.25b 0.00±0.10a 0.00±0.00a P. aeruginosa ATCC27853 4.96±0.57d 3.33±0.15c 2.36±0.25b 0.00±0.00a *B. subtilis 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a B. subtilis ATCC21332 1.96±0.57c 1.00±0.00b 2.36±0.25d 0.00±0.00a *S. aureus 3.20±0.10b 0.00±0.00a 0.00±0.00a 0.00±0.00a S. aureus ATCC43300 5.16±0.15d 2.36±0.25b 3.20±0.10c 0.00±0.00a *S. pyogenes 2.06±0.11b 0.00±0.00a 2.26±0.15b 0.00±0.00a S. pyogenes ATCC29212 3.20±0.10d 2.20±0.10c 1.10±0.17b 0.00±0.00a Values represent means ± standard deviation of triplicate readings. Superscripts of the same letter in a row are not significantly different at P≤0.05. *Clinical isolate The zones of inhibition (mm) of the extracts on the test organisms evaluated ranges from 1.00±0.00 mm to 5.16±0.15 mm. The highest zone of inhibition was observed on Staphylococcus aureus ATCC 43300 (ethanoic extract) with inhibition zone of 5.16±0.15 mm while the lowest zone of inhibition was observed on Bacillus subtilis ATCC 21332 (aqueous extract) with inhibition zone of 1.00±0.00 mm (Table 3). It is noteworthy that the clinical test organism of B. subtilis was resistance to all the extracts at this concentration. However, the clinical and type culture of Streptococcus pyogenes were susceptible to the inhibitory effects of all the B. pilosa extracts at this concentration except for the aqueous extract in which the clinical organism was resistance. 3.5. Comparative Antibacterial Activities of Bidens pilosa Extracts at 100 mg/ml on Test Organisms The antibacterial activities of crude ethanoic, aqueous and chloroform leaf extracts of Bidens pilosa at 100 mg/ml concentration on clinical and type test microorganisms after 24 hours of incubation is presented in Table 4. Copyright © 2017 by Modern Scientific Press Company, Florida, USA