Ind ry st trial Chem i us Industrial Chemistry Achika et al., Ind Chem 2016, 2:1 http://dx.doi.org/10.4172/2469-9764.1000113 ISSN: 2469-9764 Research Article Research Article Open OpenAccess Access Phytochemical Screening and Antimicrobial Studies of the Aerial Part of Aeschynomene uniflora Mey. Jonathan I Achika*, George I Ndukwe and Rachel G Ayo Department of Chemistry, Ahmadu Bello University, Zaria, Kaduna State, Nigeria Abstract The aim of the study was to investigate the phytochemical composition and antimicrobial activities of extracts of aerial part of Aeschynomene unilora Mey used in folklore medicine in order to scientiically validate some of its ethnomedicinal claims. The dried and pulverized aerial part of Aeschynomene unilora was extracted using petroleum ether, chloroform, ethyl acetate and methanol by Soxhlet extraction and each concentrated in vacuo to yield four extracts. The extracts were then subject to preliminary phytochemical screening and antimicrobial studies using standard method. Carbohydrates, cardiac glycoside, lavonoids, saponins, steroids, triterpenes and tannins were present in the crude extracts. The highest zone of inhibition of 25 mm was exhibited by the ethyl acetated extract against Staphylococcus aureus, while the petroleum ether extract show the lowest zone of inhibition of 16 mm against Streptococcus pyogenes. The chloroform and ethyl acetate extracts exhibited a minimum inhibitory concentration (MIC) of 7 mg/mL against, Bacillus subtilis and Candida stellatoidea respectively. Both the methanol and petroleum ether extracts had a MIC of 30 mg/mL against Candida stellatoidea. The minimum bactericidal/ fungicidal concentration (MBC/MFC) of the extracts ranged between 15 mg/mL to 60 mg/mL. In conclusion, the aerial parts of Aeschynomene unilora showed signiicant antimicrobial properties. This justiies the use of the in the treatment of human and animal infectious disease. Keywords: Aeschynomene unilora; Aerial part; Antimicrobial activity; Phytochemical screening; Fabaceae Introduction he plant kingdom is a treasure house of potential drugs and in the recent years there has been an increasing awareness about the importance of medicinal plants. Drugs from plants are easily available, less expensive, safe, eicient and rarely have side efects [1]. here has been an increasing interest in the study of medicinal plants as natural products in diferent parts of the world [2]. Medicinal plants represent rich sources of antimicrobial agents used medicinally in diferent countries and are a source of many potent drugs used for traditional medicine. hey contain a wide range of substances that can be used to treat chronic as well as infectious diseases [3]. Medicinal plants play important role in meeting the basic health needs in many developing countries and the industrialized countries. he use of medicinal plants has been based on the extraction which has led to the development of several drugs traditionally used in folk medicine [4]. Various drugs with medicinal properties have been attributed to natural herbs and medicinal plants which constitute the main source of new pharmaceuticals and health-care products [5]. Plants are used in the traditional medicine for treatment of various types of ailments, including microbial infections [6,7]. Many plant species are reputed to possess antimicrobial activities. Aeschynomene unilora is an erect shrub, it is rarely almost prostrate and a short-lived (2-4 years) perennial shrub. It is 0.5-2 m tall [8,9]. he plant is used in the treatment of psychotic disorders, tuberculosis, skin infections, menstrual disorders, small pox and antidote to snake venom. he aqueous extract of the whole plant is administered topically over the whole body to cure small pox in northern Nigeria. he plant is eaten as vegetable to cure fever symptoms and cough in Benue State Nigeria. he main aim of this study was to carry out the phytochemical and antimicrobial screening of Aeschynomene unilora in order to scientiically justify the ethnomedicinal usage of the plant in folklore medicine. he Aeschynomene genus belongs to the family Papilionaceae which are marshy erect herbs. In traditional medicinal system, this plant is used to treat body pain and swellings [10,11]. It is also used Ind Chem ISSN: 2469-9764 ICO, an open access journal to treat mumps [12]. he Aeschynomene genus is comprised of about 60 species which are widely distributed throughout tropical and subtropical regions. Most species are annual herbs or shrubs, but a few are small trees [13]. A. aspera aerial part juice is administered to cure cold, cough, and fever. he Dried young shoot powder with half tea spoon powdered candy is given to increase the consistency of semen; local herbalists used it for urinary troubles [14]. A. aspera is also recognized as leafy vegetable [15]. Aeschynomene indica is a swampy medicinal plant used to treat kidney stones and urinary disorders by local herbalists [16]. he leaves of A. granillora are used as tonic, diuretic, laxative, antipyretic, chewed to disinfect mouth and throat. he lower in headache, dimness of vision catarrh, headache, cooling and improving appetite, bitter, astringent, acrid, antipyretic [17]. It is used as a bitter tonic, anthelmintic, astringent febrifuge, and for curing diarrhea, small pox [18]. he fruits are bitter and acrid, used as a laxative and to cure fever, pain, bronchitis, anemia, tumors, colic, jaundice, poisoning. Root used in rheumatism, expectorant, painful swelling, catarrh [19]. he hepatoprotective activity of benzene and alcoholic extracts of root of Aeschynomene aspera was investigated in rats for carbon tetrachloride induced hepatotoxicity and reported by Ref. [20]. Saravana et al. (2012) [21] reported the preliminary phytochemical screening of methanolic extract of Aeschynomene gradilora to contain alkaloids, lavonoids, tannins, triterpenes, gums and mucilage. Phytochemical investigation of Aeschynomene luminensis leaves and *Corresponding author: Jonathan I. Achika, Department of Chemistry, Ahmadu Bello University, Zaria, Kaduna State, Nigeria, Tel: +2347036890546; E-mail: achijonile@yahoo. com Received December 24, 2015; Accepted January 18, 2016; Published January 23, 2016 Citation: Achika JI, Ndukwe GI, Ayo RG (2016) Phytochemical Screening and Antimicrobial Studies of the Aerial Part of Aeschynomene unilora Mey. Ind Chem 1: 113. doi: 10.4172/2469-9764.1000113 Copyright: © 2016 Achika JI, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Volume 2 • Issue 1 • 1000113 Citation: Achika JI, Ndukwe GI, Ayo RG (2016) Phytochemical Screening and Antimicrobial Studies of the Aerial Part of Aeschynomene unilora Mey. Ind Chem 1: 113. doi: 10.4172/2469-9764.1000113 Page 2 of 2 branches led to isolation of the lavonoidglycosides kaempferol 3,7-diO-α-L-rhamnopyranoside, kaempferol 7-O-α-L-rhamnopyranoside, kaempferol 3-O-apiofuranosil- 7-O- rhamnopyranoside, quercitin 3-O-α-L-rhamnopyranoside, quercitin 3-O-arabinofuranoside, 8-β-Dglucopyranosyl 4’,5,7 trihydroxylavanone, the isolavonoid 4’,7-dihydroxy-isolavone, the dimer epicatechin-(2β, 4β)+- epicatechin, the polyol 3-O-methyl-chiro-inositol and two steroids in sitosterol and stigmasterol mixture [22]. In 2011, Chen et al. [23] reported novel compound monotetracontane from the dry leaves of Aeschynomene indica Linn. Materials and Methods Collection of plant materials he aerial part of A. unilora was collected during the rainy season in September, 2012 from Makurdi, Benue State, Nigeria. he plant was identiied in the Herbarium unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria; and a Voucher specimen, number 2408, was deposited in the Herbarium. he plant materials were air-dried, pulverized and stored in air-tight containers until needed for further investigation. Extraction procedure he air-dried pulverized plant material (200 g) was placed in a Soxhlet extractor, where it was exhaustively and successively extracted using petroleum ether (60-80°C), chloroform, ethyl acetate and methanol. he crude extracts of the plant were concentrated in vacuo at 40°C using a rotary evaporator. he crude extracts were subjected to phytochemical and antimicrobial studies. Phytochemical screening Phytochemical screening of the extracts was carried out using the standard procedures [7,24]. he anti-microbial screening he anti-microbial activity of the petroleum ether, chloroform, ethyl acetate and the methanol extracts was determined using some pathogenic microbes, obtained from the Department of Medical Microbiology Ahmadu Bello University Teaching Hospital, Zaria. Each extracts (0.6 g) was weighed and dissolved in 10 ml dimethyl sulphoxide (DMSO) to obtain a concentration of 60 mg/ml. his initial concentration was used to determine the antimicrobial activity of the extracts. Mueller Hinton agar was the growth medium used for the microbes. he medium was prepared, sterilized at 121°C for 15 minutes and the sterilized medium was poured into sterile Petri dishes. he plates were allowed to cool and solidify. Agar difusion method was used for screening of the extracts. he sterilized medium was seeded with 0.1 ml of the standard inoculum of the test microorganism; the inoculum was spread evenly over the surface of the medium with a sterile swab. Using a standard cork borer of 6 mm in diameter a well was cut at the center of each inoculated medium. Solution (0.1 ml) of each extracts of concentration of 60 mg/ml was then introduced into each well on the medium. he inoculated medium was then incubated at 37°C for 24 h ater which each plate was observed for the zone of inhibition of growth. he zone was measured with a transparent ruler and the result recorded in millimeters [25]. Minimum inhibition concentration of extracts he minimum inhibition concentration (MIC) of the extracts was determined using broth dilution method. Mueller Hinton broth was prepared; 10 ml was dispensed into test tubes and was sterilized Ind Chem ISSN: 2469-9764 ICO, an open access journal at 121°C for 15 minutes. he broth was allowed to cool. Mc- Farland turbidity standard scale number 0.5 was prepared to give turbid solution. Normal saline was prepared and 10 ml was dispensed into sterile test tubes. he test microbes were inoculated incubated at 37°C for 6 hours. Dilution of the test microbes was done in the normal saline until the turbidity matched that of the Mc - Farlands scale by visual comparison. At this point the concentration of the microbes was about 1.5 × 108 cfu/ml. Two-fold serial dilution of the extracts in the sterile broth was made to obtain diferent concentration (60 mg/ml, 30 mg/ ml, 15 mg/ml, 7.5 mg/ml and 3.25 mg/ml). he initial concentration was obtained by dissolving 0.6 g of the extracts in 10 mL of the sterile broth. he broths were incubated at 37°C for 24 hours. he results were recorded ater 24 hours. hereater 0.1 ml of the test microorganism in the normal saline was inoculated in to the diferent concentrations, incubations was made at 37°C for 24 h for bacteria and at 30°C for 48 h for fungi, ater which each test tube of the broth was observed for turbidity (growth). he MIC was the value recorded from the test tube with the lowest concentration of the extract in the broth which showed no growth [25,26]. Minimum bactericidal concentration/Minimum fungicidal concentration he minimum bactericidal concentration/minimum fungicidal concentration (MBC/MFC) is the concentration that determines if the test microbes were killed or only their growth was inhibited. Mueller Hinton agar was prepared and sterilized at 121°C for 15 minutes, poured into Petri dishes and allowed to cool and solidify. he content of the MIC in the serial dilution was then sub-cultured onto the prepared medium and incubation was done at 37°C for 24 h. hereater each plate of the medium was observed for colony growth. he value obtained in the plate with the lowest concentration of the extract without colony growth was recorded as the MBC/MFC [7,25,26]. Results Phytochemical analysis conducted on the plant extracts revealed the presence of constituents which are known to exhibit medicinal as well as physiological activities [27]. he presence of steroids and triterpenes in all the four extracts were observed: cardiac glycosides were present in all the extracts except in petroleum ether extracts. Carbohydrates, cardiac glycoside, tannins, lavonoids, steroids and triterpenes were present in the ethyl acetate, chloroform, and methanol extracts. Saponins were present only in the methanol extract while alkaloids and anthraquinones were absent in all the four extracts (Table 1). he result of the antimicrobial screening is summarized in Tables 2 and 3. From the results of the antimicrobial screening (Table 2), the extracts showed activity against S. pyogenes, B. subtilis, C. albicans and S aureus. he petroleum ether extract was efective against S. pyogenes, B. subtilis, C. stellatoidea, K. pneumoniae, S aureus and C. albicans with a zone of inhibition of 16, 17, 17, 18 and 18 mm respectively. he MIC showed that the petroleum ether extract inhibited the growth of all the pathogenic microorganisms at a concentration of 30 mg/ml. he MBC/ MFC was found to be 60 mg/ml for the petroleum ether extract against all the test microorganism (Table 3). he chloroform extract showed activity against S. pyogenes, C. albicans, C. stallatoidea, S. aureus and B. subtilis, with zones of inhibition of 22, 22, 23, 24 and 27 mm, respectively. At the MIC of 15 mg/ml the chloroform extract inhibited the growth of S. aureus, S. pyogenes, K. pneumoniae, C. albicans and C. stallatoidea, while at 7.5 mg/ml the growth of B. subtilis was inhibited. he MBC/MFC was found to be 30 mg/ml and at this concentration the extract exhibited activity against all the test microorganisms; Volume 2 • Issue 1 • 1000113 Citation: Achika JI, Ndukwe GI, Ayo RG (2016) Phytochemical Screening and Antimicrobial Studies of the Aerial Part of Aeschynomene unilora Mey. Ind Chem 1: 113. doi: 10.4172/2469-9764.1000113 Page 3 of 2 Test AUPE AUCH AUET AUME Carbohydrates + + + + Cardiac glycosides - + + + Tannins - - + + Saponins - - - + Flavonoids - - + + Anthraquinones - - - - Steroids + + + + Triterpenes + + + + Alkaloids - - - - Key: +=present; -=absent; AUPE=Petroleum ether extract; AUCH=Chloroform extract; AUET=Ethyl acetate extract; AUME=Methanol extract Table 1: Phytochemical screening of the extracts of A. unilora. Test organism Diameter of zones of inhibition (mm) AUPE AUCH AUET AUME Ciproloxacin Fluconazole S. aureus 18 24 25 21 35 ND S. pyogenes 16 22 22 20 30 ND B. subtilis 17 27 28 22 42 ND K. pneumonia 18 24 23 20 44 ND C. albicans 18 22 24 20 ND 40 C. stellatoidea 17 23 26 19 ND 32 Key: AUPE=Petroleum ether extract; AUCH=Chloroform extract; AUET=Ethyl acetate extract; AUME=Methanol extract; ND=Not determine Table 2: Zone of Inhibition of extracts of A. unilora (mm). MIC MBC/MFC Test organism AUPE AUCH AUET AUME AUPE AUCH AUET AUME S. aureus 30 15 15 15 60 30 30 60 S. pyogenes 30 15 15 15 60 60 60 60 B. subtilis 30 7.5 7.5 15 60 30 30 30 K. pneumonia 30 15 15 15 60 30 30 60 C. albicans 30 15 15 15 60 30 30 60 C. stellatoidea 30 15 7.5 30 60 30 30 60 Key: AUPE=Petroleum ether extract; AUCH=Chloroform extract; AUET=Ethyl acetate extract; AUME=Methanol extract Table 3: Minimum inhibitory concentration and minimum bactericidal/fungicidal concentration of extracts of A. unilora (mg/ml). except S. pyogenes which had 60 mg/ml. he MIC showed that at lower concentration of 7.5 mg/ml the ethyl acetate extract inhibited the growth of B. subtilis and C. stellatoidea, but the inhibitions of S. aureus, S. pyogenes, and C. albicans occurred at 15 mg/ml. MBC/MFC (30 mg/ml) of the ethyl acetate extract was active against S. aureus, B. subtilis, K. pneumniae, C. albicans and C. stallatoidea, and the value for S. pyogenes was 60 mg/ml. he methanol extract induced signiicant zone of inhibition for C. stallatoidea, C. albicans, S. pyogenes, S. aureus and B. subtilis, with values of 19, 20, 20 and 21 mg/ml, respectively. At MIC of 15 mg/ml for S. aureus, S. pyogenes, B. subtilis and C. albicans. At MIC of 30 mg/ml, the growth of C. stellatoidea was inhibited, while other microbes were inhibited at a value of 15 mg/ml. At MBC/MFC of 60 mg/ml, the methanol extract exhibited activity against all the microbes with the exception of B. subtilis inhibited at 30 mg/ml. Discussion he presence of the secondary metabolites in the crude extracts of this plant may be responsible for some of the biological activities observed [28]. Phenolic compounds such as lavonoids and tannins which are presents in this plant are one of the largest and most ubiquitous groups of plant metabolites [29]. hey possess biological properties Ind Chem ISSN: 2469-9764 ICO, an open access journal such as antiapoptosis, antiaging, anticarcinogen, antiinlammation, antiatherosclerosis, cardiovascular protection and improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities [30]. his could explain the vast usage of this plant to manage infectious disease in folklore medicine. Several studies have described the antioxidant properties of medicinal plants which are rich in phenolic compounds [31,32]. Natural antioxidant mainly comes from plants in the form of phenolic compounds such as lavonoid, phenolic acids, tocopherols etc. his therefore implies that this plant could possess antiaging, anticarcinogenic properties [33]. Saponins are known to produce inhibitory efect on inlammation and as such, the presence of saponins in the crude extracts of this plant shows that this plant could be used as an anti-inlammatory agent [34]. he presence of saponins in the crude extracts may be responsible for the signiicant anti-bacteria activity exhibited, as these bacteria are responsible for inlammations. Saponins have the property of precipitating and coagulating red blood cells. Some of the characteristics of saponins include formation of foams in aqueous solutions, hemolytic activity, cholesterol binding properties and bitterness [35,36]. he presence of steroids in all the crude extracts account for the signiicant antibacterial activity observed especially in the ethyl acetate extracts. Steroids have been reported to have antibacterial properties [37], and they are very important compounds especially due to their relationship with compounds such as sex hormones [38]. Glycosides are known to lower the blood pressure according to many reports [39]. he results obtained in this study suggest that, the identiied phytochemical compounds may be the bioactive constituents and this plant is proving to be an increasingly valuable reservoir of bioactive compounds of substantial medicinal merit [40]. Of the four extracts tested, the ethyl acetate extract demonstrated the highest antimicrobial activity against all the microorganisms. he result demonstrated that the aerial part of the plant may contain potent active constituent which are beneicial in the treatment and prevention of microbial diseases. Based on the inding of this present study, the application of the decoction of the aerial part of A. unilora in ethnomedicine is justiied. In conclusion, the extracts of Aeschynomene unilora demonstrated signiicant antibiotic potential may be used for the development of novel antimicrobial agents for the treatment of several diseases caused by microorganisms. 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