Ojo Joseph Sunday，Shola Kola Babatunde，Adeyinka Elizabeth Ajiboye，Racheal Majekodunmi Adedayo，Mufutau Adeyemi Ajao，Busayo Isreal Ajuwon

Department of Biosciences and Biotechnology，Kwara State University，Malete，PMB 1530，Ilorin，Kwara State，Nigeria

Evaluation of phytochemical properties and in-vitro antibacterial activity of the aqueous extracts of leaf，seed and root of Abrus precatorius Linn.against Salmonella and Shigella

Ojo Joseph Sunday*，Shola Kola Babatunde，Adeyinka Elizabeth Ajiboye，Racheal Majekodunmi Adedayo，Mufutau Adeyemi Ajao，Busayo Isreal Ajuwon

Department of Biosciences and Biotechnology，Kwara State University，Malete，PMB 1530，Ilorin，Kwara State，Nigeria

ARTICLE INFO

Article history:

Accepted 15 May 2016

Available online 26 Jul 2016

Abrus precatorius

Phytochemicals

Antibacterial susceptibility Salmonella

Shigella

Objective:To investigate the phytochemical components of Abrus precatorius（A.precatorius）and the in-vitro susceptibility of Salmonella typhi and Shigella dysenteriae to the aqueous extracts of A.precatorius leaf，seed and root.

Methods:The leaf，seed and root of A.precatorius were collected and homogenized separately after drying at 40°C for seven days in hot-air oven.The aqueous extracts of each of the parts were prepared and subjected to phytochemical screening.Dilutions of 400，300，200，100 mg/mL，of each of the extracts were used for broth dilution in minimum inhibitory concentration（MIC）determination against clinical isolates of Salmonella typhi and Shigella dysenteriae，while 50，40，30，20，and 10 mg/mL dilutions were used for the agar diffusion test and 100μg/mL and 10μg/mL of gentamycin were used as controls for broth dilution in MIC determination and agar diffusion test，respectively.

Results:Qualitative study reveals that tannin，saponins，alkaloids，flavonoids，terpenoids，steroids and phenols were present in all of the plant parts.The leaf has the highest quantities of tannin and phenol.The root generally showed the lowest quantity of all the compounds.The pathogens were susceptible to aqueous extracts of the leaf，stem and root of A.precatorius at 50 mg/mL.At concentrations of 40，30 and 20 mg/mL，all the aqueous extracts of A.precatorius showed variation in MIC，but produced no minimum bactericide effect upon subculture.There were variations in diameter of zone of inhibition against the organisms at lower concentrations.

Conclusions:These findings suggest that A.precatorius is a valuable source of phytochemicals with promising antibacterial activity.Considering this bioactivity，A.precatorius could be probed further for toxicity，and to obtain some novel antibacterial molecules.

1.Introduction

Plants are important sources of chemical compounds with potential therapeutic effects.Medicinal plants have been identified and used throughout human history for combating infectious diseases［1］.They have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions，and for defense against pests，pathogens，fungi and herbivorous mammals.At least 12000 of such compounds have been isolated；a number estimated to be less than 10%of the total［2，3］.In 2001，researchers identified 122 compounds used in modern medicine which were derived from plants sources，and 80%of these have ethnomedicinal uses［4］.

Abrus precatorius（A.precatorius）belongs to the class Magnoliophyta；order Fabales；family Fabaceae； subfamily Faboidene；tribe Abreae；genus Abrus；and of precatoriusspecies.This plant is known with various names such as Abrus seed，Jequirity，Imisimisi，Aivoeiro，Crab eye，Rotary pea，and Indian bead among others in Africa，India，China and Brazil.

A.precatorius subspecies Africanus is a slender，perennial climber that twines around trees，shrubs and hedges.The leaves are glabrous with long internodes and are alternate，compound paripinnate with stipules.Each leaf is about 100-150 mm long with 20 or more leaflets.Each leaflet is about 15-25 mm long，6-8 mm wide，oblong and obtuse.Hot or cold water extracts and dried powder of the root，leaf and stem of this plant are used traditionally as medicinal herbs.Previous reports indicated that A.precatorius leaf，stem and root have human and veterinary uses as antimicrobial（including Mycobacteria tuberculosis），antiprotozoal，insecticidal and anti-snake venom remedies［4，5］. Several groups of secondary metabolites such as alkaloids，triterpenoids，isofluranoquinones，anthocyanins，starch，tannin，flavonoids，orientin have been isolated from this plant［6，7］. These compounds may be responsible for various potential medicinal properties attributed to the plant.

Acute gastroenteritis is one of the leading causes of illness and death in infants，children and aged individuals throughout the world，especially in developing countries.Asia，Africa and Latin America had an estimated 2.5 million death annually in children less than five years of age due to infectious agents［8］. Among the enteric pathogens，Salmonella and Shigella species are of particular concern as they are responsible for enteric fever，food poisoning and gastroenteritis.

Local traditional herbal specialists use aqueous infusion or extracts（cold or hot）of leaf，seed and root of A.precatorius for the treatment of intestinal illnesses that could be of bacterial，viral or protozoan origins.Very few research studies have been done on the practical application of A.precatorius parts on clinical isolates from the intestine［9，10］，these previous investigations were on different bacteria species and from other clinical sites［11］.Previous studies also used only a part of the plant［12，13］. This study therefore investigates antimicrobial activity of aqueous extracts of leaf，seed and root of A.precatorius on clinical isolates of Salmonella and Shigella species.

2.Materials and methods

2.1.Collection and maintenance of plant sample

The leaf，seed and root of A.precatorius were collected from farms in Ilorin and Igosun areas of Kwara State Nigeria.The plant was identified and authenticated by a plant taxonomist of the herbarium unit，Department of Botany，Obafemi Awolowo University，Ile-Ife，Nigeria.The parts were carefully removed，separated and dried at 40°C for 7 days in hot-air oven（Unicorn，England）.Each part was grounded to powdery form with countertop electric blender（Binatone，Japan），and stored in airtight bottles at 4°C until it is required for use.

2.2.Preparation of extracts

The aqueous extracts of leaf，seed and root of A.precatorius were prepared separately as described by previous authors［14］. Two hundred grams each of the powder was suspended in 1000mL of sterile distilled water（SDW）in a flask. Appropriately，labeled flask was used for each plant part and placed in an orbital shaker（Gem Instrument，Japan）and agitated continuously for 8 h at 25°C.It was then sieved with filter of pore size 30μm.The filtrates were concentrated using rotatory evaporator（Quickfit，UK）at 80°C，and then further evaporated in small beakers at 80°C in water bath（Unicorn instruments，UK）.The extracts were collected in airtight plastic universal bottles，labeled accordingly and stored at 4°C till further use.

2.3.Phytochemical screening of leaf，seed and root of A.precatorius

Aqueous extract of A.precatorius leaf，seed and root were subjected to phytochemical analysis using standard techniques previously established［15］.The detection of steroids，saponins，phenolics，tannins，flavonoids，terpenoids and alkaloids were carried out respectively as previously described［16-19］.Each test was qualitatively expressed as negative（-）not present or positive（+）present；the intensity of the characteristic color was expressed as（++）or（+++）or（++++）.Quantitate of steroids，saponins，phenolics，tannins，flavonoids，terpenoids and alkaloids were also determined using previously established methods［19-21］.

2.4.Dilution of leaf，seed and root extracts and gentamycin

Twenty five grams of each of the extract concentrates was weighed and dissolved in 50 mL of SDW to make a dilution of 500 mg/mL as stock solution.From the stock solution，dilutions of 400，300，200，100 mg/mL were made.These were used for broth dilution in minimum inhibitory concentration determination of the leaf，seed and root extracts.

From the stock，dilutions of 50，40，30，20，and 10 mg/mL were prepared for each of the extracts for agar diffusion test. Gentamycin injection（Mayer and Baker，Nigeria）ampules containing 40 mg/mL was diluted serially to 100μg/mL and 10μg/mL and these were used as control drug for broth dilution and agar diffusion tests，respectively.

2.5.Test organisms

Clinical isolates of Salmonella typhi（S.typhi）and Shigella dysenteriae（Sh.dysenteriae）were obtained from the Department of Microbiology and Parasitiology，University of Ilorin Teaching Hospital，Ilorin，Kwara State，Nigeria.The organisms were isolated and confirmed from stool samples of patients with intestinal illnesses.The isolates were maintained on nutrient agar plates at4°C and were sub-cultured onto nutrient agar for 24 h before in-vitro microbial test commenced.Standard inoculum was prepared in sterile normal saline to 0.5 Mcfarland standard of1×106CFU/mL.

2.6.Dilution of nutrient broth

For each of the extract，9 mL of nutrient broth were prepared in separate McCartney bottles from the serial dilution of 500，400，300，200，100 mg/mL previously prepared and 100μg/mL of gentamycin and SDW were used as controls.One milliliter of each of these was added to 9 mL of nutrient broth in the separate bottles to give a final concentration of 50，40，30，20，10 mg/mL of each extract.One milliliter of each of 100μg/mL of gentamycin and SDW were used as control.Each test organism was prepared by adding a drop of standardized organism suspension to all bottles prepared using sterile Pasteur pipette.All bottleswere incubated at 37°C for 18-24 h.Minimum inhibitory concentration was determined by checking for the lowest dilution in which there was no turbidity［22］.

All bottles with no turbidity were sub-cultured on Mueller Hinton agar and incubated at 37°C for 18-24 h to determine minimum bactericidal concentration.The lowest dilution that showed no growth on agar plate was taken as minimum bactericidal concentration［22］.

2.7.Agar well diffusion

Three plates，each for leaf，seed and root of the extracts，were used for the agar diffusion test for each of the test organisms. Mueller Hinton agar plates previously prepared and allowed to dry in the incubator at 37°C for 30 min were used.The plates were seeded with sterile swab stick dipped in diluted culture to 1×106CFU/mL of each of the test organisms and spread on the entire surface of the plate to give semi-confluent growth.Using a sterilized cork borer，six holes of 5-mm diameter were bored in circle around a central hole on the seeded agar plate at equal distance apart.Six drops of each dilution was dispensed into appropriately labeled hole while the center hole received six drops of 10μg/mL dilution of gentamycin.The same procedure was repeated for all the extracts in triplicates for each of the organism.All the plates were left on the surface of the inoculating chamber for 2 h to allow diffusion of extracts and antibiotics.The plates were then incubated at 37°C aerobically for 18-20 h.The zone sizes were measured to the nearest millimeter and averaged. It was interpreted as described by previous studies［23，24］.

3.Results

Seven phytochemicals，namely，tannin，saponins，alkaloids，flavonoids，terpenoids，steroids and phenols were assayed in aqueous extracts of leaf，seed and root of A.precatorius.The phytochemicals were detected in all the three parts.The three parts were very rich in tannin and phenols but low in alkanoids，flavonoids and steroids（Table 1）.

Quantitative analyses of the constituents of these phytochemicals showed that the leaf had the highest quantity of tannin and phenol with 27.71 mg/g and 22.84 mg/g，respectively.The root generally showed lower quantities of the seven phytochemical assayed than the leaf and seed as shown in Table 2.

There were differences in susceptibility of S.typhi and Sh. dysenteriae to various concentrations of aqueous extracts of A.precatorius leaf，seed and root.Generally，the susceptibility of both organisms was concentration dependent.The higher the concentrations used，the higher the susceptibility of the organisms.S.typhi is more sensitive to the three parts of the plants than Sh.dysenteriae.The two intestinal pathogens were sensitive to gentamycin at 10μg/mL than the highest concentration of the aqueous extracts of the three parts of A.precatorius as depicted in Table 3.

Table 1　Qualitative constituents of aqueous extract of leaf，seed and root of A.precatorius.

Table 2　Quantity of phytochemicals in aqueous extracts of leaf，seed and root of A.precatorius（mg/g）.

Table 3　Antibacterial activity of aqueous extract of leaf，seed and root of A.precatorius（mm）.

The zone diameter of inhibition of each extract concentration was measured in nearest mL［24］，and this was compared with the control drug（gentamycin 10μg/mL）.S.typhi is sensitive to aqueous extracts of leaf，seed and root at higher concentration of dilution（50 mg/mL）as shown in Table 3.The extracts were resistant to lower concentrations of the extracts of leaf，seed and root.Extracts of seed have higher inhibitory effects than the leaf and root on S.typhi.

Sh.dysenteriae was sensitive only to aqueous extract of seed，but reduced susceptibility to aqueous extracts of leaf and root at 50 mg/mL.It was，however，resistant to other concentrations tested as evidenced in Table 3.At concentrations of 50 and 40 mg/mL，the three parts of A.precatorius extracts produced inhibition in broth culture of S.typhi and Sh.dysenteriae（Table 4）.The aqueous extract of the seed produced inhibition at 30 mg/mL as exhibited in Table 4.Both organisms grew at concentrations that produced minimum inhibitory concentrations in the minimum bactericidal concentration test.

Table 4　Minimum inhibitory concentration of aqueous extract of leaf，seed and root of A.precatorius.

4.Discussion

Many useful drugs have been formulated through the exploration of whole or parts of medicinal plants.This exploration will definitely continue as long as microorganisms keep developing drug resistance.Previous works showed that A.precatorius is a unique source of many potential phytochemicals that can be explored for human use.In this study，we have found seven phytochemicals in the three parts of A.precatorius.These include alkaloids，flavonoids，phenols，saponins，steroids，tannins and terpenoids.There are slight variations in the concentrations of these phytochemicals in the plant parts.The leaf and seed of the plant have higher concentration of phytochemical than the root（Table 2）.

The plant has high quantities of tannin and phenols，but lower concentration of other phytochemicals as indicated in Tables1and 2.Similar reports were obtained by other authors［5，6］.Several other compounds such as abine，anthocyanins，abrectori，and isorienlin have been found in A.precatorius［25］.These compounds might have contributed to its various medicinal uses such as antimicrobial，antiprotozoan and antihelminthic［6］.

Antibacterial activity of A.precatorius against S.typhi and Sh. dysenteriae showed that the aqueous extract of the plant parts have antibacterial activity against them（Table 3）.The antibacterial activity of seed of A.precatorius has more profound effect than the leaf and root.At concentration of 50 mg/mL，the inhibitory activity of aqueous extracts of seed of this plant against S.typhi and Sh.dysenteriae is similar to that of the control.The extract had zone of inhibition of diameter 17 mm and 19 mm against S.typhi and Sh.dysenteriae while the control has zone inhibition diameter of 23 and 21 against the respective organism［24］.At concentration of 40 mg/mL the aqueous extracts produce varied zone of inhibition（Table 3）.These were however，lower than zone diameter sizes produced by the control the organisms. This result is comparable to the findings of previous authors［10］，who reported moderate antimicrobial activity of seed of A.precatorius against clinically important bacteria such as Enterococcus faecalis，Escherichia coli，Staphylococcus aureus，Streptococcus thermophilus，Pseudomonas aeruginosa and Micrococcus luteus.

The aqueous extract of the seed of A.precatorius has slightly higher concentration of phytochemicals than the leaf and root，which may contribute to it higher antimicrobial activity.Earlier study revealed that the seed of A.precatorius has diverse phytochemical components［26］.The phytochemical components of aqueous extracts of leaf were also slightly higher than those of root（Tables 1 and 2）.This is also reflected in the antibacterial susceptibility of the tested organisms，and this could justify the reason why local herbal practitioners prefer to use the leaf and seed of the plant for various ailment treatments.

At concentrations of 50，40 and 30 mg/mL，the aqueous extracts that produced inhibition of growth of these organisms in nutrient broth failed minimum bactericidal concentration determination test.This indicated that the aqueous extracts of the parts of A.precatorius only inhibit but does not kill the organisms at the concentrations used.

However，it has been reported that the seed of A.precatorius can be extremely poisonous，especially if cracked before consumption.High toxic level can cause severe stomach cramps，diarrhea，tachycardia，coma，cold sweat and nausea［9，27］. Generally，aqueous extracts or infusion of any part of A.precatorius must be taken with caution because prolong use of this plant has been associated with anemia and can increase the level of human white blood cell count［28-30］.Therefore，it is strongly advised that aqueous extracts or any product from A.precatorius should be taken under professional guidance.

Further research work is needed on phytochemical components present in this plant that may be injurious to human health；and there is a need to determine the bioactive components that are of medicinal value which can be extracted for human use.

In conclusion，this study has offered a scientific basis for traditional use of aqueous extracts of A.precatorius for treatment of gastrointestinal bacterial infections.Further study is necessary to determine toxicity level of the bioactive component and possibly remove the toxins and determine active phytochemical components for drug production.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

This research work was supported by Institutional Based Research grant from the Tertiary Education Trust Fund，Nigeria（TETFUND/KWASU-2014-01）.We wish to acknowledge the assistance of Medical Laboratory Scientist，Mr.Jimoh Abdularaheem of Department of Microbiology and Parasitology，University of Ilorin Teaching Hospital for supplying us Salmonella and Shigella，our research assistants Miss.Phebe M. Gboyinde and Miss.Omobolanle O.Akanbi for their diligence.

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27 Oct 2015

in revised form 22 Feb，2nd revised form 29 Feb 2016

Ojo Joseph Sunday，Department of Biosciences and Biotechnology，Kwara State University，Malete，PMB 1530，Ilorin，Kwara State，Nigeria.

Tel:+234 8033672433

E-mails:obakemi2002@yahoo.com，ojo.sunday@kwasu.edu.ng

Foundation Project:Supported by Institutional Based Research grant from the Tertiary Education Trust Fund，Nigeria（TETFUND/KWASU-2014-01）.

Peer review under responsibility of Hainan Medical University.The journal implements double-blind peer review practiced by specially invited international editorial board members.

2221-1691/Copyright?2016 Hainan Medical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http:// creative commons.org/licenses/by-nc-nd/4.0/）.