Sheila M Maregesi et al.,(2013) Int. J. Res. Phytochem. Pharmacol., 3(2),94-98

94 ©JK Welfare &Pharmascope Foundation | International Journal of Research in Phytochemistry& Pharmacology

Phytochemical analysis and antimicrobial activity of Boerhaviacoccinea

Sheila M. Maregesi*1, Rogers Mwakalukwa

1, Denis T. Mwangomo

2, Nondo R.S.O

2

1Schoo of Pharmacy, Muhimbili University College of Health and Allied Sciences,P.O. Box 65013 Dar Es Salaam,

Tanzania 2Institute of Traditional Medicine, Muhimbili University College of Health and Allied Sciences, P.O. Box 65001

Dar Es Salaam, Tanzania

ABSTRACT

This study was aimed to evaluate the phytochemical profile and antimicrobial activity of the leaf and root metha-nolic extracts of BoerhaviacoccineaP.Mill, of the family Nyctaginaceae. Phytochemical analysis confirmed the presence of tannins and flavonoids and absence saponins in both root and leaves extracts. Whereas, terpenenes-were detected in root extract and not in the leaves and glycosides were detected in leaves extract but not in the root extract. The in vitro antimicrobial testing was carried by using six pathogenic microbes that included; four bacteria (Bacilusanthracis (NCTC10073), Streptococcus pyogens (clinical isolate), Vibrio cholera (clinical isolate), Shigelladysentriae(clinical isolate)) and two fungi; Candida albicans (ATCC90028) and Cryptococcus neoformans (clinical isolate).Moderate inhibitory activity with MIC value of 0.625 mg/mL against Cryptococcus neoformans and MIC value of 1.250 mg/mL against Candida albicans were obtained from leaf and root extracts respective-ly.Both extracts exhibited weak activity of 5.00 mg/mL against Bacilusanthracis whereas, Streptococcus pyogens, Vibrio cholerae, Shigelladysentriae were resistant at the maximum concentration tested (MIC > 5.00 mg/ml). In the light of our results, phytochemicals with antifungal activity are present in both leaves and roots. The tradition-al use this plant has been validated. The activity against Cryptococcus neoformans provides is an additional data that was not indicated by information providers.

Keywords: Boerhaviacoccinea; Antibacterial; Antifungal; Phytochemical analysis

INTRODUCTION

Since ancient times, plants have been used to treat various human and animal ailments. They are rich in natural chemicals and the main sources for pharma-ceuticals, nutraceuticals, cosmetics and food supple-ments (Surivayanthana et al., 2012). Infectious diseases are the leading cause of morbidity and mortality espe-cially in the developing countries (WHO, 2004). The clinical efficiency of many existing antimicrobial drugs is threatened by the emergence of multi-drug resistant pathogens which pose a continuous need to discover new antimicrobial compounds with diverse chemical structures and possibly novel mechanisms of action for new and re-emerging infectious diseases (Rojas et al., 2003). Biomolecules of plant origin appear to be one of the alternatives, used by many societies, for the con-trol of various antibiotic resistant human pathogens (Raghavendra et al., 2006).

In developing countries especially in the tropical areas rich in plants, herbs play a key role in treatment of dis-

ease.People continue using plants that are effective. Thus, the probability of discovering novel therapeutic agents are higher when plant selection is based eth-nomedicical information from reliable sources.

Boerhaviacoccinea, P. Mill, is commonly known as Scar-let Spiderling or Red Spiderling plant, belongs to the family Nyctaginaceae. It is a stout perennial, prostrate to decumbent, multi-stemmed, many-branched herb having a diffusely paniculate presentation of flowers, with stems up to 1.5ft. (3 - 50 cm) long, radiating out-ward forming a mat of herbaceous growth (Halvorson and Guertin, 2003).

Although some sources considera plantname syn-onymous with Boerhaviadiffusa, reputable databases like IntergratedTaxanomic Information Systems (ITIS) and the w3 TROPICOS (Missouri Botanical Garden) identifies them as different taxa (Halvorson and Guer-tin, 2003). Genetical studies based on the chromosome count confirmed that, Boerhaviacoccinea and Boerha-viadiffusa are two different species (Spellenberg, 2000).

In Tanzania the leaves of Boerhaviacoccinea are used against oral candidiasis and apthous ulcers. (Maregesi et al., 2007). The methanolic extract exhibited anti-plasmodial activity of 250 µg/ml and antiHIV activity against HIV1 IIIB strain with IC50 values of 54.85 and 37µg/ml for methonalic and water extract respectively

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ISSN: 2231-010X Research Article

* Corresponding Author Email: smaregesi@hotmail.com Contact: +255716574413 Received on:08-04-2013 Revised on:28-04-2013 Accepted on:08-05-2013

Sheila M Maregesi et al.,(2013) Int. J. Res. Phytochem. Pharmacol., 3(2),94-98

©JK Welfare &Pharmascope Foundation | International Journal of Research in Phytochemistry& Pharmacology 95

(Maregesi et al., 2010). Recently the plant leaves were reported for use against fungal infection of the scalp.Also, leaves are used as vegetable by some tribes of Tanzania e.g. Rangi and Luo(Maregesi, Personal Communication, 2012).

To the best of our understanding, phytochemical data is lacking, and the biological testing is scanty compared to the related species such as B. diffusa of which vari-ous pharmacological studies such as antimicrobial against various pathogenic microbes, anti-inflamatory, antioxidant, antihepatopic activity etc. has been estab-lished and even a phytomedicinepantented(Oladele et al., 2011; Khalid et al., 2011; Muthulingum, 2008; (WO2009044040 (A1), 2009)

MATERIAL AND METHODS

Chemical reagents and media

Dimethylsulphoxide was purchased from Sigma (Poole, Dorset, England), Fluconazole was purchased from CA-DILA pharmaceutical Ltd (Dholka India), Gentamicin susceptibility test discs (10µg) were purchased from Oxoid (Oxoid, Basingstoke, Hampshire, England). Iodo-nitrotetrazolium chloride was purchased from SIGMA (Sigma-Aldrich, St Louis, USA). Tryptone soya agar and Tryptone soya broth were purchased from Himedia Laboratory pvt Ltd (Mumbai, India). Sabouraud dex-trose agar and broth were obtained from Biotec Labor-atory Ltd (Ipswich. United Kingdom). Reagents and solvents used were of analytical grade purchased from the Lab Equip Ltd. Dar Es Salaam, Tanzania.

Plant material collection authentication

Leaves and roots were collected from the Botanical Garden located at Muhimbili University of Health and Allied Sciences, Dar Es Salaam Tanzania. Authentication was done at the herbarium unit, Department of Bota-ny, University of Dar Es Salaam.

Plant material processing and extracts preparation

The collected roots were freed from soil, cut into small pieces, dried in shade and powdered using a blender. Fresh leaves were pounded using mortar and pestle to increase the surface area.

60 gm of the dry powdered root was extracted by ma-

ceration using 98% Methanol (3 x 500mL) at room temperatue with occasional shaking. The micella were pooled together and filtered through 22 μm filter pa-per followed by drying using a vacuum rotor evapora-tor at about 40°C and freeze drier to yield 4.76 gm (7.4%) dry extracts.

680 gm of pounded leaves was extracted three times by maceration by using 98% Methanol (1200 mL for 72 hours, 2 x 750 mL for 48 hours) with occasional shak-ing. The micella were pooled together filtered through 22 μm filter paper dried as above to yield 18.0 gm (2.65%) dry extracts.

Qualitative analysis of phytochemicals

Chemical tests were carried out on extracts using stan-dard procedures to identify the constituents as de-scribed by Trease and Evans (2004) and Mushi et al. (2012).

Antimicrobial testing

Tested microorganisms

The extracts were tested against authentic pure cul-tures of human pathogenic vis bacteria namely; Bacil-lus anthrancis (NCTC 10073), Streptococcus pyogens (clinical isolate), Vibrio cholera (clinical isolate) and Shigellaflexneri (clinical isolate), two fungi Candida albicans (ATCC 90028) and Cryptococcus neoformans (clinical isolate). All were obtained from the Depart-ment of Microbiology and Immunology, Muhimbili University of Health and Allied Sciences.

Both antibacterial and antifungal testing used a Broth dilution method following the protocols described by Mwangomo et al. (2012). In this method, a series of dilution of test samples were mixed with a suitable medium, which has previously been inoculated with the test microorganism.

Antibacterial testing

MIC was determined by the broth microdilution tech-nique using sterile flat bottomed 96 well polystyrene microtiter plates. Bacterial suspensions equivalent to 0.5 McFarland concentrations were prepared by sus-pending microbes’ inocula in sterile distilled water and adjusting to get the right turbidity (equivalent to 0.5

Table 1: Preliminary phytochemical analysis of B. coccinea

Phytochemicals TestPerformed Root Extract Leaves Extract

Phenols /Tannins Neutral FeCl3 Gelatin test

Present Present

Saponins Olive Oil Method Liebermann Burchard’s reaction

Absent Absent

Flavonoids Ammonia Solution + Conc. H2SO4 Present Present

Terpenoids Chloroform + c. H2SO4 Present Absent

Glycosides Conc. HCl + Ammonia Soln + Benedict’s Soln

Absent Present

Reducing sugars Fehling’s test Present Present

Sheila M Maregesi et al.,(2013) Int. J. Res. Phytochem. Pharmacol., 3(2),94-98

96 ©JK Welfare &Pharmascope Foundation | International Journal of Research in Phytochemistry& Pharmacology

McFarland concentrations). Test solution were pre-pared by dissolving 20 mg of the extracts into 0.1 ml of DMSO and diluted with 0.9 ml of distilled water to make a concentration of 20 mg/ml. The stock solution (50 µl) was pipetted and added into the first well of each row of plates pre- loaded with 50 µl of tryptone soya broth. Then serial dilution was performed by transferring the test sample from first row wells to wells of the next rows, down to the last rows. The 50 µl from the last row wells were discarded. This was fol-lowed by addition of 50 µl of solution containing the test organisms (0.5 McFarland dilutions) to each of the wells. Wells in two columns were used as growth con-trols, where no drugs were added, while other two were used as the positive controls in which gentamycin was used. The microtitre plates were incubated at 37oC for 24 hours.

After the incubation period 20µl of a 0.2% p-iodonitrotetrazolium chloride (INT) were added to the wells followed by incubation at 370C for 0.5 h. Pres-ence of microbial growth was indicated by change of INT colour to pink, while absence of growth was indi-cated by absence of colour change. The lowest concen-tration at which there was no growth was taken as the minimum inhibitory concentration (MIC).

Antifungal testing

Dilution method was used to determine the antifungal activity of the plant extracts against Candida albicans and Cryptococcus neoformans. Sabourauds Dextrose Agar was used for the preparation of fungal cultures at 370C for 24 h (agar preparation was done according to the manufacturer’s instructions).

Testing against Candida albicans and Cryptococcus neoformans followed the same procedure as the anti-bacterial testing described previous with saborauds dextrose broth used instead of tryptone soya broth. Fluconazole was used as a standard positive control.

RESULTS AND DISCUSSION

Phytochemical analysis

The preliminary qualitative analysis of methanol ex-tracts of B. coccinea indicated the presence of tannins, flavonoids, terpernoids and glycoside while saponins could not be detected and can be explained by the fact that, the solvent used for extraction i.e. 98% methanol

was not polar enough extract them (see Table 1).

Antimicrobial activity

The selection of the plant, B. coccinea for this study was based on its use to treat fungal infections; the oral thrush and scalp infection in Kiswahili known as ‘Utan-du’ and Mashilingi’ respectively.Infections caused by Candida sp. and C. neoformans are of public health concerns especially that, they most common opportu-nistic infections among the immuno-compromised in-dividuals includeoropharyngeal candidiasisand crypto-coccal meningitis (Pfaller and Diekema, 2004). Thus, C. neoformans was included in this study. Both root and leaf extracts of Boerhaviacoccineawere found to pos-sess antifungal activity as shown in table 2.

Based on the antifungal assessment suggested by Al-giannis et al. (2001), the obtained results are classified as follows; the leaf extract is described as having mod-erate inhibitory activity against Cryptoccocusneofor-mans with a MIC value of 0.625 mg/mL and weak inhi-bitory activity with MIC value of 2.50 mg/mL against Candida albicans.The root extract also observed to possess weak inhibitory activity against C. neoformans (MIC value of 2.50 mg/mL) and moderate inhibitory activity MIC of 1.25 mg/mL against Candida albicans. Anticandidal activity is in agreement with the previous report where the n-hexane extract of shoot showed antifungal activity against Candida albicans(Maregesi et al. 2008). Traditionally, water macerate of the shoot is gurgled and drunk for oral thrush while pounded fresh leaves are smeared for the scalp infection and the effi-cacy is claimed by users. Our observation may in part be influenced by the method used to prepare the ex-tract and test method employed.

Antifungal activity had been reported from previous studies of related species including; Boerhaviadiffusa against Aspergillusflavus, Aspergillusniger, and C. albi-cans(Baskaran et al., 2011) the dermatophytes; Micro-sporumgypseum, Microsporumfulvum and Microspo-rumcanis by decreasing the sporulation of these fungal species (Agrawal et al., 2003) and Boerhaviaerecta against Aspergillusflavus, and Aspergillusnig-er(Suriyavathana et al., 2012).

Both extracts showed very weak antibacterial activity with the MIC value of 5.00 mg/mL against Bacillus anthracis but Streptococcus pyogens, Vibrio Cholerae and Shigellaflexneri were resistant. Lack of activity of

Table 2: Antibacterial and antifugal activity

Extract/ Reference Drug

Minimum Inhibitory Concetration (MIC)mg/ml

Ba Sp Vc Sf Ca Cn

Rootextract 5.00 ** ** ** 1.25 2.50

Leaves extract 5.00 ** ** ** 2.50 0.625

Fluconazole - - - - * *

Gentamicin 0.00625 0.00157 0.0002 0.0125 - - Abbreviations: Ba = Bacillus anthracis, Sp = Streptococcus pyogens, Vc = VibrioCholerae, Sf = Shigellaflexineri,

Ca = Candida albicans, Cp = Cryptococcus neoformans, ** = MIC is above 5.000 mg/ml, * = MIC is above 5.00 mg/ml

Sheila M Maregesi et al.,(2013) Int. J. Res. Phytochem. Pharmacol., 3(2),94-98

©JK Welfare &Pharmascope Foundation | International Journal of Research in Phytochemistry& Pharmacology 97

the shoot extracts against Staphylococcus aureus, Ba-cillus cereus, Escherichia coli, Pseudomonas aerugino-sa, and Klebsiella pneumonia had been reported for B. coccinea(Maregesi et al. 2008). These results are con-trary with previous studies on related species that ex-hibited antibacterial activity, e.g. Boerhaviaerecta against Aeromonashydrophila, Bacillus subtilis, and Pseudomonas aeruginosa(Suriyavathana et al., 2012) and Boerhaviadiffusa against Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumo-nia). (Baskaran et al., 2011).

The protein called INDIN-SAA was isolated from the root extract of B. diffusa. This compound exhibited antimicrobial, antioxidative and antilymphoprolifera-tive activity against Scrofula adenitis (tuberculosis of the neck, or, a cervical tuberculous lymphadenopathy), caused by Mycobacterium scrofulaceum. (Khan et al., 2011).Also, the B. diffusa extract is used in the treat-ment of different skin conditions such as urticaria, ec-zema and other skin inflammatory conditions. The use of a purified B. diffusa extract that is rich in hydrox-ybenzoic compounds, such as hydroxybenzoic acid, is an active ingredient in the preparation of a cosmetic composition for skin relief and for increasing skin com-fort (WO2009044040 (A1), 2009).

CONCLUSION

Boerhaviacoccinea is a common weed growing in vari-ous parts of Tanzania and resistance to diseases.It can easily be cultivated for production of drugs when effi-cacy and safety are established. Comprehensive scien-tific study is recommended to facilitate its incorpora-tion in the medicinal and nutritional application. The obtained results support to use of the plant in tradi-tional medicine. Regarding the Cryptoccocusneofor-mans infections, the inhibition (MIC value = 625 µg/mL) of the crude leaf extract is interesting and promising for isolation of bioactive compounds or partial purifica-tion to identify the active fraction that could be stan-dardize and formulated when safety is confirmed.

ACKNOWLEDGEMENT

We are very grateful to information providers for their willingness to disclose the medicinal and nutritional uses of the plant and the Department of Microbiology and Immunology, Muhimbili University of Health and Allied Sciences for providing microorganisms used in this study.

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