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Evaluation of phytochemical composition and antimicrobial activity of Terminalia glaucescens against some oral pathogens.

Introduction

The World Health Organization has recommended and encouraged the use of chewing sticks ( Almas and Al Lafi 1995). Studies by Danielsen et al., (1989), Aderinokun et al., (1999) and Almas and Al Zeid (2004 have demonstrated chewing sticks are at least as effective as toothpaste in maintaining oral hygiene. Sathananthan et al., (1996) reported that Africans that use chewing sticks have fever carious car·i·ous
adj.
Having caries; decayed.


carious (ker´ēus),
adj pertaining to caries or decay.
 lesions than those that use toothbrushes. In a related development, Enwowu (1997) posited that chewing sticks, in addition to providing mechanical stimulation of the gums, also destroy microbes; these advantages of the chewing sticks over the conventional toothpaste and brushes has been attributed to the strong teeth of Africans (Ugoji et al., 2000).

Akande and Hayashi (1998) reported that in Nigeria, some of the chewing sicks being used are obtained from the following plants: Garcinia manni, Masularia accuminata,Terminalia glaucescens, Anogeissus leiocarpus, Pseudocedrela kotschyi, Xanthoxyllum gilletti and Azadiracta indica. T. glaucescens is perhaps one of the widely used plant for chewing stick application in Nigeria. This may have been responsible for a vast array of studies on its antimicrobial activity against some oral pathogens. Ndukwe et al., (2005) studied the antimicrobial effect of its root on Staphylococcus aureus Staphylococcus au·re·us
n.
A bacterium that causes furunculosis, pyemia, osteomyelitis, suppuration of wounds, and food poisoning.


Staphylococcus aureus Staphylococcus pyogenes
, Pseudomonas aeroginosa, Escherichia coli Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract.  and Bacillus subtilis Noun 1. Bacillus subtilis - a species of bacillus found in soil and decomposing organic matter; some strains produce antibiotics
Bacillus globigii, grass bacillus, hay bacillus
. Rotimi et al., (1988) documented the antibacterial activity of the bark extract of T. glaucescens on Bacteriodes gingivalis and B. melaninogenicus. In an earlier report, we reported a significantly higher antibacterial activity of ethanolic extract of T. glaucescens root against Staphylococcus aureus and Streptococcus pyogenes Streptococcus py·og·e·nes
n.
A bacterium that causes the formation of pus or of fatal septicemias.


Streptococcus pyogenes
A common bacterium that causes strep throat and can also cause tonsillitis.
 (Ogundiya et al., 2006).

The need to process and package indigenous medicinal plants medicinal plants, plants used as natural medicines. This practice has existed since prehistoric times. There are three ways in which plants have been found useful in medicine.  that are of oral importance into toothpaste has been proposed (Ogundiya et al., 2006). Interestingly, barely two years after this proposition, a particular Nigeria- based company released a plant based dentifrice dentifrice /den·ti·frice/ (den´ti-fris) a preparation for cleansing and polishing the teeth; it may contain a therapeutic agent, such as fluoride, to inhibit dental caries.

den·ti·frice
n.
 into Nigerian market. However, this present trend requires that the bioactivity of many of these medicinal plants against common oral pathogens be scientifically established. In this regard, the present study was aimed at providing information on the antimicrobial activity of aqueous and ethanolic extracts of the stem and root of T. glaucescens on oral pathogens such as Candida albicans Candida albicans,
n a pathogenic yeast, which is the causal agent of thrush, vaginal infections, and systemic candidiasis.

Candida albicans 
, Streptococcus mutans Streptococcus mu·tans
n.
A species of Streptococcus associated with the production of dental caries.
 and Staphylococcus saprophyticus Staphylococcus saprophyticus is a coagulase-negative species of Staphylococcus bacteria (which are catalase-positive). S. saprophyticus is often implicated in urinary tract infections. S. .

Materials and methods

Plant collection and pre-extraction preparation

Different plant parts such as leaves, stem, root and fruit of T. glaucescens were collected from Oke-Ogun axis of south Western Nigeria (a woody Savannah vegetation). The plant was identified by a plant Taxonomist at the Forestry Research Institute of Nigeria, Ibadan. Nigeria. The stem and root of the plant was sun-dried for seven days, pounded using pestle pestle /pes·tle/ (pes´'l) an implement for pounding drugs in a mortar.

pes·tle
n.
A club-shaped, hand-held tool for grinding or mashing substances in a mortar.
 and wooden mortar.

Extraction procedure

The ethanol extract preparation was done as previously described by Ogundiya et al., (2006). However, for water extraction, the procedure was basically the same except that soaking was done for 48h and the filterate was evaporated to dryness. The crude extracts were reconstituted into aqueous solution using sterile distilled water Noun 1. distilled water - water that has been purified by distillation
H2O, water - binary compound that occurs at room temperature as a clear colorless odorless tasteless liquid; freezes into ice below 0 degrees centigrade and boils above 100 degrees centigrade;
 to obtain extract concentrations of 0.4g/ml and 0.2g/ml.

Microorganisms

Pure cultures of Candida albicans, Streptococcus mutans and Staphylococcus saprophyticus isolated from patients with dental diseases were obtained from the Medical Microbiology Medical microbiology is a branch of microbiology which deals with the study of microorganisms including bacteria, viruses, fungi and parasites which are of medical importance and are capable of causing diseases in human beings.  department of the University College Hospital (UCH UCH Universidad de Chile
UCH University College Hospital
UCH Ubiquitin C-Terminal Hydrolase
UCH University Community Health
UCH University of California, Hastings College of the Law
UCH Underground Coffee House (Hartford, CT) 
) Ibadan. Nigeria. Bacterial cultures were maintained on Nutrient agar Noun 1. nutrient agar - any culture medium that uses agar as the gelling agent
agar

culture medium, medium - (bacteriology) a nutrient substance (solid or liquid) that is used to cultivate micro-organisms
 slant and the fungus on Potato dextrose dextrose: see glucose.  agar slant, both at 6-8[degrees]C.

Antimicrobial assay

The antimicrobial activity of different concentrations of both ethanol and aqueous extracts was determined by modified agar-well diffusion method of Perez et al., (1990) as described by Popoola et al., (2007). The bacterial plates were incubated at 37[degrees]C (fungal plates at 28[degrees]C) and the zone of inhibition measured in mm after 24h, 48h and 72h of growth. A control experiment was set up by using an equal amount of sterile distilled water in place of different extract concentrations.

Phytochemical phy·to·chem·i·cal
n.
A nonnutritive bioactive plant substance, such as a flavonoid or carotenoid, considered to have a beneficial effect on human health.
 studies

Both qualitative and quantitative analyses of the phytochemicals present were carried out using methods described by Fadeyi et al., (1987) and Harbone (1998).

Statistical Analysis of Data

Data were expressed as mean[+ or -]standard deviation. The data obtained were subjected to ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there
 test to determine whether there was significant difference between extract used and also between the length of incubation.

Results

The results of the antimicrobial assay of the stem extract of T. glaucescens is presented in Tables 1-3. Results obtained showed that both ethanol and aqueous extracts of the tested chewing sticks had inhibitory effect on the growth of clinical isolates Candida albicans, Streptococcus mutans and Staphylococcus saprophyticus. ANOVA test of data on the antimicrobial activity of aqueous and ethanol extracts on C. albicans revealed that the solvent used in extraction procedure had significant effect (P<0.05) on the level of inhibition observed. In addition, the root extracts exhibited a significantly higher inhibition on C. albicans compared to the effect produced by the stem extract. Results of the antimicrobial testing on C. albicans also showed that the inhibition of the organism is time dependent as the degree of inhibition decreased with increased length of incubation.

The inhibitory effect of the extracts on Streptococcus mutans showed no significant difference (P>0.05) between extract concentration, but the effect exhibited by ethanol extract was significantly higher than that produced by aqueous extract. However, the length of incubation produced no significant effect (P>0.05) on the degree of inhibition of S. mutans by the root and stem extracts. ANOVA test of the data obtained from the antimicrobial assay of the different extracts on Staphylococcus saprophyticus revealed a trend similar to that observed on Streptococcus mutans.

Table 4 shows the result of qualitative and quantitative analyses of the phytochemicals present in the investigated plant parts. Alkaloids alkaloids,
n alkaline phytochemicals that contain nitrogen in a heterocyclic ring structure. They can have powerful pharmacological effects and are more often used in traditional medicine than in herbal treatments.
, saponnins and tannins are the major phytochemicals present and the quantities present in the root were significantly (P<0.05) higher than the ones contained in the stem. Steroids were present in the investigated stem and root and phenol phenol (fē`nōl), C6H5OH, a colorless, crystalline solid that melts at about 41°C;, boils at 182°C;, and is soluble in ethanol and ether and somewhat soluble in water.  was present in trace amount. Cyanoglycoside was only present in a small amount in the root.

Discussion

The activity of plant extracts against bacteria have been studied for years, but in a more intensified way during the last three decades. During this period, numerous antimicrobial screening evaluations have been published based on the traditional use of Chinese, African and Asian plant-based drugs (Suffredim et al., 2004). In the present study, the aqueous and ethanol extracts of the root and stem of T. glaucescens inhibited the growth of all clinical isolates used; but their effectiveness varied. The root extracts were more effective than the stem extracts. This may be attributed to a significantly higher (P<0.05) concentrations of the investigated phytochemicals in the root extract (Table 4). Previous reports have indicated that the root of T. glaucescens is often used as chewing sticks (Akande and Hayashi 1998; Ndukwe et al., 2005). Result from the present study is possibly giving insight on the reason for this age long practice.

The influence of solvent for extraction on the inhibitory capacity of the extract on the test organism has been reported by Al-Bayati and Sulaiman (2008). It should be pointed out that, though ethanol extracts exhibited more pronounced inhibition than aqueous extracts, yet the effectiveness of the aqueous extract to inhibit the growth of the clinical isolates in the present study could not be contemned. This perhaps explained the effectiveness of this chewing stick in vivo.

The inhibition of Candida albicans, Streptococcus mutans and Staphylococcus saprophyticus observed in this study has confirmed that the antimicrobial principles in T. glaucescens which inhibited microorganisms like Bacteriodes gingivalis, B. melaninogenicus (Rotimi et al 1988), Staphylococcus aureus, Pseudomonas aeroginosa (Ndukwe et al., 2005), Streptococcus pyogenes (Ogundiya et al., 2006) are equally active against the tested clinical isolates in the present study. Though previous reports did not indicated antimicrobial activity of T. glaucescens against C. albicans at low concentrations (Adekunle and Odukoya 2006; Okunade et al., 2007), results from the present study have shown that at higher concentrations as used in the present study, C albicans is sensitive to T. glaucescens extract.

A recent trend in the management of periodontal periodontal /peri·odon·tal/ (per?e-o-don´t'l)
1. pertaining to the periodontal ligament or periodontium.

2. near or around a tooth.


per·i·o·don·tal
adj.
1.
 infections is to employ local antimicrobial delivery (Killoy 1998). In a related development, Ndukwe et al., (2005) stated that it is possible chewing sticks provide locally available antimicrobial agents in a manner similar to antimicrobial polymers and applications and could provide suitable substitute therapy if they can be shown to be efficacious. Results from the present study have shown that T. glaucescens has great potential in dentifrice production.

References

Adekunle, A.A. and K.A. Odukoya, 2006. Antifungal Activities of Ethanol and Aqueous Crude Extracts of Four Nigerian Chewing Sticks. EthnoBotanical Leaflet http://www.siu/~ebl/leaflets

Aderinokun, G.A., J.O. Lawoyin and C.O. Onyeaso, 1999. Effect of two common Nigerian chewing sticks on gingival gingival (jin´jv  health and oral hygiene. Odontostomatol Trop., 22: 13-18.

Akande, J.A. and Y. Hayashi, 1998. Potency of extract contents from selected tropical chewing sticks against Staphylococcus aureus and Staphylococcus staphylococcus (stăf'ələkŏk`əs), any of the pathogenic bacteria, parasitic to humans, that belong to the genus Staphylococcus. The spherical bacterial cells (cocci) typically occur in irregular clusters [Gr.  auricularis. World Journal of Microbiology and Biotechnology, 14: 235-238.

Al-Bayati, F.A. and K.D. Sulaiman, 2008. In Vitro Antimicrobial activity of Salvadora persica L. Extracts against Some isolated Oral pathogens in Iraq. Turk. J. Biol., pp: 57-62.

Almas, K. and T.R. Al Lafi, 1995. The natural toothbrush. World Health Forum, 16: 206-210.

Almas, K. and Z. Al-Zeid, 2004. The immediate antimicrobial effect of a toothrush and miswak on cariogenic cariogenic (kerēōjen´ik),
adj contributing to the advancement of caries. Often used in the context of describing sugary foods.
 bacteria: a clinical study. J. Contemp. Dent. Pract., 5: 105-114.

Danielsen, B.V. Baelum and F. Manji, 1989. Chewing sticks, toothpaste and plaque removal. Acta Odontol Scand, 47: 121-125.

Enwowu, C.O., 1997. socio-economic factors in the dental caries prevalence and frequency. Nigerian caries research, 8: 155-177.

Fadeyi, M.O., A.O. Adeoye and I.D. Olowokudejo, 1987. Epidermal Epidermal
Referring to the thin outermost layer of the skin, itself made up of several layers, that covers and protects the underlying dermis (skin).

Mentioned in: Antiangiogenic Therapy, Histiocytosis X


epidermal
 and phytochemical:Studies in the genus Bohervia (Nyctaginaceae) in Nigeria. International Journal of Crude Drug Research, 27: 178-184.

Harbone, J.B., 1998. Phytochemical methods: Aguide to modern techniques of plant analysis. 3 Edition. Chapman rd and Hill, London. pp: 279.

Killoy, W., 1998. Chemical treatment of periodontitis periodontitis

Inflammation of soft tissues around the teeth (see tooth). Poor dental hygiene leads to deposition of bacterial plaque on the teeth below the gum line, irritating and eroding nearby tissues.
: local delivery of antimicrobials. Int. Dent. Journal, 48(Suppl 1): 305-315.

Ogundiya, M.O., M.B. Okunade and A.L. Kolapo, 2006. Antimicrobial activities of some Nigerian Chewing sticks. EthnoBotanical Leaflet http://www.siu/~ebl/leaflets.

Okunade, M.B., J.A. Adejumobi, M.O. Ogundiya and A.L. Kolapo, 2007. Chemical, Phytochemical compositions and antimicrobial activities of some local chewing sticks used in South Western Nigeria. Journal Phytopharmacotherapy and Natural products, 1(1): 49-52.

Rotimi, V.O., B.E. Laughon, J.G. Bartlett and H.A. Mosadomi, 1988. Activities of Nigerian chewing sticks extracts against bacteriodes-gingivalis and bacteriodes-melaninogenicus. Antimicrobiological Agents and Chemotherapy, 32(4): 598-600.

Ndukwe, K.C., W. Okeke, A. Lamikanra, S.K. Adesina and O. Aboderin, 2005. Antibacterial activity of aqueous extracts of selected chewing sticks. J. Contemp. Dent. Pract., 6(3): 086-094.

Perez, C., A. Pauli and P. Bazerque, 1990. An antibiotic assay by agar-well diffusion method. Acta Biol. Med. Exp., 15: 113-115.

Popoola, T.O.S., O.D. Yangomodu and A.K. Akintokun, 2007. Antimicrobial Activity of Cassava cassava (kəsä`və) or manioc (măn`ēŏk), name for many species of the genus Manihot of the family Euphorbiaceae (spurge family).  Seed Oil on Skin Pathogenic Microorganisms. Research Journal of Medicinal Plant, 1(2): 60-64.

Sathananthan, K., T. Vos and G. Bango, 1996. Dental caries, fluoride levels and oral hygiene practices of school children in Matebeleland South Zimbabwe. Community Dent. Oral Epidemiol., 24: 21-24.

Suffredini, I.B., H.S. Sarder and A.G. Goncalves, 2004. Screening of antibacterial extracts from plants native to Brazillian amazon Rain Forest and Atlantic Forest. Brazillian J. Med. Biol. Res., 37: 379-384.

Ugoji, E., L.O. Egwari and B. Obisesan, 2000. Antibacterial activities of aqueous extracts of ten African chewing sticks on oral pathogens. Nig. Journal of Internal Medicine, 3(1): 7-11.

Corresponding Author: A.L. Kolapo, Department of Biology, The Polytechnic, Ibadan. Ibadan. Nigeria E. Mail: adelodunkolapo@yahoo.com

(1) M.O.Ogundiya, (1) A.L. Kolapo, (2) M.B.Okunade and (2) J.A.Adejumobi

(1) Department of Biology, The Polytechnic, Ibadan. Ibadan. Nigeria (2) Department of Chemistry, The Polytechnic, Ibadan. Ibadan. Nigeria

(1) M.O. Ogundiya, (1) A.L. Kolapo, (2) M.B. Okunade and (2) J.A. Adejumobi, Evaluation of Phytochemical Composition and Antimicrobial Activity of Terminalia Glaucescens Against Some Oral Pathogens, Adv. in Nat. Appl. Sci., 2(2): 89-93, 2008
Table 1: Inhibition of Candida albicans by aqueous and
ethanol extract of T. glaucescens

                              Aqueous              Extract

                              Extract (g/ml)       Concentration

Plant part   Time of          0.4                  0.2
             incubation (h)

Root         24               28.5 [+ or -] 0.0    28.0 [+ or -] 1.0

             48               17.0 [+ or -] 0.0    16.5 [+ or -] 1.5

             72               14.0 [+ or -] 0.5    11.0 [+ or -] 1.7

Stem         24               32.0 [+ or -] 4.0    21.5 [+ or -] 0.5

             48               30.5 [+ or -] 1.5    17.5 [+ or -] 3.0

             72               20.5 [+ or -] 0.5    12.5 [+ or -] 0.5

                              Ethanol              Extract

                              Extract (g/ml)       Concentration

Plant part   Time of          0.4                  0.2
             incubation (h)

Root         24               40.0 [+ or -] 0.0    38.0 [+ or -]  0.0

             48               37.0 [+ or -] 1.0    35.0 [+ or -] 1.0

             72               35.0 [+ or -] 5.0    32.0 [+ or -] 2.0

Stem         24               32.5 [+ or -] 0.0    30.0 [+ or -] 0.0

             48               25.0 [+ or -] 0.0    19.5 [+ or -] 0.5

             72               21.5 [+ or -] 2.5    19.5 [+ or -] 4.5

Values are mean [+ or -] standard deviation (n=3)

Table 2: Inhibition of Streptococcus m utans by aqueous and ethanol
extract of T. glaucescens

                              Aqueous             Extract

                              Extract (g/ml)      Concentration

Plant part   Time of          0.4                 0.2
             incubation (h)

Root         24               28.5 [+ or -] 0.5   27.0 [+ or -] 0.0

             48               27.0 [+ or -] 2.0   26.0 [+ or -] 2.0

             72               26.5 [+ or -] 2.5   25.0 [+ or -] 1.0

Stem         24               27.5 [+ or -] 2.5   24.0 [+ or -] 0.0

             48               27.5 [+ or -] 3.5   24.5 [+ or -] 1.5

             72               22.5 [+ or -] 2.5   17.0 [+ or -] 3.0

                              Ethanol             Extract

                              Extract (g/ml)      Concentration

Plant part   Time of          0.4                 0.2
             incubation (h)

Root         24               36.0 [+ or -] 4.0   36.9 [+ or -]  4.0

             48               33.5 [+ or -] 0.5   32.5 [+ or -] 0.5

             72               34.5 [+ or -] 5.5   31.5 [+ or -] 6.5

Stem         24               35.5 [+ or -] 0.0   25.0 [+ or -] 0.0

             48               28.5 [+ or -] 4.5   20.5 [+ or -] 3.5

             72               23.0 [+ or -] 0.2   18.5 [+ or -] 0.5

Values are mean [+ or -] standard deviation (n=3)

Table 3: Inhibition of Staphylococcus saprophyticus by aqueous
and ethanol extract of T. glaucescens

                              Aqueous             Extract

                              Extract (g/ml)      Concentration

Plant part   Time of          0.4                 0.2
             incubation (h)

Root         24               29.0 [+ or -] 1.0   28.0 [+ or -] 2.0

             48               28.0 [+ or -] 1.0   26.5 [+ or -] 2.5

             72               18.0 [+ or -] 2.0   15.0 [+ or -] 0.0

Stem         24               30.0 [+ or -] 0.0   26.0 [+ or -] 0.0

             48               19.5 [+ or -] 0.5   20.0 [+ or -] 1.0

             72               15.0 [+ or -] 0.0   12.0 [+ or -] 2.0

                              Ethanol             extract

                              Extract (g/ml)      Concentration

Plant part   Time of          0.4                 0.2
             incubation (h)

Root         24               40.0 [+ or -] 0.0   39.5 [+ or -]  0.5

             48               38.0 [+ or -] 2.0   37.0 [+ or -] 5.0

             72               37.5 [+ or -] 2.5   27.5 [+ or -] 0.5

Stem         24               35.0 [+ or -] 0.0   35.0 [+ or -] 0.0

             48               24.5 [+ or -] 0.5   23.0 [+ or -] 4.0

             72               17.5 [+ or -] 2.5   19.5 [+ or -] 0.5

Values are mean [+ or -] standard deviation (n=3)

Table 4: Results of the quantitative estimation of the
phytochemicals (mg/100g) present in the ethanol extracts
of T. glaucescens

        Alkaloid   Steroid          Phenol

Stem    62.7       9.8              Trace

Root    95.5       8.5              Trace

        Tannin     Cyanoglycoside   Saponin

Stem    33.9       Trace            33.5

Root    39.6       2.8              46.6

Values are mean of triplicate determinations.
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Title Annotation:Original Article
Author:Ogundiya, M.O.; Kolapo, A.L.; Okunade, M.B.; Adejumobi, J.A.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Date:May 1, 2008
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