into the Folkloric Antimicrobial and Antiinflammatory Properties of Nauclea latifolia Leaves and Stem Bark Extracts and Fractions

Antimicrobial and Antiinflammatory
Properties of Nauclea latifolia Leaves
and Stem Bark Extracts and Fractions

P.O. Osadebe^*, U. Ajali, F.B.C. Okoye and C. Diara

Department of Pharmaceutical and Medicinal Chemistry,
Faculty of Pharmaceutical Sciences, University of Nigeria,
Nsukka, Enugu State, Nigeria

ABSTRACT

Nauclea latifolia leaves and stem bark are used traditionally as tonic and fever medicine, and in the management of toothache, dental caries, septic mouth and malaria. The antimicrobial and antiinflammatory properties of these plant materials are investigated. The crude methanolic extracts and its various solvent fractions L1-L5 for leaves and S1-S4 for stem bark were screened for antimicrobial activities against selected clinical strains of microorganisms. The crude extracts were also subjected to antiinflammatory screening using egg-albumen-induced rat paw edema as a model of inflammation. At 200 mg/kg, the crude methanolic extracts of Nauclea latifolia leaves and stem bark showed mild antiinflammatory activity with edema inhibitions of 33.3 and 13.3 per cent at 4 h respectively. The crude extracts showed antimicrobial activity against Bacillus subtilis, Eschericia coli and Salmonella parathyphi. All the fractions showed activity against Eschericia coli. Only fractions L4, S2 and S4 have activity against Salmonella parathyphi, fractions L1 and L2 have activity against Bacillus subtilis, while fractions L4, S2 and S3 have activity against Staphylococcus aureus. The antimicrobial activity of S2 against Staphylococcus aureus, Escherichia coli and Salmonella parathyphi is comparable to that of standard drug, Gentamicin. Phytochemical tests revealed the presence of alkaloids, terpens, flavonoids, saponins and tannins in the crude extracts and in the fractions. S2 contains only flavonoids, which were found on UV analysis to lack free hydroxyl groups at positions 7, 31 and 41 of the flavonoid nucleus. In conclusion, the antimicrobial activity of the leaves may be due to the flavonoids and tannins found in L4 while that of the stem bark may be due the flavonoids in S2. The mild antiinflammatory effect could be an added advantage in management of toothache, which is normally associated with microbial inversion and mild inflammation.

Keywords:Antiinflammatory, Antimicrobial, Folkloric, Nauclea latifolia.

Introduction

Infectious disease is the number one cause of death accounting for approximately one-half of all deaths in tropical countries (Iwu, 1999). Several efforts towards combating the menace of infectious disease are yet to achieve the desired goals. The situation even becomes worrisome with the emergence of newer forms of respiratory tract infections, HIV/AIDS, and increase in antibiotic resistance in nosocomial and community acquired infections. Plant derived products have shown great promise in the treatment of intractable infectious diseases including opportunistic AIDS infections. Numerous and diverse classes of plants metabolites have been isolated and their structures characterized in the past century (Kirkpatrick, 2002). Efforts are still directed in current times towards generating novel lead antiinfective agents from plants.

Nauclea latifolia Smith (Rubiaceae) is a shrub or small spreading tree widely distributed in Savanna. It is found in forests and fringe tropical forests. The plant has been used traditionally as a tonic and fever medicine, as a chewing stick, and in the management of toothache, dental caries, septic mouth, malaria, diarrhoea and dysentery (Lamidi et al., 1995). Owing to these rich folkloric uses, several attentions have been directed in recent times towards investigating some pharmacological activities of the plant with the view of validating the claimed ethnomedicinal uses. Some of these studies are: hepatoprotective effects of the leaf extract (Akpanabiatu et al., 2005a, 2005b), effects of the leaf and root extract on purinergic neurotransmission in rat bladder and on cardiovascular systems (Udoh, 1995, 1998) and effect of leaf extracts on blood glucose levels (Gidado et al., 2005). Other studies include anthelminthic effect of the stem bark extract (Onyeliyi et al., 2001), amoebicidal activity (Moundipat et al., 2005), trypanosomidal activity of the root bark extract (Madubuenyi, 1996) antimalarial activity (Iwu, 1993). Recently, Otimenyi and Uguru (2006) investigated the acute toxicity, antiinflammatory and analgesic activities of the methanolic extracts of the stem bark. The key phytoconstituents which have been isolated from the plant are indole-quinolizidine alkaloids, glycolalkaloids, monoterpene indole alkaloids and saponins (Iwu et al., 1999; Shigemiri et al., 2003).

In spite of the numerous attentions directed to this plant in the recent times, there is yet no scientific report on the antimicrobial properties of the leaf and stem bark extracts. In the present study, the antimicrobial activity of the extracts and fractions of Nauclea latifolia leaves and stem bark is reported. Since the plant is used traditionally in the management of septic mouth, toothache and dental caries, disease conditions which are usually associated with microbial inversion and inflammatory response, we also investigated the antiinflammatory effect of the leaf and stem bark extracts.

Materials and Methods

Plant Material

The leaves and stem bark of Nauclea latifolia were collected an identified by Mr Ozioko Alfred of BDCP, Nsukka. The materials were cleaned, air-dried and reduced to course powder.

Chemicals

Analytical grades n-hexane, chloroform, ethylacetate, acetone and methanol (BDH) were used. Other materials used are silica gel (70-230) (May and Baker), Sabouraud’s Dextrose Agar (SDA), Nutrient Agar (Merck). Freshly distilled water was used when required. Phytochemical reagents were freshly prepared.

Test Microorganisms

Clinical strains of Salmonella paratyphi, Bacillus subtilis, Klebsiella pneumonia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus niger obtained from the pharmaceutical microbiology unit of the Department of Pharmaceutics, University of Nigeris were used.

Animals

Wistar rats weighing 100±20 g were purchased from the laboratory animal house of Department of Pharmacology and Toxicology.

Extraction and Fractionatin

The powder leaves and stem bark (100 g each) were extracted for 48 h by cold maceration in methanol. Another 100 g portions were extracted by soxhlet for 4 h in methanol. All the crude extracts were subjected to antimicrobial studies.

A 15–g portion of the soxhlet methanol extract of the leave (LS) was adsorbed onto silica gel and eluted successively with hexane, chloroform, ethylacetae, acetone and methanol to yield hexane (L1), chloroform (L2), ethylacetate (L3), acetone (L4) and methanol (L5) fractions. Similarly 13 g of the soxhlet methanolic extract of the stem bark (SS) was adsorbed onto silica gel and eluted in succession with chloroform, ethyl acetate, acetone and methanol to yield chloroform (S1), ethyl acetate (S2), acetone (S3) and methanol (S4) fractions. All the fractions were subjected to antimicrobial screening

Antimicrobial Screening

The sensitivity of the extracts and fractions were determined by Agar well diffusion method (Lovian, 1991). The minimum inhibitory concentration (MIC) of the active fractions was determined by 2–fold serial dilution according to reported procedure (Esimone et al, 1999).

Antiinflammatory Tests

The antiinflammatory activity of the leave and stem bark extracts of Nauclea latifolia was determined using a variation of egg-albumen induced rat paw edema as a model of inflammation as previously described (Osadebe and Okoye, 2003).

Phytochemical Tests

The phytochemical constituents of the extracts and fractions were determined using standard procedure (Harbourne, 1998). In each case a small quantity of the extracts or fractions is dissolved in appropriate solvent and the required reagent added. Colour changes or precipitate were observed to indicate the presence or absence of alkaloids, tannins, flavonoids, terpens, steroids etc.

UV/Visible Spectral Analysis of the Active Flavonoid Fraction (S2)

UV/VIS Scan of S2 was determined in Ethanol and Methanol. The effect of addition of drops of 2 M NaOH on the spectrum of S2 in Ethanol was investigated. The effects of the addition of Sodium acetate and Boric acid in succession on the spectrum of S2 in Ethanol were also investigated.

Statistical Analysis

Data were analyzed using student’s t-test and expressed as mean±SEM. Differences between means were considered significant at p<0.05.

Results and Discussion

The result of the preliminary antimicrobial screening of the crude extracts of Nauclea latifolia leaves and stem bark is shown in Table 27.2. For both the stem bark and leaves, the soxhlet extracts (LS and SS) showed better antimicrobial activity than the extracts obtained by cold maceration (LC and SC). Soxhlet erxtraction usually gives better yield of plant secondary metabolites than cold maceration. This might account for the observed better antimicrobial activity of LS and SS. It is possible that higher quantities of the antimicrobial constituents are extracted using soxhlet. It is also possible that the antimicrobial constituents are not thermolabile or volatile else the soxhlet extracts would have given very poor activity.

Table 27.1: Crude extracts and their phytochemical constituents