PHYTOTHERAPY RESEARCH Phytother. Res.

21, 6771 (2007) Published online 9 November 2006 in Wiley InterScience ALCHORNEA CORDIFOLIA AGAINST S. AUREUS (www.interscience.wiley.com) DOI: 10.1002/ptr.2003

67

A Study of the In Vivo Activity of the Leaf Extract of Alchornea cordifolia against Multiply Antibiotic Resistant S. aureus Isolate in Mice
O. A. Igbeneghu1, E. O. Iwalewa2 and A. Lamikanra1*
1 2

Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria

The effect of a 50% aqueous ethanol extract of Alchornea cordifolia (Schum and Thonn) Muell. Arg. leaf was investigated in mice which had been infected intraperitoneally with 5.0 109 cfu of Staphylococcus aureus. Dose-dependent antibacterial activity was demonstrated and the rate of survival of infected mice was improved signicantly by doses between 25 and 200 mg/kg of injected extract when compared with untreated infected controls. The intraperitoneal median lethal dose of the extract was found to be 800 mg/kg. Copyright 2006 John Wiley & Sons, Ltd.
Keywords: Alchornea cordifolia; ethanol extract; antibacterial activity; in vivo; Staphylococcus aureus.

INTRODUCTION The aqueous extract of Alchornea cordifolia (Schum and Thonn) Muell. Arg. leaf has been reported to be used traditionally in the treatment of a broad spectrum of infections, including diarrhoea, dysentery, gonorrhoea, cough and infected wounds (Ayensu, 1978; Iwu, 1983; Food and Agriculture Organization of the United Nations, 1986; Laurent Ake Assi and Sita Guinko, 1991; Van Medendach de Roy, 1994). The leaves have also been reported (Iwu, 1983) to be used internally for the management of gastrointestinal, respiratory and urinary tract infections. Ogunlana and Ramstad (1975) reported activity of the methanol extract of this plant against Staphylococcus aureus, Staphylococcus albus and Escherichia coli while Lamikanra et al. (1990) demonstrated that various fractions of the extract of A. cordifolia had considerable activity against type cultures of Staphylococcus aureus, E. coli, Bacillus subtilis and Pseudomonas aeruginosa and it was proposed that this activity was caused by various constituents including phenolic acids and triisopentenyl guanidine. Okeke et al. (1999) have further shown that the 50% aqueous ethanol extract of the leaves of this plant possess signicant activity against a broad spectrum of clinical isolates including multiply antibiotic resistant aerobic and anaerobic bacteria as well as fungi. Other pharmacological activities of the leaves already investigated are antiamoebic, antidiarrhoeal and spasmolytic effects (Tona et al., 2000) as well as the antiinammatory effects (Osadebe and Okoye, 2003). The reported activity of the extract of A. cordifolia as well as its extensive use in traditional medical practice for the treatment of various infectious diseases
* Correspondence to: A. Lamikanra, Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. E-mail: alamikanra@yahoo.com Copyright 2006 John Wiley & Sons, Ltd. Copyright 2006 John Wiley & Sons, Ltd.

suggests that this plant has the potential to be a very useful antiinfective agent and one which can be used in the treatment of systemic infections including those caused by organisms which have acquired resistance to antibiotics. Antimicrobial investigations of A. cordifolia would be incomplete and conclusions concerning its use in the development of chemotherapeutic agents, especially against antibacterial resistant pathogens, cannot be made without an investigation into the in vivo antibacterial activity of this plant in an animal infection model. Such a study will be in line with the current search for natural substances to augment or replace the antibacterial drugs in current clinical use, which because of the spread of resistant organisms have become less useful than before. In view of the potential usefulness of this plant in antiinfective therapy, there is a need to provide evidence for its in vivo activity, hence the importance of this study.

MATERIALS AND METHODS Plant collection. Alchornea cordifolia (Schum and Thonn) Muell. Arg. leaves were collected in April 2004 from various locations within the Obafemi Awolowo University (OAU) campus, Ile-Ife, Nigeria and were authenticated in the Herbarium Section, Faculty of Pharmacy, OAU in which samples were deposited. Preparation of aqueous extract. The leaves were air-dried at room temperature, ground to powder and 500 g of the powder was exhaustively extracted with aqueous ethanol (50%) in a Soxhlet extractor. The extract obtained was slowly evaporated to dryness using a rotary evaporator at 35 C to yield an almost crystalline powder residue (3.5% w/w). This was stored
Received 21, 6771 (2007) Phytother. Res.13 February 2006 Revised 6 July 2006 DOI: 10.1002/ptr Accepted 12 August 2006

68

O. A. IGBENEGHU ET AL.

in a coloured powder jar and kept in the freezer at 20 C until required. Test organisms. The reference strain was from stock culture collections maintained in our laboratory. Clinical isolates were obtained from the anterior nares of primary school children by inoculating their nasal swabs onto blood agar and mannitol salt agar. Those colonies that haemolysed red blood cells and fermented mannitol were selected and conrmed as S. aureus using the tube coagulase test. The antibiotic susceptibility of the isolates to different antibiotics was determined using the agar disc diffusion method NCCLS (1990). Capsule staining was carried out using the combined positive and negative method of Okeke and Lamikanra (1995). None of the isolates was encapsulated. Organisms were maintained by cryopreservation according to the method of Gibson and Khoury (1986). Animals. Swiss albino mice of both sexes weighing 1824 g and aged 810 weeks were used. They were obtained from the Animal House of the Faculty of Pharmacy, Obafemi Awolowo University and were fed on mouse cubes (protein 21.0% (minimum); fat 3.5% (minimum); bre 6.0% (maximum); calcium 0.8% and phosphorus 0.8%) (M. O. Ladokun and Sons Livestock Feeds Ltd, Ibadan). The animals had unlimited access to water throughout the period of the study. In vitro bioassay. The hole-in-plate bioassay method was used and the extract was tested against all the S. aureus strains isolated as well as a type culture strain, S. aureus NCTC 6571. 0.5 mL volumes of overnight nutrient broth cultures of the organisms were mixed with 15 mL portions of molten nutrient agar in a sterile petri dish, allowed to solidify and incubated at 37 C for 30 min. Different concentrations of the plant extract were then lled into holes of 8 mm diameter cut into the agar with a sterile cork borer following which the plates were refrigerated at 4 C for 30 min before they were incubated at 37 C for 24 h. Zones of inhibition were measured at the end of the incubation period. Acute toxicity bioassay. The acute toxicity test (LD50) of the ethanol extract of A. cordifolia leaves was determined according to the procedure described by Lorke (1983). The method involved an initial dose nding procedure, in which the animals were divided into three groups of three animals per group. Doses of 10, 100 and 1000 mg/kg were administered intraperitoneally (i.p.), one dose for each group. The treated animals were monitored for 24 h for mortality and general behaviour. From the result of the above step, four different doses of 200, 400, 800 and 1600 mg/kg were chosen and administered (i.p.) respectively to four groups of one mouse per group. The treated animals were again monitored for 24 h. The LD50 was then calculated as the geometric mean of the lowest dose showing death and the highest dose showing no death. A total of 13 mice were used for this determination. In vivo antibacterial activity. The method was a modication of the methods of Asahi et al. (1995) and
Copyright 2006 John Wiley & Sons, Ltd.

Yamaguchi et al. (1998). Albino mice were used in groups of ve. The selected test organism was cultured overnight at 37 C on nutrient agar slope. The organism was harvested and suspended in sterile saline and each mouse was challenged intraperitoneally with a single 0.5 mL portion of bacterial suspension containing 10 109 cfu of Staphylococcus aureus per mL. This inoculum was sufcient to cause 100% mortality in untreated mice with death occurring between 6 and 10 h after challenge. 5.0, 10.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 100.0 and 200.0 mg/kg of A. cordifolia leaf extract were administered subcutaneously 15 min after bacterial challenge. The total number of mice surviving at each dose level was recorded on day 7 after infection. The length of time before the death of each infected animal was also noted. Statistical analysis. Statistical evaluation of the results was done using Students t-test to determine the signicance of difference in the mean values.

RESULTS In vitro activity The 50% aqueous ethanol extract of the leaves of A. cordifolia showed antibacterial activity against all the S. aureus isolates. The minimum inhibitory concentrations of the extract, obtained by interpolation of the graph of the diameter of zones of inhibition against the log concentration was found to be 6.3 mg/L for isolate A, 12.6 mg/mL for B, 8.1 mg/mL for C, 5.1 mg/mL for D, 7.6 mg/mL for E and 3.5 mg/mL for F. Against the reference organism, S. aureus NCTC 6571, the minimum inhibitory concentration obtained was 6.0 mg/mL (Table 1). The antibiotic susceptibility pattern of each of the isolates is shown in Table 2. The isolates possessed multiple resistance to the antibiotics tested. Acute toxicity study The result of the acute toxicity studies is shown in Table 3. The LD50 of the aqueous ethanol extract was calculated to be 800 mg/kg. Course of S. aureus infection Based on the fact that S. aureus isolate F had the lowest infective dose (colony forming units) that could cause 100% mortality in untreated mice and also had the lowest MIC from the in vitro test above, it was chosen as the test strain. After intraperitoneal injections of the mice with S. aureus isolate F, the mice were active for the rst 23 h after which they slept huddled together. Within 34 h of infection, the animals became very sick and their fur was observed to have become rufed. This was followed by a period during which they experienced increased difculty in breathing as shown by their gasping for breath. Death occurred, following a brief convulsion 610 h after infection.
Phytother. Res. 21, 6771 (2007) DOI: 10.1002/ptr

ALCHORNEA CORDIFOLIA AGAINST S. AUREUS

69

Table 1. In vitro antibacterial activity of the ethanol extract of A. cordifolia leaves on S. aureus isolates
A. cordifolia extract Conc. (mg/mL)
100 50 25 12.5 MIC (mg/mL) Zone of inhibition (mm) S. aureus isolate Log con 2.0 1.7 1.4 1.1 A 16 14 12 10 6.3 B 20 15 12 10 12.6 C 20 17 13 10 8.1 D 21 17 15 13 5.1 E 18 16 12 10 7.6 F 18 16 14 12 3.5 NCTC 6571 22 18 15 12 6.0

Table 2. Antibiotic susceptibility pattern of isolated S. aureus strains

Zone of inhibition (mm) S. aureus isolate Antibiotic (units) Ampicillin (10 g) Penicillin V (10 g) Gentamicin (30 g) Streptomycin (30 g) Chloramphenicol (30 g) A 8R 8R 18R 16R 25R B 8R 8R 14R 16R 24S C 27S 19I 15R 24S 24S D 32S 8R 15R 24S 25S E 8R 8R 20S 15R 24S F 8R 8R 25S 17R 25S

I, Intermediate; R, Resistant; S, Sensitive.

Table 3. Acute toxicity of the ethanol extract of A. cordifolia leaf

Death pattern at 24 h after injection

Table 4. Effect of A. cordifolia leaf extract on the survival of mice infected with S. aureus F
Log dose 0.70 1.00 1.18 1.40 1.48 1.54 1.6 1.65 1.7 2.0 2.3 NA NA Average survival time (h) 7.00 8.00 10.00 16.60 30.30 44.40 46.20 45.80 45.20 43.00 41.80 168.00 0.4 0.5 0.6 0.9 2.6 3.5 3.5 3.5 3.5 3.5 3.6 0.0 Survival rate (%) 0 0 0 0 0 20 20 20 20 20 20 100 0

Dose (mg/kg) Stage I 10 100 1000 Stage II 200 400 800 1600 LD50 = (400 1600)1/2 = 800 mg/kg.

Dose (mg/kg) 5 10 15 25 30 35 40 45 50 100 200 Chloramphenicol (50 mg/kg) Negative control NA, not applicable.

0/3 0/3 2/3 0/1 0/1 1/1 1/1

In vivo activity Table 4 shows the effect of the A. cordifolia leaf extract on the survival of bacteraemic mice compared with negative control animals. The extract increased the period of survival of the infected mice from a minimum average of 8 h in negative control animals to an average of 46 h at a dose of 40 mg/kg body weight. The dose-survival time curve (dose-response curve) (Fig. 1) shows a dose-dependent bioactivity against S. aureus infection in mice. Furthermore the extract increased the survival rates of infected mice to 20% compared with 0% in the negative control animals. All the mice that received A. cordifolia leaf extract and that recovered from the infection showed signs of respiratory distress and decreased activity with recovery of normal breathing and activity occurring after about 24 h. The positive control animals that were given chloramphenicol subcutaneously, showed similar behaviour but with complete recovery observed within 8 h of injection.
Copyright 2006 John Wiley & Sons, Ltd.

8.00 0.6

DISCUSSION The extensive use of Alchornea cordifolia leaf decoctions in traditional medicine for the treatment of various infectious diseases and the reported in vitro activity of the extract against a broad spectrum of microorganisms suggest that A. cordifolia leaf extract has a great potential as an antiinfective agent. However, all the antimicrobial bioassays that have been carried out on this plant hitherto by Lamikanra et al. (1990) and Okeke et al. (1999) have been in vitro studies using the hole-in-plate agar diffusion and the agar dilution methods. These in vitro studies show that A. cordifolia is indeed active as an antibacterial agent but these studies have not given any insight into the ability of
Phytother. Res. 21, 6771 (2007) DOI: 10.1002/ptr

70

O. A. IGBENEGHU ET AL.

Figure 1. Log dose response curve of bacteraemic mice (1824 g) treated with A. cordifolia leaf extract.

the plant to exert its antibacterial effect in vivo. This omission is important because it is not always certain that the degree of in vitro activity shown by a plant extract is matched by its activity in vivo. In the rst instance, the antibacterial components of plant extracts may be metabolized in vivo to yield inactive metabolites, which result in the loss of antibacterial activity in vivo. In addition, in vivo tests establish the toxicity or safety of agents that have shown activity in vitro. Since A. cordifolia is often taken orally in the form of a decoction as reported by Laurent Ake Assi and Sita Guinko (1991), in vivo tests are of particular importance because such tests simulate the actual condition of use of the plant materials. In vivo studies to determine toxicity are also of importance when considering the future formulation of A. cordifolia leaf extract or any other plant of medicinal value into dosage forms for human or veterinary use. From the study of acute toxicity, the intraperitoneal LD50 of the ethanol extract of A. cordifolia obtained falls within the practically non-toxic range according to the classication by Loomis (1978). This result is in agreement with those of Osadebe and Okoye (2003) who reported an intraperitoneal LD50 of 1131.4 mg/kg, still falling within the practically non-toxic range. Based on the result obtained in this present determination, the dose for treatment of infected mice was chosen up to a maximum of 200 mg/kg. This method of acute toxicity determination was used because it utilizes fewer mice (usually 13) and provides a 24 h LD50 value. The results of the antibacterial study show that the aqueous extract of the leaves of A. cordifolia possesses a demonstrable level of in vivo antibacterial effects in S. aureus infection in mice at doses which are much smaller than the dose at which 50% of the test animals are killed by the extract of the leaves. The leaf extract produced a noticeable prolongation of the lives of multiply antibiotic-resistant S. aureus-infected mice beyond those of the infected untreated mice in a dosedependent manner (p < 0.05). Statistically signicant
Copyright 2006 John Wiley & Sons, Ltd.

prolongation of survival time was shown at a dose as low as 25 mg/kg of the animals up to a dose of 200 mg/ kg when compared with the infected untreated animals. Statistical evaluations between doses in the A. cordifolia treated animals showed dose-dependent prolongation of life of infected animals up to a dose of 35 mg/kg beyond which increase in dose produced no signicant change in response. The investigation into the antiinammatory activity of A. cordifolia leaf extract carried out by Osadebe and Okoye (2003) showed that the leaf extract showed signicant (p < 0.05) antiinammatory activity at a dose of 50 mg/kg in rats. This may contribute signicantly to the observed in vivo antibacterial activity of this plant. On comparing the activity of A. cordifolia in vivo with that of the positive control drug, chloramphenicol, it was observed that at a dose of 50 mg/kg body weight, chloramphenicol produced a 100% survival rate against 20% produced by A. cordifolia. It is worth noting however, that the fact that the extract has shown activity in vitro and in vivo against a S. aureus strain that was found to be resistant to penicillin V, ampicillin and streptomycin in vitro indicates the potential of A. cordifolia leaf extract as an antiinfective agent. From this point of view further studies on A. cordifolia leaf extract (total extract and pure isolated active constituent) may lead to the discovery of alternative antibacterial agents against antibiotic resistant pathogens. In the light of the rising incidence of antibiotic resistance in this environment as noted by Okeke et al. (2000), there is a need to carry out more investigation on this and other plants with a demonstrated potential for use as antiinfective agents.

Acknowledgement
The authors thank the International Program in the Chemical Sciences (IPICS) of the Uppsala University, Uppsala, Sweden for material support.
Phytother. Res. 21, 6771 (2007) DOI: 10.1002/ptr

ALCHORNEA CORDIFOLIA AGAINST S. AUREUS

71

REFERENCES
Asahi Y, Miyazaki S, Yamaguchi K. 1995. In vitro and in vivo antibacterial activities of Bo-2727, a new carbapenem. Antimicrob Agents Chemother 39: 10301037. Ayensu ES. 1978. Medicinal Plants of West Africa. Reference Publications Inc.: Algonac, MI, 117121. Food and Agriculture Organization of the United Nations. 1986. Some Medicinal Plants of Africa and Latin America. FAO Technical Paper 67. FAO: Rome, 1567. Gibson L, Khoury J. 1986. Storage and survival of bacteria by ultrafreeze. Lett Appl Microbiol 3: 127129. Iwu MM. 1983. Traditional Igbo Medicine. Institute of African Studies, University of Nigeria: Nsukka, 75. Lamikanra A, Ogundaini AO, Ogungbamila FO. 1990. Antibacterial constituents of Alchornea cordifolia leaves. Phytother Res 4: 198200. Laurent Ake Assi, Sita Guinko. 1991. Plants Used in Traditional Medicine in West Africa. Hoffmann-La Roche: Basel, 78 79. Loomis JA. 1978. Essential of Toxicology, 3rd edn. Lea and Febiger: Philadelphia, 241. Lorke D. 1983. A new approach to acute toxicity testing. Arch Toxicol 54: 275 287. NCCLS. 1990. Performance Standards for Antimicrobial Disk Susceptibility Tests, 4th edn. Approved standard, M2-A5. National Committee for Clinical Laboratory Standards: Villanova, PA. Ogunlana EO, Ramstad E. 1975. Investigations into the antibacterial activities of local plants. Planta Med 27: 354 360. Okeke IN, Fayinka ST, Lamikanra A. 2000. Antibiotic resistance in Escherichia coli from Nigerian students, 1986 1998. Emerg Infect Dis 6: 393 396. Okeke IN, Lamikanra A. 1995. Bacterial capsule: a simple method for demonstration under the light microscope. Br J Biomed Sci 52: 321 322. Okeke IN, Ogundaini AO, Ogungbamila FO, Lamikanra A. 1999. Antimicrobial spectrum of Alchornea cordifolia leaf extract. Phytother Res 13: 7 9. Osadebe PO, Okoye FBC. 2003. Anti-inammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves. J Ethnopharmacol 89: 19 24. Tona L, Kambu K, Ngimbi N et al. 2000. Anti-amoebic and spasmolytic activities of extracts from some anti-diarrhoeal traditional preparations used in Kinshasa, Congo. Phytomedicine 7: 3138. Van Medendach de Roy JM. 1994. Antidiarrhoeal plants of the sundi of Kinamba (Congo). In The Biodiversity of African Plants, Bitsindu M, Lejoy J, Bargt XM, Vander G (eds). Proceedings of the 14th AETFAI Congress, 2227 August 1994, Wageninggen, Netherlands, 722727. Yamaguchi K, Domon H, Miyazaki S et al. 1998. In vitro and in vivo antibacterial activities of Cs-834, a new oral carbapenem. Antimicrob Agents Chemother 42: 555 563.

Copyright 2006 John Wiley & Sons, Ltd.

Phytother. Res. 21, 6771 (2007) DOI: 10.1002/ptr