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1 Introduction Cola nitida and C. acuminata are plants belonging to the family Sterculiaceae, and indigenous to tropical Africa. (1) They have major economic, social and cultural importance in West Africa, especially, Nigeria where about 70% of kola nuts are produced. (2) The nuts of Cola nitida and C. acuminata contain between two to three percen t caffeine and are chewed by many people as a stimulant. (3) Long distance lorry drivers, university students as well as others chew the nuts in place of drinking coffee, to keep awake and alert. The two types of kola nuts are distinguished on the basis of their cotyledons; while C. nitida has two cotyledons, C. acuminata has three or more cotyledons. Kola nuts are very special and important items used in social, cultural and ceremonial activities by Africans. For example, among the Igbos of Nigeria, kola nut is ascribed ritual powers of peace, long life, prosperity and unity. (4) It is also used as a medium for communicating between people and gods during sacrifices and spirit worship. No welcome or marriage ceremony is complete without the presentation o f kola nuts to the visitors and celebrants. (4) Kola nuts are also used in industry for the production of drugs, soft drinks, wines, candies, and beverages. (2) Extracts from red kernels are used as food colorant. The plant is used medicinally for the treatment of diarrhea, dysentery, vomiting, hemorrhoids, headache, and stomach ulcers. (2) Another type of nut, Garcinia kola (bitter kola) from a flowering plant found mostly in the tropical rain forest regions of Central and West Africa is also widely consumed in West Africa. Garcinia kola differs from C. nitida and C. acuminata in that it has only one cotyledon, almost always whitish and very bitter. The bark of G. kola can be taken orally for fever, cough, in flammation, respiratory tract infection and as an anti -helminthic. (5) The dried root soaked in alcohol is taken orally for the treatment of cough, inflammation, liver cirrhosis, tooth decay and gonorrhea. (5) The seed enjoys a folk reputation in Africa as a poison antidote; additionally, the plant possesses anti -hepatotoxic properties. (6, 7) Garcinia kola has been used as a purgative, anti -parasitic, and antimicrobial agent for the treatment of throat infections, diarrhea and bronchitis. [7] Aqueous extracts of Garcinia kola have also been reported as equally effective as timolol in the treatment of glaucoma. (8) Cola acuminata Cola nitida Garcinia kola 2 The studies highlighted above indicate that Cola nitida, Cola acuminata and Garcinia kola have great potential for the treatment of various diseases including the treatment of bacterial infections. For example, the Centers for Disease Control and Prevention (CDC) states that e ach year in the United States, at least 2.8 million people are infec ted with antibiotic -resistant bacteria. (9) In the United States alone, antibiotic -resistant pathogen -associated hospital acquired infections cause an estimated 99,000 deaths annually. (10) In 2006, about 50,000 people died of pneumonia and sepsis alone, with medical cost of about 8 billion dollars. (10) Treatment costs per patient for antibiotic resistant bacteria are estimated at about $29,000. (10) In total, economic losses due to antibiotic resistant bacteria are estimated at about $20 billion for tre atment, while losses due to lost productivity are estimated at $35 billion annually. (10) The situation is so critical that it attracted the attention of President Barack Obama who ordered the National Security Council to draft a comprehensive national act ion plan to tackle antibiotic drug resistance. (10) In order to tackle the problem of containing bacterial resistance to antibiotics, one of the strategies is to discover new sources of antibiotics including plants such as Garcinia kola, Cola nitida and Cola acuminata . Some reports indicate that 75% of test bacteria are susceptible to ethanolic extract of Garcinia kola; 37.5% of test bacteria are susceptibility to aqueous extract of the plant, while methanolic extract of Garcinia kola inhibited the growth of 62.5% of the tested organisms. (11) A study on the antibacterial activity of Garcinia kola showed that methanolic extract showed antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae but no activity against Escherichia coli and Salmonella typhi . (12) The aqueous extract of Garcinia kola showed no antibacterial activity against E. coli but showed activity against Staphylococcus aureus , Klebsiella pneumoniae and Salmonella typhi . (12) The same study showed that methanolic extract o f Cola nitida showed antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Salmonella typhi . (12) Aqueous extract of Cola nitida also exhibited antibacterial activity against Escherichia coli , Staphylococcus aure us , Salmonella typhi and Klebsiella pneumoniae. (12) Another study has also shown that methanolic extract of Cola nitida exhibited antibacterial activity on Bacillus cereus, Serratia marcescens, Staphylococcus epidermidis and Proteus vulgaris .(13) A diff erent study on the antibacterial activity of methanolic, ethanolic and aqueous extracts of G. kola and C. nitida showed that all extracts of G. kola had antibacterial activity against E. coli, Salmonella, Pseudomonas spp and Shigella spp.(14) C. nitida significantly inhibited Salmonella species, Shigella species and Pseudomonas species, but not E. coli .(14) Aqueous and methanolic extracts of Cola acuminata have also been shown to have a wide range of anti bacterial activity against Gram -positive and Gram -negative bacteria, and Candida albicans . (15) The studies cited above indicate that all three types of kola nuts, Garcinia kola , Cola. nitida and Cola acuminata have antibacterial properties. This study was therefore 3 carried out to corroborate the results of other studies and to seek ways to utilize these nuts for alleviating or preventing bacterial infections. Methods and materials Samples of Cola nitida, Cola a cuminata and Garcinia kola were purchased from the African market in Bear, Delaware. The samples were first sliced into smaller pieces before drying at 45 oC. Dried samples were then ground using a Waring blender, and stored at 4 oC until used. Two and half (2.5) grams of each of the ground samples of Cola nitida, Cola acuminata and Garcinia kola were placed into sterile plastic tubes. Sterile distilled water, 95% ethanol and 95% methanol was added to each respective tube to achieve a volume of 10ml and dilu tion of ¼ (250mg/ml). Extraction was achieved by placing the tubes in a shaking incubator set at 25 oC and 150 rpm for 48 hours. Extracts were obtained by centrifuging the mixture at 25 oC and 3,000 rpm for 20 minutes. The supernatant obtained after centrifu gation was sterilized by passing it through a 0.22 membrane filter. All bacterial and fungal cultures used in the study were purchased from Ward ’s Natural Science, Rochester, New York. Overnight cultures of Staphylococcus aureus (85W 1941) , Staphylococcus epidermidis (85W 1940) , Enterococcus faecalis (85W 1943) , Bacillus cereus (85W 1815), Enterobacter aerogenes (85W 1805), Escherichia coli (85W 1860) , Salmonella enteritidis (85W 1910), Pseudomonas aeruginosa (85W 1903) and Candida albicans (85W 4150) were each suspended in tubes of sterile saline solution to a turbidity equivalent to 0.5 McFarland. Sterile cotton swabs were dipped in each suspension, and used to thoroughly smear the surfaces of Mueller -Hinton agar until dry. The antibacteri al effect of aqueous, ethanolic and methanolic extracts of Garcinia kola, Cola nitida and Cola acuminata was determined on each smeared bacterial sample using the agar well diffusion technique. For each bacterial smear on Muller -Hinton agar, four holes wer e punched using 6mm cork -borer. The agar within the holes was carefully and aseptically removed, and 50 l of the appropriate solvent (distilled water; 95% ethanol; 95% methanol) was pipetted into the center well. 50 l each of Garcinia kola, Cola nitida and Cola acuminata extracts of same solvent were pipetted into the remaining three wells. The plates were allowed to sit at room temperature for one hour before incubation at 37 oC for 24 hours. Zones of inhibition were measured with a caliper and metric ruler . Minimum bactericidal concentration (MBC) on extracts showing antimicrobial activity was determined by double dilution [1/4 (12.5mg) to 1/2048 (0.0245mg)] of the extracts using same solv ent of extraction and the agar w ell diffusion technique as indicated above. Results Results from this study show that aqueous extracts of Garcinia kola and Cola nitida had no antimicrobial effect on all the test organisms; however, the aqueous extract of Cola acuminata exhibited antimicrobial effect against all the Gram positive bacteria; Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus and Enterococcus faecalis. Ethanolic extracts of Cola acuminata also showed antimicrobial activity against 4 all the test organisms including Candida albicans ; however, the methanolic extract showed antimicrobial activity against all the test organisms except Enterobacter aerogenes and Candida albicans. (Table 1) The methanolic extracts of Cola nitida showed antimicrobial activity on Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Enterococcus faecalis and Pseudomonas aeruginosa; however, the ethanolic extracts inhibited all test organisms except Candida albicans and Enterobacter aerogenes . (Table 1) Methanolic extracts of Garcinia kola inhibited the growth of Enterococcus faecalis, Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis, while the ethanolic extracts of Garcinia kola inhibited the growth of Pseudomonas aeruginosa , Staphylococcus au reus , Staphylococcus epidermidis , Enterococcus faecalis and Bacillus cereus. (Table 1) Table1: Zones of inhibition (mm) of kola nut extracts on test microorganisms Methanolic extracts of Garcinia kola inhibited the growth of Enterococcus faecalis, Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis, while the ethanolic extracts of Garcinia kola inhibited the growth of Pseudomonas aeruginosa , Staphylococcus aureus , Staphylococcus epidermidis , Enterococcus faecalis and Bacillus cereu s. Test microorganism Kola nuts Water (mm) Methanol (mm) Ethanol (mm) St. aureus C. acuminata 27 21 34 C. nitida 0 19 30 G. kola 0 22 33 St. epidermidis C. acuminata 24 24 31 C. nitida 0 17.5 28 G. kola 0 21 36 B. cereus C. acuminata 12.5 18.5 30 C. nitida 0 16.5 30 G. kola 0 17 30 E. faecalis C. acuminata 27 17.5 26 C. nitida 0 14 30 G. kola 0 18 28 E. coli C. acuminata 0 10 10 C. nitida 0 0 12 G. kola 0 0 0 S. enteritidis C. acuminata 0 15 13 C. nitida 0 0 9 G. kola 0 0 0 E. aerogenes C. acuminata 0 0 10 C. nitida 0 0 0 G. kola 0 0 0 Ps. aeruginosa C. acuminata 0 16 22 C. nitida 0 9 15 G. kola 0 0 10 C. albicans C. acuminata 0 0 10 C. nitida 0 0 0 G. kola 0 0 0 5 Table 2 and Figure 1 show the minimum bactericidal concentration (MBC) of aqueous, methanolic and ethanolic extracts of Cola acuminata, Cola nitida and Garcinia kola on Staphylococcus aureus. Our results show that the MBC of aqueous extract of Cola acuminata was 3.125mg. The MBC of the methanolic extracts of Cola acuminata and Cola nitida was identical at 0.1956mg; the ethanolic extracts of both kola nuts was also identical at 0.3913mg. The MBC of methanolic and ethanolic extracts of Garcinia kola wa s however lower, at 0.0978mg for the methanolic extract, and 0.0245mg for the ethanolic extract. Table 2 : MBC of aqueous, methanolic and ethanolic extracts on Staphylococcus aureus Figure1: MBC of aqueous, methanolic and ethanolic extracts on Staphylococcus aureus Table 3 : MBC of aqueous, methanolic and ethanolic extracts on Staphylococcus epidermidis Figure 2 : MBC of aqueous, methanolic and ethanolic extracts on Staphylococcus epidermidis 05101520253035 C. ACUMINATA C. NITIDA G. KOLA C. ACUMINATA C. NITIDA G. KOLA C. ACUMINATA C. NITIDA G. KOLA WATER (MM) METHANOL (MM) ETHANOL (MM) MBC of extracts on S. aureus 12.50mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.1956mg 0.0978 0.0489 0.0245Ex tra ct Kola type 12.50mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.1956mg 0.0978 0.0489 0.0245 Wa te r (mm) C. acuminata 27 24 22 0 0 0 0 0 0 0 C. nitida 0 0 0 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 0 0 0 Me tha nol (mm) C. acuminata 21 19 17 15 12 10.5 8 0 0 0 C. nitida 19 18 15 12 11 9 8 0 0 0 G. k ola 22 19.5 17 15 13 11 10 9 0 0 Etha nol (mm) C. acuminata 34 32 28 26 23 21 0 0 0 0 C. nitida 30 27 25 22 21 18 0 0 0 0 G. k ola 33 30 28 26 21 18 12 10 9 8 Ex tra ct Kola type 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.1956mg 0.0978 0.0489 0.0245 Wa te r (mm) C. acuminata 24 20 18 0 0 0 0 0 0 0 C. nitida 0 0 0 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 0 0 0 Me tha nol (mm) C. acuminata 24 21 17 12.5 11 10 9 9 0 0 C. nitida 17.5 14 12.5 11 9.5 9 8 0 0 0 G. k ola 21 19 16 15.5 11.5 9.5 0 0 0 0 Etha nol (mm) C. acuminata 31 28 22 20 18 0 0 0 0 0 C. nitida 28 26 23 23 18 16 0 0 0 0 G. k ola 36 34 30 25 18 16 0 0 0 0 6 Table 3 and Figure 2 show the MBC of the aqueous, methanolic and ethanolic extracts of the three kola nuts on Staphylococcus epidermidis . The results show that only the aqueous extract of Cola acuminata showed any antimicrobial activity against Staphylococcus epi dermidis with MBC of 3.125mg. The methanolic extract of Cola acuminata had the lowest MBC of 0.0978mg, followed by the methanolic extracts of Cola nitida (0.1956mg) and G. kola (0.3913mg). The ethanolic extracts of C. nitida and G. kola had identical MBC o f 0.3913mg while that of C. acuminata was higher at 0.7813mg. Table 4 : MBC of aqueous, methanolic and ethanolic extracts on Bacillus cereus Figure 3 : MBC of aqueous, methanolic and ethanolic extracts on Bacillus cereus 0 5 10 15 20 25 30 35 40 C. ACUMINATA C. NITIDA G. KOLA C. ACUMINATA C. NITIDA G. KOLA C. ACUMINATA C. NITIDA G. KOLA WATER (MM) METHANOL (MM) ETHANOL (MM) MBC of extracts on S. epidermidis 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.1956mg 0.0978 0.0489 0.0245Ex tra ct Kola type 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.1956mg 0.0978 0.0489 0.0245 Wa te r (mm) C. acuminata 12.5 11 10 9 0 0 0 0 0 0 C. nitida 0 0 0 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 0 0 0 Me tha nol (mm) C. acuminata 18.5 18 15 13.5 12 11 8 0 0 0 C. nitida 16.5 15 14.5 12.5 11 10.5 9 8 0 0 G. k ola 17 16 14 13.5 13 12 12 11 9 8 Etha nol (mm) C. acuminata 30 26 25 24 21 18 0 0 0 0 C. nitida 30 26 24 21 19 16 9 0 0 0 G. k ola 30 29 26 24 19 16 13 11 9 8 7 Table 4 and Figure 3 show the MBC of the aqueous, methanolic and ethanolic extracts of C. acuminata, C nitida and G. kola on Bacillus cereus . Only the aqueous extract of C. acuminata showed antibacterial activity on B. cereus with MBC of 1.565mg. The methanolic a nd ethanolic extracts of G. kola were most effective against B. cereus with MBC of 0.0245mg. The methanolic extract of C. nitida was the next effective with MBC of 0.0978mg, followed by the methanolic extract of C. acuminata and ethanolic extract of C. nitida (0.1965mg), and the ethanolic extract of C. acuminata with MBC of 0.03913mg. Table 5 : MBC of aqueous, methanolic and ethanolic extracts on Enterococcus faecalis Figure 4 : MBC of aqueous, methanolic and ethanolic extracts on Enterococcus faecalis Ex tra ct Kola type 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.19561mg W a te r (mm) C. acuminata 27 24 22 0 0 0 0 C. nitida 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 Me tha nol (mm) C. acuminata 17.5 15.5 14.5 12.5 10.5 9.5 0 C. nitida 14 13 12 10 0 0 0 G. k ola 18 16.5 13.5 11.5 10.5 9.5 0 Etha nol (mm) C. acuminata 26 24 21 19 16 10 0 C. nitida 30 26 24 22 21 0 0 G. k ola 28 25 22 20 20 0 0 8 Table 5 and Figure 4 show the MBC of the aqueous, methanolic and ethanolic extracts of C. acuminata, C. nitida and G. kola on E. faecalis . Only the aqueous extract of C. acuminata exhibited antibacterial activity on Enterococcus faecalis with MBC of 3.125mg. The MBC of methanolic and ethanolic extracts of C. acuminata and methanolic extract of G. kola was identical at 0.3913mg. This wa s followed by the MBC of ethanolic extracts of C. nitida and G. kola (0.7813mg), and the methanolic extra ct of C. nitida (1.563mg). Table 6 shows the MBC of the methanolic and ethanolic extracts of C. acuminata, C. nitida and G. kola on Pseudomonas aeruginosa . The results show that the aqueous extracts of C. acuminata, C. nitida and G. kola had no antibacterial activity on Pseudomonas aeruginosa . The ethanolic extract of C. nitida had the lowest MBC of 0.7813mg, followed by the ethanolic extract of C. acuminata (1.563mg). The MBC of the methanolic extract of C. acuminata was 3.125mg while tha t of C. nitida was 6.25mg. The methanolic extract of G. kola had no antibacterial activity against Pseudomonas aeruginosa. Table 6 : MBC of aqueous, methanolic and ethanolic extracts on Pseudomonas aeruginosa 0 5 10 15 20 25 30 C. ACUMINATA C. NITIDA G. KOLA C. ACUMINATA C. NITIDA G. KOLA C. ACUMINATA C. NITIDA G. KOLA WATER (MM) METHANOL (MM) ETHANOL (MM) MBC of extracts on E. faecalis 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.19561mgEx tra ct Kola type 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.19561mg W a te r (mm) C. acuminata 0 0 0 0 0 0 0 C. nitida 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 Me tha nol (mm) C. acuminata 16 8 8 0 0 0 0 C. nitida 9 8 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 Etha nol (mm) C. acuminata 22 10 9 9 0 0 0 C. nitida 15 12 10 10 8 0 0 G. k ola 10 10 10 0 0 0 0 9 Table 7 shows the MBC of aqueous, methanoli c and ethanolic extracts of C. acuminata, C. nitida and G. kola on Salmonella enteritidis . Only the methanolic and ethanolic extracts of C. acuminata and the ethanolic extract of C. nitida showed antibacterial activity against Salmonella enteritidis . The MBC of the methanolc extract of C. acuminata was 3.125mg while that of the ethanolic extract was 6.25mg. The MBC of the ethanolic extract of C. nitida on Salmonella enteritidis was 12.5mg. Ta ble 7 : MBC of aqueous, methanolic and ethanolic extracts o n Salmonella enteritidis The ethanolic extract of C. nitida was active against Escherichia coli at a minimum bacterial concentration of 6.25mg; while the methanolic and ethanolic extracts of C. acuminata were active against E. coli at MBC of 12.5mg. The methanolic extract of C. nitida , and the methanolic and ethanolic extracts of G. kola had no antibacterial activity against Escherichia coli . No aqueous extract of the kola nuts was effective against E. coli . The aqueous, methanolic and ethanolic extracts of C. nitida and G. kola had no antibacterial activity against Enterobacter aerogenes . The ethanolic extract of C. acuminata was active against Enterobacter aerogenes at 12.5mg; the methanolic extract had no antibacterial activity against this ba cterium. Candida albicans , the only fungus used in this study was only susceptible to the ethanolic extract of C. acuminata at a concentration of 12.5mg. It was not susceptible to any of the extracts of C. nitida and G. kola . Discussion The results of this study corroborate that of other researchers that Cola acuminata , Cola nitida and Garcinia kola have antimicrobial activity against a variety of microorganisms. However, it would appear that all extracts of C. acuminata have broader antimicrobial activity than both C. nitida and G. kola . For example, only the aqueous extract of C. acuminata was active against all the Gram -positive bacteria used in the study; and only the ethanolic extract of C. acuminata was active agains t Candida albicans . Our study does not support the results of other studies, (11) reporting that 37.5% of test bacteria are susceptibility to aqueous extract of Garcinia kola . It also does not corroborate another study, (12) that the aqueous extract of Garcinia kola showed activity against Staphylococcus aureus , Klebsiella pneumoniae and Salmonella typhi . Ex tra ct Kola type 12.5mg 6.25mg 3.125mg 1.563mg 0.7813mg 0.3913mg 0.19561mg W a te r (mm) C. acuminata 0 0 0 0 0 0 0 C. nitida 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 Me tha nol (mm) C. acuminata 15 10 9 0 0 0 0 C. nitida 0 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 Etha nol (mm) C. acuminata 13 9 0 0 0 0 0 C. nitida 9 0 0 0 0 0 0 G. k ola 0 0 0 0 0 0 0 10 This study also does not corroborate the results from other research, (12) indicating that aqueous extract of Cola nitida exhibited antibacterial activ ity against Escherichia coli , Staphylococcus aureus , Salmonella typhi and Klebsiella pneumoniae. The only aqueous extract from our study showing antimicrobial activity was that of C. acuminata. Our study does not also agree with the conclusion of another s tudy, (14) indicating that methanolic, ethanolic and aqueous extracts of G. kola showed antibacterial activity against E. coli, Salmonella, Pseudomonas spp and Shigella spp. This is surprising, given the fact that most users of Garcinia kola believe that it is the most suitable of the kola nuts for alleviating upper respiratory tract infections. We were surprised at the resistance of enteric bacteria like Escherichia coli and Salmonella enteritidis to methanolic, ethanolic and aqueous extracts of G. kola, C. acuminata and C. nitida. We can speculate that there is a possibility of these enteric bacteria acquiring resistance due to mutation resulting from the wide scale consumption of these kola nuts. We are still in the process of elucidating the mechanism s of action of C. acuminata, C. nitida and G. kola . In spite of this, one can speculate that it is possible that the aqueous extract of C. acuminata probably targets the cell walls of bacteria. This may be responsible for its antibacterial activity against all the Gram -positive bacteria used in the study. The aqueous extract of C. acuminata can therefore be exploited in the production of lozenges for alleviating upper respiratory tract infections. The ethanolic and methanolic extracts can be exploited as antiseptics for swabbing and other medical uses. Acknowledgements This study wa s supported by Lincoln’s Excellent Academic Program in Science – Transformation (LEAPS -T) funded by the National Science Foundation Grant # 92 -1583 . References 1. Dah -Nouvlessounon D, Baba -Moussa F, Adjanohoun A, Sina H, Noumavo PA, Adoukonou -Sagbadja H, Dia rrassouba N, N’tcha C , Anago FS, Baba -Moussa L. Phytochemical screening and biological activities of Garcinia kola (bark, leaves and seeds) collected in Benin. 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