Antimicrobial and Anti-Inflammatory Activities of Pterygota macrocarpa and Cola gigantea (Sterculiaceae)

Pterygota macrocarpa and Cola gigantea are African medicinal plants used in traditional medicine for the treatment of sores, skin infections, and other inflammatory conditions including pains. This study therefore aims at investigating the antimicrobial properties of ethanol leaf and stem bark extracts of P. macrocarpa and C. gigantea using the agar diffusion and the micro-dilution techniques and also determining the anti-inflammatory properties of the extracts of these plants in carrageenan-induced foot edema in seven-day old chicks. The minimum inhibitory concentration of both ethanol leaf and bark extracts of P. macrocarpa against the test organisms was from 0.125 to 2.55 mg/mL and that of C. gigantea extracts was 0.125 to 2.75 mg/mL. Extracts with concentration of 50 mg/mL were most active against the test organisms according to the agar diffusion method. All the extracts of P. macrocarpa and C. gigantea at 30, 100, and 300 mg/kg body weight except ethanol leaf extract of C. gigantea exhibited significant anti-inflammatory effects (P ≤ 0.001).


Introduction
The search for newer antimicrobial agents from various sources has become imperative because of the emergence of resistance strains of microorganisms against orthodox antibiotics especially difficulty to treat infections from resistant strains of bacteria [1] and also the fact that the number of scientists who are developing new antibacterial agents has dwindled, even as bacteria evolve ever more clever mechanisms of resistance to antibiotics [2]. The recent search for new antibiotics includes various sources such as the synthetic compounds, bioactive agents from aquatic microorganisms, and natural products including medicinal plants. In Africa and other developing countries, it is estimated that 70 to 80% of people rely on traditional healers and herbal practitioners for their health needs [3,4] and medicinal plants are the main source of remedies used in this therapy. Some of these medicinal plants are used for the management of several different disease conditions such as bacterial infections, parasitic infections, skin diseases, hypertension, pains, and inflammation such as rheumatoid arthritis [5][6][7][8].
Several medicinal plants including their isolated compounds have been found to exhibit biological activities related to their traditional uses, for example, geraniin and furosin isolated from Phyllanthus mellerianus (Kuntze) Exell. have been found to possess wound healing properties ascribed to this plant as wound healing agent [9]. Cryptolepine, an alkaloid from Cryptolepine sanguinolenta, has been shown to possess antimicrobial and antiplasmodial activities which have gone to confirm its medicinal uses as antiinfective and antimalarial agent [10,11].
Pterygota macrocarpa K. Schum. belongs to the family Sterculiaceae and is known in local Asante-Twi language as kyereye in Ghana. It is a large tree that grows in dense 2 Evidence-Based Complementary and Alternative Medicine semideciduous forests usually distributed in West Africa from Sierra Leone to Cameroun. The soaked leaves are used to treat stomachache, pains, and disorders of digestion. Leaf decoctions are used for the treatment of gonorrhea and other urinary tract infections [12][13][14]. Traditionally, the bark is used in the management of haemorrhoids, dropsy, swellings, edema, gout, leprosy, and pain [15]. The seeds of P. macrocarpa have been found to contain phytate, oxalate and tannins [16].
Cola gigantea A. Chev. belongs to the family Sterculiaceae and is commonly known as giant cola and local Asante-Twi name is watapuo in Ghana. It is a large tree in dry semideciduous forests in West Africa and the West Indies. The nuts (mostly called kola) are often used to treat whooping cough, asthma, malaria, and fever. Other traditional uses include increasing the capacity for physical exertion and for enduring fatigue without food, stimulating a weak heart, and treating nervous debility, weakness, lack of emotion, nervous diarrhea, depression, despondency, brooding, anxiety, and sea sickness [12,14,15,17]. Kola nut is the name of the mature fruits of the Cola species [18] and has a bitter flavour and high caffeine content [19,20], and when the fruit is ingested, it acts as stimulants and thus creates an ecstatic and euphoric state [20]. The caffeine present acts as a bronchodilator, expanding the bronchial air passages [21]. These fruits are also chewed in communities during traditional ceremonies and also are known to reduce hunger pangs. The ethanol leaf extract of C. gigantea has been shown to be active against Candida albicans and phytochemical screening of the leaf extract indicated the presence of alkaloids, saponins, tannins, anthraquinones, and cardenolides [22]. The aim of this study is to investigate the antimicrobial and anti-inflammatory activities of ethanol stem bark and leaf extracts of P. macrocarpa and C. gigantea.

Preparation of Extracts.
The plant materials were air dried and powdered, and 200 g each of the dried powdered material of P. macrocarpa and C. gigantea was extracted, respectively, with 70% ethanol (1.5 L) using Soxhlet apparatus. The ethanol extracts obtained were evaporated to dryness under reduced pressure and kept in a dessicator. The yields of the stem and leaf extracts of P. macrocarpa were 4.2 and 12.4% w/w, respectively. And the yields of C. gigantea were 3.6 and 16.5% w/w for stem bark and leaf extracts, respectively. Various quantities of the ethanol leaf extract (CGLE) and ethanol stem bark extract (CGBE) of C. gigantea and ethanol leaf extract (PMLE) and ethanol stem bark extract (PMBE) of P. macrocarpa were dissolved in normal saline and methanol for acute anti-inflammatory and antimicrobial determinations, respectively.

Preliminary Phytochemical Screening.
Phytochemical screening was conducted on both leaf and stem bark of P. macrocarpa and C. gigantea to ascertain the presence of carbohydrates, tannins, sapogenetic glycosides, flavonoids, steroids, and alkaloids [23,24]. The tannins content was determined according to the method of Glasl [25] using pyrogallol (Merck, Darmstadt, Germany, purity 99.5%, HPLC) as reference compound.

Determination of Antimicrobial Activity
The antimicrobial activities of the extracts (PMLE, CGLE, PMBE, and CGBE) and reference drugs (chloramphenicol and clotrimazole (Sigma, Deisenhofen, Germany)) were determined using the agar diffusion method [26]. Nutrient agar (Oxoid Limited, United Kingdom) and Sabouraud agar (Oxoid Limited, United Kingdom) media were used for both determinations of antibacterial and antifungal activities, respectively. 0.1 mL of 18 h culture of the test organisms (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis NCTC 10073, and clinical fungal agent, Candida albicans, were used to seed nutrient agar and Sabouraud agar plates, respectively. In each of these plates, 4 equidistant wells (10 mm) were cut out using sterile cork borer and were filled with 200 µL each of the different concentrations of extracts and reference drugs and allowed to diffuse at room temperature for 1 h. The zones of inhibition were measured after 24 h incubation at 37 • C (for bacteria) and after 72 h at 30 • C (for fungi). The activities of the methanol (solvent) alone were also determined.

Determination of Minimum Inhibitory Concentration (MIC) Using Microdilution
Technique. The MICs of the extracts (PMLE, CGLE, PMBE, and CGBE) against the test bacteria were determined using the microdilution technique as described by Eloff [27] and modified by Agyare and Koffuor [26]. Test solutions (100 mg/mL) of both extracts were prepared, test solution (25-100 µL) was serially diluted with distilled water to 100 µg/mL, and 50 µL of an 18 h old culture of one of the test bacteria grown in nutrient broth (Oxoid Limited, United Kingdom) was added to each well in the microplates. The covered microplates were incubated at 37 • C for 24 h. To indicate growth, 30 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, thiazolyl blue) dissolved in distilled water was added to the microplate wells and incubated at 37 • C for 30 min. C. albicans was cultivated in Sabouraud dextrose broth (Oxoid Limited, United Kingdom) and then incubated for 3 days at 30 • C. The MICs of PMLE, CGLE, PMBE, and CGBE against the test fungus were determined according to the guidelines described in the National Committee for Clinical Laboratory Standards [28] for filamentous fungi. The minimum inhibitory concentration of the each extract against the test organisms was detected as the minimum concentration of extracts where there was no microbial growth, that is, nonformation of blue color after the addition MTT to the medium [29]. The previous experiment was repeated three times.

Determination of Acute Anti-Inflammatory Activity.
The carrageenan-induced inflammatory model in seven-day-old chicks [30] was employed and the responsiveness of these chicks to anti-inflammatory drugs/extracts was determined.

Experimental Design.
At the beginning of the experiment the chicks (7 days old) were randomly assigned to one of fifteen groups (n = 5). The initial foot volumes of the chicks were measured using plethysmometer (IITC Life Science Inc., CA, USA) after which 0.01 mL of 2% carrageenan was injected into the plantar of the right foot to induce inflammation. The inflammation produced was measured, the increase in foot volumes was calculated, and those with an increase between 15 and 40% were selected and put into thirteen groups of five after which they were injected intraperitoneally with either diclofenac (Sigma, purity 98% HPLC) (

Preliminary Phytochemical Screening.
Both the leaf and stem bark of P. macrocarpa and C. gigantea were found to contain tannins (with varying amounts), alkaloids, steroids, saponins, and carbohydrate while the leaves of the two plants contain flavonoids. The stem bark and leaves of P. macrocarpa were found to contain sapogenetic glycosides (Table 1).     Figures 1, 2, and 4). Similar effects were observed after treating the animals with diclofenac (F 3,80 = 79.81, P < 0.0001) and dexamethasone (F 3,80 = 90.49, P < 0.0001) used as positive controls ( Figure 5).

Discussion
The present studies indicate the antimicrobial and antiinflammatory properties of ethanol extracts of P. macrocarpa (PMLE and PMBE) and C. gigantea (CGLE and CGBE). These findings were similar to our previous work [     the MICs for the previous test bacteria were comparable to the MICs of the C. gigantea extracts. This observation goes to support the assumption that size of inhibition halos of different extracts cannot be used for the determination of the relative antimicrobial potency since a more diffusible but less active extract could give a bigger diameter than a nondiffusible but more active extract [27,31]. The antimicrobial activities exhibited by the ethanol extracts of leaves and stem bark of C. gigantea (CGLE and CGBE) are in line with previous antimicrobial works on the leaves of different species of Cola [22,32,33] where different extracts of cola were found to exhibit inhibitory activities against certain bacteria and fungi with respect to the leaf extract. The MIC range of both the leaf and stem bark extracts of C. gigantea against the test bacteria is from 0.125 to 2.75 mg/mL and the mean zones of inhibition of the different concentration (10-50 mg/mL) extracts (crude) were almost the same as those of the reference antibacterial agent, chloramphenicol at concentration of 1000 µg/mL. However, with the agar diffusion technique, the leaf extracts of C. gigantea (10 to 50 mg/mL) showed more activity against both test bacteria and fungus compared to the extracts from      the stem bark. P. aeruginosa was found to be generally less susceptible to all extracts from P. macrocarpa and C. gigantean, respectively (Table 3), and this was not surprising since it has been found to be resistant to most orthodox antibiotics [34]. The antimicrobial properties may justify the use of these plants for the treatment of various bacterial and fungal infections such as gonorrhea and urethral infections and sores.
The study also establishes the anti-inflammatory activity of the ethanol extracts of the leaves and stem bark of P. macrocarpa and C. gigantean, respectively. Carrageenaninduced oedema has been commonly used as an experimental animal model for acute inflammation and is established to be biphasic. The early phase (1 to 2 hours) of the carrageenan model is chiefly mediated by serotonin, histamine, and increased synthesis of prostaglandins in the  damaged tissues. The late phase is sustained by prostaglandin release and mediated by bradykinin, leukotrienes, polymorphonuclear cells, and prostaglandins produced by tissue macrophages [35]. The extracts (PMLE, PMPE, and CGBE) inhibited the inflammation induced with carrageenan in both phases indicating the ability of these extracts to inhibit the synthesis or release of inflammatory mediators such as histamine, serotonin, bradykinin, and leukotrienes.
In comparing the four extracts (PMLE, CGLE, PMBE, and CGBE), only CGLE had insignificant anti-inflammatory activity in the carrageenan-induced hind paw oedema. However the CGBE had significant activity. The reason for this observation may be due to different chemical composition of the stem bark and leaves of the same plant. Traditionally, the bark is used in the management of haemorrhoids: dropsy, swelling, edema, gout, leprosy, and pain [15], and hence these results may confirm the medicinal uses of the bark. The CGBE extract may have relatively high amounts of the bioactive constituents in relation to the leaves. Phytochemical screening of C. gigantea showed the presence of alkaloids and this confirms the findings of Sonibare et al. [22]. Members of the cola family are closely related to Theobroma family of South America which have the methylxanthine alkaloids such as caffeine, theobromine, and theophylline as secondary metabolites. Caffeine is one of the alkaloids of genus cola and it is used as an analgesic and anti-inflammatory adjuvant [36,37].
There was no significant difference in anti-inflammatory activity between the leaf and stem bark extracts (PMLE and PMBE) of P. macrocarpa. However, the leaves are traditionally used as diuretics, antiflatulence, and a remedy for stomach, bladder, and urinary problems [15].
Powdered dried leaves and stem bark of P. macrocarpa and C. gigantea showed the presence of tannins and with different tannin contents (1.02-1.57% w/w). Amoo and Agunbiade [38] reported on the high tannin content of the seeds of P. macrocarpa. Similar results were found in the samples of leaves and bark of P. macrocarpa. Tannins form a class of polyphenolic compounds which can act as antioxidants, antiviral, antibacterial, antiparasitic, and antiinflammatory activity [39][40][41][42]. During the inflammatory cascade, antioxidants act as scavengers for free radicals protecting cells against oxidants which are mostly reactive oxygen and nitrogen species [43][44][45]. The aforementioned findings may justify the traditional medicinal uses of the P. macrocarpa and C. gigantea and hence there is the need to perform bioactivity fractionation of the active extracts to isolate the compounds that may be responsible for the antimicrobial and anti-inflammatory properties.

Conclusion
The minimum inhibitory concentration ranges of both ethanol leaf and bark extracts of P. macrocarpa against the test organisms were from 0.125 to 2.55 mg/mL and those of C. gigantea extracts were from 0.125 to 2.75 mg/mL. Extracts (10, 25, and 50 mg/mL) of P. macrocarpa and C. gigantea exhibited antimicrobial activity with concentrations of 50 mg/mL showing the highest zones of inhibition against the test organisms. All the extracts of P. macrocarpa and C. gigantea at 30, 100, and 300 mg/kg except leaf extract of C. gigantea exhibited significant anti-inflammatory activity. The aformentioned activities may confirm the ethnobotanical uses of these two plants as antimicrobial and antiinflammatory agents. It is recommended that the bioactive 8 Evidence-Based Complementary and Alternative Medicine extracts should be fractionated and the active compounds responsible for the previous pharmacological properties should be isolated.