According to World Health Organization estimation, nearly 80% of the world population relies on traditional medicine for primary health care and most of which involves the use of medicinal plants extract [1].

Population rise, inadequate supply of drugs, prohibitive cost of treatments, side effects of several synthetic drugs and development of resistance to currently used drugs for infectious disease have led to increased emphasis on the use of plant materials as a source of medicine for a wide variety of human ailments. These plants are cooked and eaten as soups, crushed into powdered form and sprinkled in meals or even applied on the surface of wounds on the skin etc.

The medicinal value of plant lies in the chemicals called phytochemicals and they include alkaloids, flavonoids, saponins, phenolic compounds, tannins, terpenoids, phytosterols, proteins, gum, lignin [2]. 

This research work attempts to give an overview on the medicinal plant species Afrofittonia silverstris Lindau employed by the indigenous people of Akwa Ibom State to treat respiratory problems especially cough and also used to treat diseases such as digestive tract disorders, sores, skin diseases and constipation [3-5].

Afrofittonia silverstris Lindau belongs to the Acanthacea family, the plant trails on moist ground and grows throughout the year [6]. Its natural habitat is sub-tropical or tropical moist lowland forest.

Reagents and Chemicals

Dichloromethane, Ethanol, Ammonia solution, ethyl acetate, sulphuric acid, glacial acetic acid, Mayer’s reagent, Hager’s reagent, Wagner's reagent, Dragendorf reagent, Ferric chloride, Bromine water, 1% aqueous Hydrochloric acid, Chloroform, n-hexane and saline water, Nutrient agar. 

Collection and Authentication of Plant Material

Fresh plant of Afrofittonia silvesteris Lindau was collected from OboyoIkotIta village in Nsit Ibom Local Government of Akwa Ibom State during the early hours of the morning on the 10^th of October 2018. The plant was identified and authenticated by Pharmacist Chimezie Osuala of the Department of Pharmacognosy and Herbal Medicine, University of Uyo.

Plant Preparation

The fresh leaves of Afrofittonia silvestris Lindau were washed with tap water and then rinsed with distilled water, after which it was air dried for the period of 14 days. The dried leaves were then transferred into an oven at 60⁰C to aid complete dryness for 2 hours. The dried leaves were powdered using an electric blender.

Extraction of Plant Material

Total weight of the powdered plant was obtained as 220g. 110g each were macerated in 800ml of dichloromethane and 1000ml of ethanol respectively at room temperature for 120hours. The extracts were filtered using a filter paper and concentrated using the rotary evaporator.

Phytochemical Screening

• Test for Tannins: 0.5g each of the ethanol and dicloromethane extract were dissolved in 10ml each of distilled water and filtered. A drop of 5% ferric chloride was added to 1ml of the filtrate of the respective extract. A blue-black or blue-green precipitate is evident of tannins. 

• Test for Saponins: 10ml of distilled water was added to 0.3g of the individual extract and shaken vigorously. It was observed for stable persistent frothing even on warming.

• Test for Alkaloids: 0.5g of the individual plant extract was diluted in 1% aqueous hydrochloric acid and heated on a steam bath it was cool and filtered. 1 ml of the filtrate was transferred into 4 different test tubes and treated with few drops of the following reagents; Meyer's reagent (Potassium Mercuric Iodide solution), Dragendorff's reagent (solution of Potassium bismuth iodide), Wagner's reagent (solution of iodine in potassium iodide) and Hager's reagent (Picric acid solution). Precipitation shows that there's the presence of Alkaloids.

• Test for Terpenoids: 2ml of chloroform was added to 0.5g each of the ethanolic and DCM plant extracts. 3ml of concentrated sulphuric acid was also added to the test tubes containing the extracts and chloroform. They were shaken and observed for Reddish brown colouration at the interface which indicates presence of Terpenoids.

• Test for Flavonoids: 10ml of ethyl acetate was added to 0.5g of the concentrated ethanol and DCM plant extract in the test tube and were boiled for 3 minutes in a water bath. After 3mins, the mixtures were allowed to cool and then filtered to obtain clear filtrate. 4ml aliquots of the filtrate was treated with 1ml of dilute ammonia solution and then shaken and observed for a yellow colouration at the interface. 

• Test for Cardiac Glycoside (Keller-Killiani Test): 0.5g each of the concentrated ethanolic and DCM extracts were transferred into different test tubes and treated with 2ml glacial acetic acid containing a drop of ferric chloride solution, 1ml of concentrated sulphuric acid was gently added down the side of the test tubes. They were both observed for violet ring at the interface indicating the presence of deoxy sugar which is a characteristic of cardiac glycoside (Cardenolides).

• Test for Anthraquinone: 0.5g each of the concentrated ethanolic and DCM extracts were transferred into clean test tubes and were treated with 5ml of chloroform and 5ml of 100% ammonia. The reaction was observed for a pink, violet or red colour in the ammonia layer (lower layer) which is an indication of the presence of anthraquinones. 

Thin Layer Chromatography 

0.5g each of the Ethanolic and Dichloromethane extracts were subjected to chromatographic studies. A developing chamber was obtained and 5ml of the TLC developing solvent (7:3 mixtures of n-hexane and ethyl acetate) was introduced and then covered for saturation to occur. The plate was allowed to dry and thereafter visible spots were circled and then viewed under UV lamp to locate other spots. This method was repeated for various ratios as follows: 100% n-hexane, 3:1, 9:1, 3:2, 8:2, 4:3.

Antibacterial Testing

The antibacterial potential test was carried out using the agar disc diffusion method [7]. All the agar media to be used for the process were all prepared following manufacturer's instructions. The test organisms (Staphylococcus aureus, Klebsiella pnuemoniae, Pseudomonas aeruginosa and Escherichia coli ATCC 25922) were grown in nutrient agar to ensure they were viable and then standardized using saline solution to match the turbidity of MacFarlane standard (1.5x108 CFU/ml). 

The plates were allowed to stand for some time and then incubated at 37°C for 24hours. This method was repeated for Dichloromethane extract. Zones of inhibition were observed, measured and then compared with the controls.

The percentage yield of the extracts was calculated, the yield (10%) of ethanol extract was more when compared with the yield of the dichloromethane extract which was (3%). 

The Phytochemical Screening carried out on A. silvestris showed the presence of Tannins, Alkaloids, Saponins, Cardiac glycosides, Flavonoids in the ethanolic extract and anthraquinone in the DCM extracts. Terpenoids was absent in both extracts. These compounds as earlier stated confer activity on the plant including antibacterial activity. 

The TLC results obtained showed the different proportions of compound present in the leaves and the R_f value was determined for the individual component. Low R_f indicates the similarity of the substance with the stationary phase implying that the compounds are polar while high R_f value indicates non polarity which is similar to the mobile phase.

Antibacterial activity of A. silvestris has been evaluated in vitro against four clinical bacterial isolates which are frequently incriminated in human infections. The results obtained showed that the ethanol extract of the leaves of A. silvestris had antibacterial activity on E. coli ATCC25922, K. pneumoniae, P. aeruginosa and S. aureus. The microorganisms showed varying degree of sensitivity to the extract at the various concentration used. These findings have been found to be consistent with those obtained in previous studies done by [8].

Extraction of antimicrobial compound of A. Silvertris with DCM was poor as the microorganisms weren’t susceptible at all except for Pseudomonas with relatively small zone of inhibition.

Previous studies reports that ethyl acetate, butanol, aqueous extracts of the plant demonstrated antibacterial activity on typed Pseudomonas aeruginosa NCIB 950 (8mm, 5mm and 3mm) E. coli. NCIB 86 (7mm, 4mm and 3mm) and S. aureus NCIB 85 (8mm, 5mm and 3mm) respectively [8].

Pseudomonas aeruginosa was susceptible to the ethanol extract of the plant with a zone of inhibition of 15mm at the concentration of 8mg/0.4ml. Comparing the result with that of standard antibiotics, gentamicin had an inhibition of 25mm. Klebsiellapneumoniae had an intermediate susceptibility with zone of inhibition 21mm. The zone of inhibition for gentamicin was 26mm.

The zone of inhibition for E. coli ATCC 25922 was 18mm and 34mm for Gentamicin. The activity on S. aureus at this concentration was evident with a zone of inhibition of 18mm and the activity of gentamicin was 34mm.It was observed that the microorganisms were highly susceptible to gentamicin evidenced by a large zone of inhibition. The activity of the extracts was not as high as that of the reference standard probably due to the fact that gentamicin is a pure compound that has already been isolated but the extract contained both active and inactive compounds. Another reason could be as a result of the concentration used. The zone sized increased with increased concentration (8mg/0.4ml). In other words, if the concentration of this extract is increased, the level of activity may increase accordingly. 

In conclusion, activity of the ethanol and DCM extract of the leaves of A. silvestris against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E. coli which are among the organisms indicated in upper respiratory tract infections was studied in vitro. The ethanolic leave extract inhibited the growth of these bacteria hence justifying the claims and beliefs of the Akwa Ibom people. This leave extract could be isolated to determine the active antimicrobial component which may serve as a future alternative for combating upper respiratory tract infections.

Table 1: Phytochemical Screening results for the plant Afrofittonia silvestris L.

DCM = Dichloromethane, — = Not Present, + = Present in small quantity ++ = Present in moderate quantity, +++ = Present in very high quantity

Table 2: Showing thin layer chromatography R_f values of Afrofittonia silvestris Lindau using n- Hexane and Ethyl acetate as solvent system at different ratio

A = Spots for DCM extract, B = Spots for Ethanol extract

Table 3: Antibacterial activity of the leaves of Afrofittonia silvestris extracted using different solvents compared with standard antibiotics

CIP5: Ciprofloxacin, C30: Chloramphenicol, COT25: Cotrimoxazole, CN: Gentamicin, LEV: Levofloxacin, FOX30: Cefoxitin, TZP: Piperacilin/tazobactam, MEOH: Methanol, DCM: Dichloromethane, ETOH: Ethanol - Stock concentration = 20mg/ml

1. World Health Organization. Global Tuberculosis Report 2015. World Health Organization, 2015.

2. Malikaharajuna, P.B. et al. “Phytochemical studies of Strychnos potatorum L.f., a medicinal plant.” E-Journal of Chemistry, vol. 4, 2007, pp. 510–518.

3. Burkill, H.M. The Useful Plants of Wet Tropical Africa. 2nd ed., vol. 1, Families A–D, Royal Botanic Gardens, Kew, 1985.

4. Etukudo, I. Ethnobotany: Convention and Traditional Uses of Plants. Verdict Press, 2003.

5. Ajibesin, K.K. et al. “Ethnobotanical survey of Akwa Ibom State of Nigeria.” Journal of Ethnopharmacology, vol. 115, no. 3, 2008, pp. 387–408.

6. Taylor, R.S.L. et al. “Antimicrobial activities of southern Nepalese medicinal plants.” Journal of Ethnopharmacology, vol. 50, 1996, pp. 97–102.

7. Kokate, C.K. Practical Pharmacognosy. Vallabh Prakashan, 2000, pp. 218.

8. Ajibesin, K.K. “Antibacterial effects of the flavonoids of the leaves of Afrofittonia silvestris.” Antibacterial Effects of Flavonoids, vol. 10, 2009, pp. 315–320.