Nutritive Value, Phytochemical and Antifungal Properties of Pergularia tomentosa L. (Asclepiadaceae)

MATERIALS AND METHODS

Chemicals: All chemicals were of analytical grade.

Plant material: The leaves, roots and stems of P. tomentosa (Fatakko; Hausa language) were collected in May 2006, around Usmanu Danfodiyo University Campus, Sokoto, Nigeria. The voucher specimens of the plant were deposited in the departmental herbarium (Botany unit) for reference. The study was undertaken in June to July 2006. The portions collected were open air-dried under the shade, pulverized into a moderately coarse powder (using pestle and mortar) and stored in plastic containers until required for use.

Organisms: The fungal organisms used were Trichophyton rubrum, Microsporum gypseum, Aspergillus niger and Aspergillus flavus. They were clinical isolates obtained from Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria. The organisms were maintained on Sabouraud Glucose Agar (SGA) medium and re-identified by microscopic examination of a portion of the colony for spores and characteristic hyphae (Cheesbrough, 1982).

Extraction and fractionation procedure: This was done by activity-guided fractionation using ethanol-water (1:1) and different (hexane, petroleum ether and chloroform) organic solvents as described by Morris and Aziz (1976) and Springfield and Weitz (2006). The powdered leaves of P. tomentosa (40 g) were extracted with ethanol-water (1:1, 400 mL) at room temperature overnight. The extract was filtered and partitioned in Hexane (HX) separately (250 mL) and clarified by further filtration. Evaporation of HX fraction to dryness in an oven at 45°C yielded residues (Table 1). The aqueous filtrate (ethanol-water) of the extract fraction was further partitioned (to obtain fractions of different polarities) with petroleum ether (250 mL) and chloroform (250 mL) separately. Evaporation of the Petroleum Ether (PE), Chloroform (CHL) and Last remaining water-ethanol fractions (LWEF) yielded residues (Table 1).

Table 1:	Amount of residues obtained after extraction
HX = Hexane, PE = Petroleum Ether, CHL = Chloroform, LWEF = Last remaining water-ethanol fractions

The stems and roots were subjected to the same procedure. All procedures were repeated to obtain more residues. Another 40 g of each powdered leaf, root and stem were extracted with 400 mL of distilled water for 24 h and filtered. All the residues obtained were reconstituted in sterilized distilled water and screened for antifungal and phytochemical properties. This procedure enabled us remove the possible contributory effects of the organic solvents.

Antifungal activity: The antifungal activities of the aqueous and organic solvent extracts of P. tomentosa were evaluated according to reported procedures of Zacchino et al. (1999), using the agar dilution method with some modifications. The fungal species were maintained on Sabouraud Dextrose Agar (SDA) medium in 90 mm petridishes. Five milliliter of filter-sterilized reconstituted water solution of each plant extract (HX, PE and CHL) at concentrations of 10 to 120 mg mL^-1 (stems, roots and leaves) were aseptically mixed with 15 mL of SDA (Liquefied and maintained at melting point in waterbath at 45°C) to give final concentrations ranging from 0.67 to 8.00 mg mL^-1. The crude water extracts of the plant parts (leaf, stem and root) at 10 to 120 mg mL^-1 were also prepared using above procedure to give final concentrations of 0.67 to 8.00 mg mL^-1. The Petri-dishes (90 mm) were filled to 20 mL final volume with Sabouraud Dextrose Agar containing the requisite amounts of diluted extract solution. The Petri-dishes were then inoculated at their center with a disk (2x2 mm) cut from the periphery of a 14-day-old (T. rubrum and M. gypseum) and 7-day-old (A. niger and A. flavus.) fungal colonies. Griseofulvin (0.67 and 4.00 mg mL^-1), Clarion Medicals Ltd. Lagos, Nigeria, measured from the pulverized 500 mg tablet was also included to serve as positive control. Water (5 mL) in place of the extract and media (15 mL) were mixed together as negative control. The treated and the control Petri-dishes were incubated at room temperature for 14 days for superficial mycosis (dermatophytes) and 48 h for A. niger and A. flavus. Growth was observed each day to the last day. From these, the percentage inhibitions were calculated using the following formula:

Phytochemical screening: This was done using standard procedures of Wall et al. (1954), Harborne (1973), Trease and Evans (1978) and El-Olemyl et al. (1994).

Proximate analysis and nutrients composition: The extracts were analysed for moisture content using the method of Oyeleke (1984) and crude protein by modified Kjeldhal method. The method of Yawas and Obi (2001) was employed in the analysis of carbohydrates. Ash content was determined by the method of Samuel et al. (1997). Crude lipids and fibre content were determined by the procedures of Association of Official Analytical Chemists (AOAC, 1980).

RESULTS AND DISCUSSION

The nutritive value, antifungal and phytochemical properties of leaf, stem and root extracts of P. tomentosa are shown in Table 2-7. The leaf and stem extracts revealed the presence of alkaloids, cardiac glycosides, cyanogenic glycosides, saponins, flavonoids, tannins and anthraquinones while the roots contain trace amount of these phytochemicals with absence of alkaloids and flavonoids (Table 2).

Higher amount of phosphorus and potassium were present in root and stem with sodium, magnesium and calcium in the leaf extracts (Table 6). Higher contents of carbohydrates (53.27±1.75 to 61.31±2.84%) and crude fibre (16.33±0.29 to 23.17±0.58%) were observed in all the plant parts but lipids, ash and crude protein were higher in the leaf compared to the other extracts. The moisture (10.67±0.76%) content was higher in stem extracts with leaves having the lowest (0.69±0.29%) amount (Table 7). All the organic solvents extracts of the plant parts were active against the tested fungal isolates with percentage inhibition of 41.90±5.63 to 97.52±0.28% (Table 3-5) with near complete inhibitions at 8.00 mg mL^-1. All the aqueous extracts of the plant parts at this concentration had also shown near complete inhibition (97.45±0.21%) of the isolates.

The present study presents systematic report on the antifungal properties of P. tomentosa against multiple drug resistant common pathogenic fungi and its nutritive value. This supports the view that P. tomentosa is a potent antifungal agent with valuable nutritional information. In recent years, multiple drug resistance in human pathogenic microorganisms has developed due to indiscriminate use of conventional antimicrobial drugs commonly used in the treatment of infectious diseases (Subramanian et al., 2006). This forced scientific researchers to find alternative antimicrobial drugs from other sources including medicinal plants. The mechanism of action of the AQ, HX, PE and CHL extracts of P. tomentosa against the pathogenic fungal isolates may be due to inhibition of fungal cell wall (due to pore formation in the cell and leakage of cytoplasmic constituents by the active components such as alkaloids), protein, amino acid and sphingolipid biosynthesis and electron transport chain (Shelton, 1991; Lartey and Moehle, 1997; Ueki and Taniguchi, 1997; Dominguez and Martin, 1998).

Table 2:	Phytochemical analysis of water and organic solvent extracts of P. tomentosa
- = Absence, + = Trace amounts, +++ = Presence, I = Water, II = Hexane, III = Petroleum ether, IV = Chloroform, V = Last remaining water-ethanol extract, ALK = Alkaloids, GLY = Glycosides, CG = Cardiac Glycosides, CYG = Cyanogenic Glycosides, SAP = Saponins, FLV = Flavonoids, TA = Tannins, ATG = Anthraquinone Glycosides

Table 3:	Percentage inhibitions of fungal isolates by aqueous and organic solvent leaf extracts of P. tomentosa
Diameter of water control petridishes of all the organisms used is 90 mm, Values are mean±standard deviation of percentage inhibitions, 0-25% = None or little inhibition; 25-50% = Average inhibition; 50-75% = Strong inhibition; 75-100% = Very strong inhibition. HX = Hexane, PE = Petroleum ether, CHL = Chloroform, AQ = Aqueous, GS = Griseofulvin, n = 3

Table 4:	Percentage inhibitions of fungal isolates by organic solvent stem extracts of P. tomentosa
Diameter of water control petridishes of all the organisms used is 90 mm, Values are mean±standard deviation of percentage inhibitions, 0-25% = None or little inhibition; 25-50% = Average inhibition; 50-75% = Strong inhibition; 75-100% = Very strong inhibition. HX = Hexane, PE = Petroleum Ether, CHL = Chloroform, AQ = Aqueous, GS = Griseofulvin, n = 3

Table 5:	Percentage inhibitions of fungal isolates by organic solvent root extracts of P. tomentosa
Diameter of water control petridishes of all the organisms used is 90 mm, Values are mean±standard deviation of percentage inhibitions, 0-25% = None or little inhibition; 25-50% = Average inhibition; 50-75% = Strong inhibition; 75-100% = Very strong inhibition, HX = Hexane, PE = Petroleum Ether, CHL = Chloroform, AQ = Aqueous, GS = Griseofulvin, n = 3

Table 6:	Mineral element content of extracts of P. tomentosa
Values are mean±standard deviation, n = 3

Table 7:	Proximate composition of extracts of P. tomentosa
Values are mean±standard deviation, n = 3

The present study highlights the use of P. tomentosa in folk medicine for the treatment of fungal diseases and underscores the importance of the ethnobotanical approach for the selection of P. tomentosa in the discovery of new bioactive compounds.

The presence of alkaloids, cardiac glycosides, cyanogenic glycosides, saponins, flavonoids, tannins and antraquinones (Table 2) in the extracts may be attributed to the antifungal actions of P. tomentosa. The antifungal actions of these phytochemicals have been associated with antimicrobial properties (Scalbert, 1991; Favel et al., 1994; Cook and Samman, 1996; Oyagade et al., 1999; Shale et al., 1999).

The level of dietary fibre is quite high (23.17±0.58%) in stem when compared to root and leaf extracts. The presence of high fibre levels in diet can cause intestinal irritation, lower digestibility and overall decreased nutrient utilization (Oyenuga and Fetuga, 1975). The low crude protein content of P. tomentosa (3.35±0.48 to 6.39±0.17%) coupled with the fact that it is abundant in northern Nigeria and with antifungal properties may still contribute to the sources of proteins. Higher carbohydrates content of the extracts (53.27±1.75 to 61.31±2.84%) may encourage its use as high carbohydrates sources in some food formulations and as fodder for animals. The higher moisture content of the root and stem extracts (8.67±0.58 to 10.67±0.76%) implies that their shelf life will be longer than that of leaves. The ash content of P. tomentosa was higher than that of the medicinal plants P. thonningii (3.50%) and S. bicolar (5.34%) (Jimoh and Oladiji, 2005; Adetuyi, 2004).

The levels of calcium and magnesium were low in the extracts. Magnesium is an antioxidant micronutrient (Talwar et al., 1989) and its presence may boost the immune system and aid in removing magnesium deficiencies which could lead to severe metabolic disorders and compromise the health of the organism.

Higher contents of phosphorus were observed in stem (7.07±0.06 ppm) and root (8.13±0.06 ppm) compared to leaves extracts. However, it is worth noting that calcium in conjunction with magnesium, chlorine and proteins are involved in the formation of bone (Abulude, 2001). It also plays an important role in blood clotting, coordination of inorganic elements present in the body and balancing of calcium and phosphorus. The root (167.30±5.03 ppm) and stem (215.00±10.00 ppm) of P. tomentosa are also good sources of potassium. Higher potassium with low sodium as presented in this study is protective against excessive sodium intake. The higher potassium content in the stem and root extracts may be an added advantage over the leaf samples for therapy and are vital for bone (Dzomeku et al., 2006). Increasing dietary potassium lowers blood pressure in humans and reduces the risk of stroke. Thus, maintaining a high potassium intake may be achieved by consuming the stems and roots of P. tomentosa.

In view of the present study, the leaf, root and stem extracts of P. tomentosa could be utilized as a cheap source of nutrients and antifungal agent(s). Further studies on isolation of the active antifungal agent(s) and toxicological evaluations of the leaf root and stem extracts of P. tomentosa are recommended.

ACKNOWLEDGMENTS

Authors are thankful to Abdulrahman Barau, Mycology Laboratory, Department of Biological Sciences and Malam Ahmad Modi Bodinga, Agric Physical Laboratory, Faculty of Agriculture, Usmanu Danfodiyo University, Sokoto, Nigeria, for providing laboratory assistance.