Phytochemical composition and antioxidant and antimicrobial activities of Pergularia daemia

The oldest form of treatment known to humans is the application of herbs to cure ailments and diseases. The prehistoric medical systems such as Ayurveda, Siddha and Homoeopathy use herbal formulations to treat various ailments (Kelly 2009). According to the World Health Organization (WHO), more than 80% of the global population in poor and underdeveloped countries rely primarily on traditional plant-based medicines for their primary health care needs because pharmaceuticals are not available or are relatively expensive (WHO 2002). Even in developed countries where pharmaceuticals are readily accessible, many still prefer herbal drugs over the pharmaceutical drugs (Sridevi, Prema & Sekar 2014).


Introduction
The oldest form of treatment known to humans is the application of herbs to cure ailments and diseases.The prehistoric medical systems such as Ayurveda, Siddha and Homoeopathy use herbal formulations to treat various ailments (Kelly 2009).According to the World Health Organization (WHO), more than 80% of the global population in poor and underdeveloped countries rely primarily on traditional plant-based medicines for their primary health care needs because pharmaceuticals are not available or are relatively expensive (WHO 2002).Even in developed countries where pharmaceuticals are readily accessible, many still prefer herbal drugs over the pharmaceutical drugs (Sridevi, Prema & Sekar 2014).

Phytochemical composition and antioxidant and antimicrobial activities of Pergularia daemia
Read online: Scan this QR code with your smart phone or mobile device to read online.

Extract preparation
Stems and leaves of the plant were air-dried separately at room temperature (25 °C) and were pulverised after drying.
Of the pulverised sample, 400 g was kept in contact with 1 L n-hexane in a stoppered glass container for 5 days with frequent agitation (Ncube, Afolayan & Okoh 2008).The extract was decanted and filtered through a cotton plug funnel and Whatman no.1 filter paper.The filtrate from the n-hexane extraction was concentrated under vacuum using rotary evaporator.Subsequent extraction using the above procedure was also carried out to obtain crude ethyl acetate and ethanol extracts independently.

Quantitative analysis of phytochemicals
Total phenolic content: The total phenolic content in the plant extracts was determined using the spectrophotometric method (Singleton, Orthofer & Lamuela-Raventos 1999).
Concentrations of 1 mg/mL, 5 mg/mL and 10 mg/mL of methanolic solution of the extract were prepared and used in the analysis.The reaction mixture was prepared by adding 0.5 mL of methanolic extract to 2.5 mL 10% Folin-Ciocalteu's reagent and 2.5 mL 7.5% NaHCO 3 .Blank was concomitantly prepared, containing 0.5 mL methanol, 2.5 mL 10% Folin-Ciocalteu's reagent and 2.5 mL 7.5% of NaHCO 3 .The mixture was thereafter incubated in a 45 o C thermo-stated water bath in a dark cupboard for 45 min.The absorbance was then determined at a wavelength of 765 nm using a spectrophotometer (Deckmann GU750).The mixture was prepared in triplicate for each analysis and the mean value of absorbance was obtained.A solution of gallic acid, which serves as a standard, was prepared in methanol to achieve the concentration of 0.5 mg/mL.This solution was then diluted to obtain concentrations of 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.13 µg/mL, 1.56 µg/mL and 0.78 µg/mL, and the calibration line was constructed.Based on the measurement of the absorbance, the concentration of phenolics was determined (µg/mL) from the calibration line and the total phenolic content in the extracts was expressed in terms of gallic acid equivalent (mg of GA/g of extract).

Determination of flavonoid content:
The total flavonoids in the plant extracts were determined using the spectrophotometric method (Quettier et al. 2000).Methanol was used to prepare the extracts with concentrations of 1 mg/mL, 5 mg/mL and 10 mg/mL, which were used in the analysis.Of each solution of the extracts, 1 mL was added to 1 mL of 2% AlCl 3 solution, which was also prepared in methanol.The samples were incubated for an hour at room temperature and the absorbance was then read using a spectrophotometer at a wavelength of 415 nm (Deckmann GU750).The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained.The quercetin equivalent and calibration curve were evaluated by preparing quercetin solutions at concentrations varying from 10 µg/mL to 100 µg/mL in methanol.Based on the measured absorbance, the concentration of flavonoids was determined (µg/mL) on the calibration line and the flavonoid contents in the extracts were expressed in terms of quercetin equivalent (mg of QE/g of extract).

Antioxidant activity 2,2-diphenyl-1-picrylhydrazyl free radical scavenging
The ability of the plant extracts to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was assessed following the method of Chang et al. ( 2001) and Kumarasamy et al. (2007).The stock solution of the extracts was prepared in methanol to achieve the concentration of 1 mg/mL.Dilutions were made to obtain concentrations of 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.62 µg/mL, 7.81 µg/mL, 3.90 µg/mL, 1.99 µg/mL and 0.97 µg/mL.The prepared solution (1 mL) was mixed with 1 mL of methanolic solution of DPPH of concentration 1 mg/mL.After 30 min of incubation in the dark at room temperature, the absorbance was recorded at 517 nm with a spectrophotometer.A control sample containing 1 mL of methanol and 1 mL of methanolic DPPH solution was set alongside.Percentage inhibition was calculated using the equation below, while IC 50 (inhibitory concentration at 50%) was estimated from the percentage inhibition versus log concentration plot, using a non-linear regression algorithm:

Antimicrobial assay: Antibacterial and antifungal activities
Cultures of six human pathogenic bacteria made up of four gram-negative and two gram-positive species were used for the antibacterial assay.The sample extracts that were used for the antimicrobial sensitivity test were prepared by dissolving 1 mg of the extract in 5 mL of the extracting solvents, that is, n-hexane, ethyl acetate and ethanol, to make a 200-µg/mL solution.
In the antibacterial assay, an overnight culture of the organism was prepared by taking a loop full of the organism from stock and inoculating into a sterile nutrient broth of 5 mL and then incubated for 24 h at 37 °C.From the overnight culture, 0.1 mL of the organism was taken and put into 9.9 mL of sterile distilled water to get 1:100 (10 -2 ) dilution of the organism.From the diluted organism (10 -2 ), 0.2 mL was taken into the prepared sterile nutrient agar, which was maintained at 45 °C, and aseptically poured into sterile petri dishes and allowed to solidify for 60 min.Using a sterile cork borer of 8 mm diameter, wells were made according to the number of graded concentrations.In each well, the different graded extract concentrations were poured, and this was performed in duplicates.The plates were allowed to stay on the bench for 2 h to allow for pre-diffusion.The plates were then incubated upright in the incubator for 24 h at 37 °C (Oloyede et al. 2010;Onocha & Ali 2010).
In the fungal activity inhibition study, surface plate method was used.Sterile sabouraud dextrose agar (62 g/L) was prepared accordingly and aseptically poured into the sterile plates in duplicates and allowed to solidify properly.A volume of 0.2 mL of the organism was spread on the surface of the agar using a petri dish to cover the entire surface of the agar.Then, eight wells were bored using a sterile cork borer of 8 mm diameter.
The graded concentrations of the extract were put into the wells accordingly, including the controls.All the plates were left on the bench for 2 h to allow the extract to diffuse properly into the agar, that is, pre-diffusion.The plates were incubated at 25 °C for 72 h.Solvents of extraction were used as control, while gentamycin (5 µg/mL) and tioconazole (0.7 µg/mL) were used as standard reference drugs in the study (Bayer et al. 1986).

Statistical analysis
All experimental measurements were carried out in triplicate and are expressed as an average of three analyses ± standard error.SPSS (Chicago, IL) statistical software package (SPSS for Windows, ver.II, 2004) was used to determine the magnitude of correlation between the variables.

Phytochemical screening of the crude extract
The phytochemical screening results (Table 1) showed the presence of a high content of glycosides, steroids, terpenoids and flavonoids in all the extracts of both the stem and leaf of P. daemia.
Tannins and saponins were observed in the ethanol extracts of the stem and leaf.Anthraquinones were detected in small quantities in n-hexane extracts of both the stem and the leaf.The presence of flavonoids, steroids, terpenoids and glycosides was high in all the extracts.Tannins, alkaloids, saponins and phenols are present in polar solvents extracts, that is, ethyl acetate and ethanol extracts.It is worth mentioning that the high concentration of all the metabolites in the ethanol extract, except for anthraquinone, confirms ethanol as the better extracting solvent of the three solvents.Ethanol, being a polar solvent and close in polarity to water, lends credence to the efficiency of the aqueous extracts of this plant as used in folk medicine.The phytochemicals are responsible for the therapeutic efficiencies and efficacies of medicinal plants (Shoge, Ndukwe & Amupitan 2011) and are present in high concentrations in ethanol, a polar solvent.The presence of this phytochemical in the extracts of the plant may therefore be responsible for the detoxifying action and antimicrobial, antioxidant and anti-inflammatory activities of the plant (Li et al. 2004;Yen, Duh & Chuang 2000).It is therefore not surprising that P. daemia has found extensive application as an antioxidant, an analgesic, a hepatoprotective substance and as an anticancer herb in traditional medicine (Karthishwaran & Mirunalini 2010).The presence of some of the above-mentioned metabolites in P. daemia had earlier been detected by Sridevi et al. (2014) in a similar study.

Total phenolic content of the plant extracts
The total phenolic content of the P. daemia extracts using Folin-Ciocalteu's reagent is expressed in terms of gallic acid equivalent (the standard curve equation: y = 0.0052x, R 2 = 0.9905).The total phenolic content in the examined extracts ranged from 15.898 ± 0.111 mg GA/g to 54.679 ± 0.675 mg GA/g.Ethanol extracts from the stem recorded the presence of high concentrations of phenolic compounds, while ethyl acetate and n-hexane extracts recorded medium concentration.The polarity of solvents is directly related to extraction efficiency; therefore, polar solvents give better extraction than non-polar solvents (Mohsen & Ammar 2008;Zhou & Yu 2004).Ethanol affords 54.679% and 34.167% GA/g for leaf and stem extracts, respectively, while 18.654% and 15.888% were recorded for n-hexane extracts of leaf and stem, respectively.The total phenolic content of 1 mg/mL, 5 mg/mL and 10 mg/mL of each extract was determined in triplicate.
Phenolic compounds play a key role as antioxidants or free radical scavengers.The antioxidant activity of the phenolic compounds is mainly because of their redox properties, which absorb and neutralise free radicals, triplet oxygen or decomposing peroxides (Osawa 1994).It has also been noted that phenolic compounds provide antimutagenic and anticarcinogenic actions in humans when approximately 1.0 g of mixed antioxidant compounds were consumed daily from vegetable and fruit diet over a short period of time (Tanaka et al. 1998).
The gallic acid equation was used as a standard curve equation for the estimation of the total phenolic compound in the samples.In the standard curve equation y = 0.0052x, R 2 = 0.9905, y denotes the absorbance and x denotes the concentration of the The values obtained for the concentration of total phenols are expressed as mg of GA/g of extract in Figure 1.
The pairs of n-hexane stem extract and ethyl acetate stem extract were significantly different.Ethyl acetate stem extract was significantly different from that of ethanol stem extract, with p < 0.05.All other pairs were not different.

Flavonoid content
The concentration of flavonoids was expressed in terms of quercetin equivalent (mg of QE/g of extract).The concentration of flavonoids in plant extracts from P. daemia ranged from 72.549 ± 0.449 mg/mL to 400.196 ± 0.339 mg/mL (Figure 2).
The leaf has a higher concentration than that of the stem.Ethanol extracts of the leaf have the highest concentration of the flavonoids and, generaly, ethanol extracts recorded significantly higher concentrations of flavonoids.The concentration of flavonoids in ethanol extracts of the leaf was 400.196 mg QE/g, followed by that of ethyl acetate with 388.627 mg QE/g and the n-hexane extract with 338.725 mg QE/g at 10 mg/mL.The lowest flavonoid concentration was recorded in the n-hexane extract of the stem.The quantities of the flavonoids extracted increases with the polarity of the extraction solvent.This result is in agreement with the observation of Min and Chun-Zhao (2005) when they compared extraction techniques in flavonoid extraction.
Our result revealed that the aerial part of the plant is highly loaded with flavonoids than the stem.Several studies reported that flavonoids present in herbs significantly contributed to their antioxidant properties and healing ability.Also, flavonoids are found to be effective scavengers of most oxidising molecules, including single oxygen and various free radicals (Manik et al. 2014).
The data obtained were subjected to statistical analysis.It was found that only the n-hexane leaf extract and ethyl acetate leaf extract were different significantly at p < 0.05.

Antioxidant activity 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity
The percentage inhibition of P. daemia over DPPH free radicals varied from 34.65% to 84.90%.The activity was expressed as IC 50 (µg/mL), that is, the minimum concentration of the extract capable of scavenging 50% of the DPPH radicals.
The IC 50 value of ascorbic acid with the minimum concentration at 5.67 µg/mL was enough to scavenge the DPPH radical, which is considered to be chemotherapeutically significant (IC 50 < 10 µg/mL) with potency that could be used as a reference (Tian et al. 2013).The parallel examination of ascorbic acid as a reference with antioxidant activity of plant extracts is illustrated in Figure 3 and Figure 4.
Figure 3 shows the antioxidant activity of an ethanolic stem extract of P. daemia at 10.19 µg/mL in close range with ascorbic acid obtained at 5.67 µg/mL and 26.99 µg/mL for ethyl acetate stem extract, followed by 58.20 µg/mL for n-hexane stem extract, which was observed to be very far from the standard.
Figure 4 shows IC 50 activity of leaf extracts at 6.27 µg/mL (ethanol), 17.78 µg/mL (ethyl acetate) and 22.49 µg/mL (n-hexane).When compared with the standard at 5.67 µg/mL, the ethanolic extract shows a very close antioxidant activity, followed by ethyl acetate, and the least antioxidant activity was observed in n-hexane.This study reveals that by consuming P. daemia, the free radicals that might be present in humans can be eliminated to get relief from oxidative stress and degenerative sicknesses.Ethanolic extract from P. daemia manifested the strongest capacity for scavenging DPPH radicals and was even effective at low concentration.

Antimicrobial sensitivity
The antibacterial sensitivity inhibition increases with the polarity of the solvent of extraction and with extract concentration.n-Hexane extract from the stem of P. daemia does not show sensitivity on the microorganisms at concentrations lower than 25 µg/mL.Ethyl acetate stem extract shows sensitivity at 12.5 µg/mL and even at 6.25 µg/ mL on S. aureus, P. aeruginosa and K. pneumoniae (Table 2).
Meanwhile, full sensitivity was observed in ethanolic stem extract at all concentration levels.The ethyl acetate and ethanol extracts showed active inhibition on the growth of all tested microorganisms and the activities were comparable to that of gentamycin, the reference antibacterial drug, particularly at high concentration (Table 2 and Table 3).n-Hexane leaf extract shows sensitivity at 200 µg/mL, 100 µg/ mL, 50 µg/mL and 25 µg/mL on all organisms but is slightly sensitive at 12.56 µg/mL on S. aureus and B. subtilis (Table 3).
Four clinical strains of human pathogenic fungi were used in the study.Candida albicans, A. niger, R. stolon and P. notatum.Candida albicans and A. niger were the only fungi that were sensitive to the n-hexane stem extract inhibitory action at high concentration, although all the four fungi were sensitive to the leaf extract (Table 4 and Table 5).
Rhizopus stolonifier and P. notatum were resistant to n-hexane leaf extract but were inhibited by other extracts, particularly at high concentration.Ethanol extracts for both leaf and stem were active in curbing the activities of the fungi, but they were not comparable to that of tioconazole, even at high concentration of extracts.
All fungi were resistant to the action of extracts at low concentrations, but ethanol extract of the leaf, showed inhibition at all levels.Also, inhibition activity increases with the polarity of extracts as well as concentration.
In the analysis of the zones of inhibition produced by the extracts, it was observed that ethanolic extracts of P. daemia showed prominent antimicrobial activity against all microorganisms.Thus, the ethanolic extracts were more potent compared to n-hexane and ethyl acetate extracts.From Table 1, the preliminary phytochemical studies reveal the presence of tannins, terpenoids, alkaloids and flavonoids in high concentration in ethanol extracts, which may contribute to the inhibition of microbial activities as observed.
The antimicrobial inhibition results of P. daemia extract buttress the literature on the plant usage to combat bacterial     and fungal infections such as sexually transmitted diseases and skin diseases (Kokwaro 1981).

Conclusion
The results show that P. daemia has a high content of flavonoids and other phenolic compounds but can only be efficiently extracted with polar solvents.The high content of phenols and flavonoids in the plant extracts contributed to the strong antioxidant activity exhibited by the plant.
Consequently, extracts of polar solvent showed more appreciable sensitivity against human pathogens as reported than the less polar extracts.In folk medicine, extraction is carried out with water or liquor (polar solvent), which is highly efficient in the extraction of the active ingredients from the studied plant.The results of this study present P. daemia as a potential plant for drug discovery.The plant is therefore recommended for further phytochemical analysis in order to isolate the active compounds responsible for the biological activities exhibited by the P. daemia.

FIGURE 1 :FIGURE 2 :
FIGURE 1: Estimated phenolic contents in the stem and leaf extracts of Pergularia daemia at 1 mg/mL, 5 mg/mL and 10 mg/mL of each extract.