A comparative in vitro study on antifungal and antioxidant activities of Nervilia aragoana and Atlantia monophylla

Abstract

Ethyl acetate extract of the whole plant of Nervilia aragoana Gaud. (Orchidaceae) and ethanol extract of the leaves of Atlantia monophylla Linn. (Rutaceae) were evaluated for antifungal and antioxidant activities. At 5 mg/mL concentration of the extracts, the former exhibited more inhibitory activity than the latter against fungi. The order of MIC values for Nervilia aragoana were Saccharomyces cerevisiae (1.4 mg/mL) > Aspergillus niger (1.2 mg/mL) > Aspergillus fumigatus (0.95 mg/mL) > Cryptococcus neoformans (0.75 mg/mL). In the case of Atlantia monophylla values were Cryptococcus neoformans (1 mg/mL) > Candida albicans (0.95 mg/mL) > Aspergillus niger (0.65 mg/mL). TLC-DPPH method assay was carried out to evaluate the antioxidant potential. Further DPPH radical, superoxide, nitric oxide, H₂O₂ scavenging, and reducing power activities were carried out. N. aragoana (85%) extract exhibited more scavenging activity than that of A. monophylla (66%) by DPPH free radical scavenging method. A. monophylla extract exhibited more superoxide, nitric oxide, H₂O₂ scavenging activities than that of N. aragoana. The acute toxicity studies of both extracts have shown no mortality rate even up to 3 g/kg body weight in albino rats. Screening for secondary metabolites showed the presence of carbohydrates in both extracts. Flavonoids were found only in the ethyl acetate extract of N. aragoana. Tannins, alkaloids, triterpenoids and steroids were present in A. monophylla. Total phenols present in N. aragoana and A. monophylla were 340 and 560 mg/g extract of gallic acid equivalents, respectively.

Keywords::

• Antifungal activity
• antioxidant activity
• acute toxicity
• Nervilia aragoana
• Atlantia monophylla

Introduction

Fungi are the eukaryotic, opportunistic pathogens mostly seen in immuno-compromised patients. Disseminated fungal infections are on the increase (Denning, 1991) and the therapeutic difficulties encountered in their management have prompted investigators and clinicians to be more creative in the development of antifungal drugs. Increased fungal infections are commonly observed in patients with immuno-suppressed conditions such as AIDS, many of whom are surviving longer with supportive therapy, and increased numbers of transplant patients as well as cancer patients undergoing aggressive chemotherapy (de Marie et al., 1994). Medicinal plants have been the basis for the treatment of various infectious diseases in traditional medicine, and possess a variety of compounds of known therapeutic properties (Tempone et al., 2008). Plant-derived compounds are gaining much importance in pharmaceutical and therapeutically applications as there are usually fewer side effects and are not very toxic. Much attention has been paid to plant-derived antifungal compounds, (Gurgel et al., 2005) based on the knowledge that plants have their own defense systems against fungal pathogens (Fontenelle et al., 2007; Wojtaszek, 1997).

Oxidative stress, induced by oxygen radicals, is believed to be a primary factor in various degenerative diseases such as cancer (Muramatsu et al., 1995), atherosclerosis (Steinberg et al., 1989), gastric ulcer (Das et al., 1997) and other conditions (Oliver et al., 1987). Antioxidants play an important role in inhibiting and scavenging radicals, thus providing protection to humans against infection and degenerative disorders. Antioxidants are compounds that help to inhibit many oxidation reactions caused by free radicals such as singlet oxygen, superoxide, peroxy radicals, hydroxyl radicals and peroxy nitrate, thereby preventing or delaying damage to the cells and tissues (Karthikumar et al., 2007). Although there are some synthetic antioxidant compounds such as butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT), which are commonly used in processed foods, it has been reported that these compounds may have side effects (Pourmorad et al., 2006; Witschi, 1981; Kehrer & DiGiovanni, 1990; Yamamoto et al., 1980).

Nervilia aragoana Gaud. (Orchidaceae) is distributed in hilly wet areas of Western Ghats of Kerala, Karnataka, Tamil Nadu and in some rare places of Andhra Pradesh (India).

Atlantia monophylla Linn. (Rutaceae) has been earlier reported for insecticidal activity against pests of agricultural importance (Sukumar et al., 1991; Sivagnaname & Kalyanasundaram, 2004).

The aims of the present study were to investigate: 1) antifungal activity; 2) in vitro antioxidant activity and 3) acute toxicity studies of ethyl acetate extract (whole plant) of N. aragoana and ethanol extract (leaves) of A. monophylla. This is probably the first scientific report on antifungal and antioxidant activities of both the plants.

Materials and methods

Plant collection

Nervilia aragoana was collected from Western Ghats of India during the month of September 2007 and authenticated by P. Sudhakaran, Curator at Raiyrath Nursery, Thrissur, Kerala. Leaves of Atlantia monophylla were collected from Tirumala hills of Eastern Ghats of India during the month of December 2007 and authenticated by N. Yasadomma, Department of Botany, S.V. University, Tirupati. The whole plant of N. aragoana and leaves of A. monophylla were shade dried, powdered and used in the present studies.

Solvent extraction

Whole plant powder (100 g) of N. aragoana was extracted by Soxhlet for 6-8 h with ethyl acetate, and the extract was distilled to remove the solvent. The yield of the ethyl acetate extract was 2.25 g and soluble in 10% DMSO.

Leaf powder (100g) of A. monophylla was placed in a Corning jar, 300 mL of ethanol was added and soaked for 48 h. After that the ethanol extract was collected and Rota vaporized at 40°C to drive off the ethanol. The yield was 9 g which was also soluble in 10% DMSO.

Fungal strains

Fungal cultures used in the present study were procured from IMTECH, Chandigarh, India. These include Candida albicans ATCC 10231 (MTCC 227), Aspergillus niger ATCC 16404 (MTCC 1344), Aspergillus fumigatus (MTCC 1811), Cryptococcus neoformans ATCC 32045 (MTCC 1347) and Saccharomyces cerevisiae ATCC 9763 (MTCC 171).

Antifungal activity

Antifungal activity of both the plant extracts was tested using the diffusion method according to Bauer et al. (1966) and NCCLS (2000). The fungal strains were maintained on potato dextrose agar (PDA) medium (Hi-Media). Sterile discs of Whatman No.1 filter paper of about 6 mm diameter were impregnated on the surface of the media. Different concentrations of both the extracts were prepared and applied on the discs and incubated for 48–72 h at 28°C. The results were recorded by measuring the inhibitory zone around the discs.

Minimum inhibitory concentration

Different concentrations from 0.05 mg to 2 mg/ml were prepared. The cells were adjusted so that 0.1 mL of culture contains 1x10⁸ CFU/mL. Inoculum (0.1 mL) was added to each test tube and incubated at 28°C for 48–72 h. The lowest concentration (highest dilution) of the extract that produced no visible signs of fungal growth (no turbidity) was regarded as the minimum inhibitory concentration (MIC).

Antioxidant activity

TLC-DPPH antioxidant screening

Each extract (10 µL) was loaded as a 1 cm band on the origin of TLC (Merck, Silica gel 60 F₂₅₄ plates). Plates were developed using methanol and ethyl acetate (5:5) for Nervilia aragoana and 100% methanol for Atlantia monophylla. To detect antioxidant activity, chromatograms were sprayed with 0.2% DPPH in methanol as an indicator (Deby & Margotteaux, 1970) until just wet, and dried in a fume hood. The presence of antioxidant compounds were detected as yellow spots against a purple background on TLC plates.

Free radical scavenging activity

Free radical scavenging activity was determined by using the 2,2′-diphenly-1-picrylhydrazyl (DPPH) method followed by Burits and Bucar (2000). One mL of various concentrations of the extracts in methanol was added to 4 mL of 0.004% methanol solution of DPPH. After a 30-min incubation period at room temperature, the absorbance was read against blank at 517 nm. The inhibition of free radicals by DPPH in percentage terms (I%) was calculated by using the following equation.

I % = [(A control – A sample)/A blank] × 100

where A control is the absorbance of the control reaction (containing all reagents except the test compound), and A sample is the absorbance of the test compound.

Superoxide anion scavenging activity

Measurement of superoxide anion scavenging activity of both plant extracts was carried out by the modified Liu et al. (1991) method (Gulcin et al., 2004). 1.5 mL of Tris-HCl buffer (pH 8.0, 32 mM), 0.5 mL of Nitroblue tetrazolium (NBT) (300 µM), 0.5 mL of nicotinamide adenine dinucleotide (NADH) solution of 468 µM and different concentrations of plant extracts (10-100 µg/mL) were taken. The reaction was initiated by the addition of 1 mL phenozine methosulphate (PMS) (30 µM) to the mixture. The reaction mixture was incubated at 25°C for 5 min and the absorbance was measured at 560 nm. Decreased absorbance of the reaction mixture indicates the increased superoxide anion scavenging activity. Ascorbic acid was used as a standard. The % of inhibition was calculated.

Nitric oxide scavenging activity

Nitric oxide scavenging activity was measured by the method of Marcocci et al. (1994). Nitric oxide radicals (NO) were generated from sodium nitroprusside. 1 mL of sodium nitroprusside (10 mM), 1.5 mL of phosphate buffer saline (0.2 M, pH 7.4) was added to the different concentrations of the both plant extracts and incubated for 150 min at 25°C. After incubation 1 mL of the reaction mixture was treated with 1 mL of Griess reagent (1% sulfanilamide, 2% H₃PO₄, and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromatophore was measured at 546 nm. Butylated hydroxyl toluene was used as a standard. The % of inhibition was calculated.

H₂O₂ scavenging activity

The H₂O₂ scavenging activity of both plant extracts was determined according to the method of Ruch et al. (1989). A solution of H₂O₂ (40 mM) was prepared in phosphate buffer (pH 7.4). Different concentrations of both plant extracts in 3.4 mL phosphate buffer were added to a H₂O₂ solution (0.6 mL, 40 mM). The absorbance value of the reaction mixture was recorded at 230 nm. The % of inhibition was calculated.

ABTS radical scavenging activity

The antioxidant activity of both the extracts was determined with stable ABTS⁺ cation radical method of Re et al. (1999). ABTS (2 mM) was prepared by dissolving in 50 mL of phosphate buffered saline (pH 7.4). ABTS⁺ was produced by reacting 50 mL of stock solution with 200 µL of 70 mM potassium per sulfate (K₂S₂O₈) water solution. The mixture was left to stand in the dark at room temperature for 15-16 h before use. For the evaluation of antioxidant activity, the ABTS⁺ solution was diluted with PBS to obtain the absorbency of 0.8 ± 0.03 at 734 nm. Different concentrations of both the extracts were prepared and mixed with 3 mL of ABTS solution. The absorbance was read at room temperature after 10 min at 734 nm. PBS solution was used as a blank sample. The percentage of inhibition was calculated.

Reducing power

The reducing power was determined according to the Oyaizu (1986) method. Different concentrations of both extracts were prepared in methanol mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe (CN) ₆] (2.5 mL, 1%). The mixture was incubated at 50°C for 20 min and 2.5 mL of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl₃ (0.5 mL, 0.1%). The absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid was used as a standard.

Screening for secondary metabolites

The qualitative analyses for screening of secondary metabolites performed according Harborne and Baxter (1995). Various tests were carried out to confirm the presence of phenols, carbohydrates, tannins, alkaloids, triterpenoids, and steroids.

Determination of total phenols

Total phenols in both plant extracts were determined by Folin-Ciocalteau reagent using gallic acid as standard (Singleton & Rossi, 1965). The absorbance was measured at 650 nm. The concentration of total phenols was expressed in terms of grams of gallic acid equivalents.

Acute toxicity studies

The Nervilia aragoana ethyl acetate extract and Atlantia monophylla ethanol extract were dissolved in 10% DMSO. The extract was orally administered to different group of male albino rats in doses ranging from 500 mg to 3 g/kg body weight for the LD₅₀ study using the method of Ghosh (1984). No mortality was observed in the groups even after 7 days treatment.

Results and discussion

Screening for antifungal and antioxidant activities of both plants was carried out with different solvents (complete data not presented). We have observed that ethyl acetate extract of Nervilia aragoana and ethanol extract of Atlantia monophylla were good in terms of antifungal and antioxidant activities.

Antifungal activity

Medicinal plants possess a variety of compounds of known therapeutic properties (Chopra et al., 1992; Harborne & Baxter, 1995; Ahmad & Beg, 2001). Hence, much attention has been paid to plant-derived antifungal compounds based on the knowledge that plants have their own defense system (Fontenelle et al., 2007). The therapeutic use of such plant products is an alternative strategy to prevent the spread of diseases (Elkovich, 1988). Both the plant extracts showed good antifungal activity. The ethyl acetate of Nervilia aragoana showed a wider range of antifungal activity on fungi used in the present study compared to the ethanol extract of Atlantia monophylla. At 5 mg/mL both the plant extracts exhibited antifungal activity. The results of antifungal activity of both plants are presented in Table 1. The following is the order of susceptibility of the test fungi, namely: C. neoformans (26 mm) > A. fumigatus (24 mm) > S. cerevisiae (16 mm) > A. niger (12 mm) > C. albicans (8 mm) at 5 mg/mL concentration in the case of N. aragoana. A. monophylla exhibited the order of susceptibility of the test fungi namely, A. niger (18 mm) > C. albicans (16 mm) > C. neoformans (14 mm). But A. fumigatus and S. cerevisiae have not shown susceptibility.

Table 1.  Antifungal activity and MIC values of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

Inhibitory zones (mm) and concentration (mg/mL)
	N. aragona						A. monophylla
Fungi	C	0.5	1	2	5	MIC*	0.5	1	2	5	MIC*
C. albicans	18	–	–	–	8	nt	8	11	14	18	0.95
A. niger	22	–	–	ft	12	1.2	10	13	15	18	0.65
A. fumigatus	–	–	12	14	16	0.95	–	–	–	–	nt
C. neoformans	12	–	14	18	26	0.75	9	12	14	16	1
S. cerevisiae	12	ft	8	10	16	1.4	–	–	–	–	nt

C, control (ketcanazole 10µg/disc); ft, faint traces; nt, not tested; MIC* values expressed as mg/mL.

MIC values of both the extracts were also represented in Table 1. In the case of N. aragoana, the order of MIC values were S. cerevisiae (1.4 mg/mL) > A. niger (1.2 mg/mL) > A. fumigatus (0.95 mg/mL) > C. neoformans (0.75 mg/mL). In the case of A. monophylla, the order of MIC values were, C. neoformans (1 mg/mL) > C. albicans (0.95 mg/mL) > A. niger (0.65 mg/mL).

Antioxidant activity

Results of TLC-DPPH chromatograms (forming yellow spots) indicated the presence of antioxidant compounds in both the plant extracts (Figures 1A and 1B). Free-radical reactions have been implicated in the pathology of many human diseases/disease conditions like atherosclerosis, ischemic heart disease, aging process, inflammation, diabetes, immune-suppression, neurodegenerative diseases, etc. (Maxwell, 1995; Droge, 2002; Beckman & Ames, 1998). The disturbance in “redox homeostasis” occurring when antioxidant defenses are inadequate can damage lipids, proteins, carbohydrates, and DNA. Drugs with multiple protective mechanisms, including antioxidant activity, may be one way of minimizing tissue injury (Halliwell, 1991).

Figure 1.  Screening of antioxidant activity by TLC-DPPH method (A) Ethyl acetate extract of N. aragoana and (B) Ethanolic extract of A. monophylla).

The hydrogen atom or electron donation abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-colored methanol solution of 2,2′-diphenly-1-picrylhydrazyl (DPPH). At 250 µg/mL concentration, the ethyl acetate extract of N. aragoana exhibited 85% of DPPH radical scavenging activity than ethanol extract of A. monophylla that exhibited 66% (Figure 2). Sanchez-Moreno et al. (1998) classified the kinetic behavior of antioxidant as follows: <5 min (rapid) 5–30 min (intermediate) and >30 min (slow). According to this classification both the extracts decolorizes rapidly within 5 min.

Figure 2.  DPPH free radical scavenging activity of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

It is well known that superoxide anions damage biomolecules directly or indirectly by forming H₂O₂, OH, peroxy nitrite or singlet oxygen during aging and pathological events such as ischemic reperfusion injury. Superoxide has also been observed to directly initiate lipid peroxidation (Yen & Duh, 1994). The superoxide scavenging activity of both extracts was increased markedly with the increase in concentrations. At 250 µg/mL, the inhibition of SOD activity was 65% in case of N. aragoana and 78% in case of A. monophylla (Figure 3).

Figure 3.  Superoxide scavenging activity of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

Nitric oxide is a diffusible free radical which plays many roles as an effector molecule in diverse biological systems including neuronal messenger, vasodilation and antimicrobial and antitumor activities (Miller et al., 1993). Nitric oxide inhibitors have been shown to have beneficial effects on some aspect of inflammation and tissue damage seen in inflammatory diseases. At 250 µg/mL, the inhibition of both extracts on nitric oxide activity was 25% (N. aragoana) and 42.5% (A. monophylla) (Figure 4).

Figure 4.  Nitric oxide scavenging activity of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H₂O₂ can probably react with Fe²⁺ and possibly Cu²⁺ ions to form hydroxyl radical and this may be the origin of many of its toxic effects (Halliwell & Gutteridge, 1993). It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. At 250 µg/mL the inhibition of H₂O₂ scavenging activity of A. monophylla was 75% and N. aragoana was 65% (Figure 5).

Figure 5.  Hydrogen peroxide scavenging activity of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

ABTS is a relatively stable free radical which involves in the direct generation of ABTS radical monocation without any involvement of intermediary cation. Here, the radical cation is formed prior to addition of the antioxidant test system, rather than the generation of the radical taking place continually in the presence of antioxidant. This method, used for the screening of antioxidant activity, is applicable to both lipophilic and hydrophilic antioxidants (Long et al., 2000). The free radicals were scavenged by concentration-dependent manner. Maximum scavenging activity observed at 250 µg/mL and minimum activity was found to be 10 µg/mL for both the extracts. At 250 µg/mL, the inhibition of ABTS scavenging activity was found to be 91% in Nervilia aragoana and 93% in Atlantia monophylla (Figure 6). Increased absorbance with the increased concentrations of the reaction mixture indicated the increased reducing power (Figure 7). The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity (Meir et al., 1995).

Figure 6.  ABTS scavenging activity of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

Figure 7.  Reducing power of ethyl acetate extract of N. aragoana and ethanolic extract of A. monophylla.

Acute toxicity test gives clues on the range of doses that could be toxic to the animal. It could also be used to estimate the therapeutic index (LD₅₀/ED₅₀) of drugs and xenobiotics (Rang et al., 1999). The acute toxicity studies have showed no mortality rate even after 7 days treatment with both the extracts up to 3 g/kg body weight.

Phytochemical analysis

Screening for secondary metabolites revealed the presence of carbohydrates and flavonoids in the ethyl acetate extract of Nervilia aragoana. Steroids, triterpenoids, alkaloids and tannins are present in the ethanol extract of Atlantia monophylla (data not presented). Flavonoids are known to be synthesized in plants in response to microbial infections; they have been found in vitro to be effective antimicrobial substances against a wide range of microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins. Tannins, which are present as secondary metabolites in plants, may exhibit their mode of antimicrobial action by inactivating microbial adhesions, enzymes, cell envelope transport protein and complex with cell wall (Cowan, 1999). Scalbert (1991) reviewed the antimicrobial properties of tannins. The mechanism of action of terpenes is not fully understood but it is speculated to involve membrane disruption by the lipophilic compounds. Heterocyclic nitrogen compounds are called alkaloids. Recent in vitro studies have shown activity against bacteria, specifically enteric pathogens, and some activity against Candida spp. (Sawer et al., 1995). The concentration of total phenols present in the N. aragoana and A. monophylla are 340 mg/g and 560 mg/g extract of gallic acid equivalents.

Conclusions

Nervilia aragoana and Atlantia monophylla possess distinct antifungal and antioxidant properties which have been attributed to the presence of polyphenolic and other phytochemical constituents. As the plants are being used in traditional medicine there is ample opportunity to examine how they cure diseases on a more scientific basis. Thus there is a need to isolate and identify the compounds and use them in modern medicine. Investigations are in progress towards this goal.

Acknowledgements

We also thank Dr. S.C. Basappa, former Deputy Director and Scientist, Central Food Technological Research Institute (CFTRI), Mysore (India), for his suggestions, encouragement and critical comments on the manuscript.

Declaration of interest

We thank the University Grants Commission, New Delhi for financial assistance.