Anti-inflammatory, antioxidant and antifungal activity of Chuquiraga spinosa

Abstract

Context: Stem and leaves infusion of Chuquiraga spinosa (R&P) Don. (Asteraceae) is used in the Peruvian traditional medicine for its anti-inflammatory properties and for the treatment of vaginal infections.

Objective: This study evaluated the antioxidant, anti-inflammatory and antifungal activities of C. spinosa for the first time.

Materials and methods: Extracts of methanol, 50% methanol and water were obtained from C. spinosa aerial parts. Antioxidant activity of the extracts was evaluated (DPPH˙, ABTS˙⁺ and superoxide radical-scavenging activity). The correlation between these results and total polyphenolic content was determined by Pearson’s Correlation Coefficient. Anti-inflammatory activity of 50% methanol extract was evaluated with the rat model of carrageenan-induced acute inflammation and mouse model of TPA-induced acute inflammation. The antifungal activity of the extracts against Cladosporium cucumerinum and Candida albicans was studied by direct bioautography, and antifungal activity against phytopathogenic fungi was performed by culture in potato dextrose agar plates.

Results: All the extracts showed high antioxidant activity, and there was correlation between the activity and total polyphenolic compounds. As 50% methanol extract was administered orally, the paw edema in rats was reduced significantly (52.5%). This extract, by topical administration, produced a reduction of 88.07% of the edema TPA-induced in ear of mice. The aqueous and 50% methanol extracts were active against C. albicans (minimum inhibitory concentration of 2.5 and 6.25 µg, respectively). The aqueous extract showed antifungal activity against C. cucumerinum (MIC: 2.5 µg).

Discussion and conclusion: Preliminary phytochemical screening and the analysis of the three extracts by high-performance liquid chromatography diode-array detection showed the majority compounds are flavonoids and phenolic acid derivatives. These compounds may be responsible of the radical-scavenging activity of these extracts as well as responsible of anti-inflammatory effect in vivo of 50% methanol extract. Several authors have demonstrated the antioxidant and anti-inflammatory properties of some flavonoids and phenolic acids. The antifungal activity of the extracts obtained from aerial parts of C. spinosa has been investigated here for the first time. Other studies are necessary to determine the mechanism of action and to identify the bioactive compounds of this plant.

Keywords::

• Chuquiraga spinosa
• antifungal
• anti-inflammatory
• antioxidant
• flavonoid
• phenolic acid

Introduction

Peru is one of the 12 mega-diverse countries of the world, and it has 5354 endemic species (Brack, 1999). The Peruvian flora offers great possibilities for the discovery of new compounds bioactives and as source of lead compounds in drug development. Traditional medicine based on the use of medicinal plants is an excellent source of information in this area of research. In Peru, just as in many countries of the world, traditional medicine has represented and still represents an important role in the primary attention of the health (Bussmann & Sharon, 2006; De la Cruz et al., 2007).

Chuquiraga spinosa (R&P) Don. (Asteraceae), commonly called huamanpinta or care-sirve, grows in the Andes between 2650 and 4500 m of altitude. Stem and leaves infusion is used for the treatment of gonorrhea and vaginal infections (Girault, 1990; De Feo, 1992). In addition to this use, it is employed for its anti-inflammatory properties (Rojas et al., 2003). Seventy components were detected by GC and GC-MS in the essential oil (Senatore, 1996). Senatore et al. (1999) described the presence of a p-hydroxyacetophenone and three flavonol glycosides in the methanol extract of the leaves: kaempherol-3-O-glucoside, kaempherol-3-O-rutinoside and quercetin-3-O-rutinoside. In addition to these flavonoids, in a previous work carried out in our department, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, kaemperol-3-O-glucuronide, isorhamnetin-3-O-glucuronide, isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-glucoside were identified (Landa et al., 2009). Some of these flavonoids are present in other species of the genus Chuquiraga and this fact suggests that flavonoids are useful phylogenetic markers in this genus (Juárez & Mendiondo, 2002).

Flavonoids and phenolics compounds show a significant role as antioxidants and they have become object of multiples investigations for nutritional and therapeutic interest. Antioxidants provide protection against damage caused by an uncontrolled production of free radicals and oxidative stress associated with several human diseases (Montoro et al., 2005; Ismail et al., 2010).

In this study, 50% methanol extract of C. spinosa was evaluated for the therapeutic potential as anti-inflammatory agent. At the same time, in our research, based on the discovery of new antifungal agents from higher plants, the extracts of C. spinosa were evaluated against human pathogenic yeast Candida albicans and phytopathogenic fungi C. cucumerinum, Botrytis cynerea, Alternaria alternata, Penicillium expansum and Rhizopus stolonifer.

Materials and methods

Chemicals and reagents

All chemicals and biochemicals were purchased from Sigma-Aldrich (St. Louis, MO). Solvents for analysis were obtained from Panreac (Barcelona, Spain) and solvents for mobile phases were from Merck (Darmstadt, Germany).

Plant material and preparation of the extracts

Aerial parts of C. spinosa were collected in May of 2001 in the north of Yauyos (Peru) and the specie was identified by Luz Amanda Vivas (Agronomist Engineer). A voucher specimen (number 43) was deposited in the Herbarium of the Rural Institute Valle Grande, placed in San Vicente de Cañete (Peru), and plants were dried at room temperature. An amount of 500 g of pulverized plant material was macerated three times for 2 days at room temperature with 500 mL of different solvents: methanol, 50% methanol and water. The extracts were dried in a rotary evaporator and then were lyophilized (Virtis BT3-SL, NY). The dry extracts were stored in glass vials at −40°C until tested and analyzed. The yields of methanol, 50% methanol and aqueous extracts were 18.02, 17.40 and 17.82%, respectively.

Phytochemical screening

The phytochemical screening of powdered aerial parts of C. spinosa was performed to identify the presence or absence of different compounds: alkaloids with Mayer and Dragendorff’s reagents; flavonoids with AlCl₃/HCl; tannins with 1% gelatin and 10% NaCl solutions; cardiac glycosides with FeCl₂ and H₂SO₄; cyanogenic glycosides with picrate paper; terpenoids with the Liebermann-Burchard method, anthraquinones with the Borntrager reaction and saponins with the ability to produce foam (Trease & Evans, 1983; Khandelwal, 2004).

Analysis of extracts by HPLC-DAD

The extracts were analyzed using a HPLC system Waters (Milford, MA) equipped with a separations module (Waters 2695) with column heater and degasser online, photodiode array detector (Waters 2996) and Empower Software. Separation was achieved using a reversed-phase column C18 Nova-Pak Waters (150 × 3.9 mm, 4 µm) at temperature of 25°C. DAD detection was employed at the wavelength range between 210 and 500 nm. Samples were dissolved in the corresponding solvent of the extract at 10 mg/mL followed by filtration using Millipore^® (unit type HV 0.45 µm). The volume of sample injected was 10 µL. The mobile phase was a mixture of acetonitrile (A) and water (B) containing 0.5% (v/v) acetic acid and the flow rate was 1 mL/min. The elution system was in mode gradient: 0–15 min, 90–80% of B; 15–20 min, 80–75% of B; 20–30 min, 75–70% of B.

Quantification of total polyphenols

Total polyphenols content of the three extracts was determined using a method, based on Prussian Blue method, described by Pueyo and Calvo (2009) for the quantification of these compounds using 96-well microplates.

Antioxidant activity

DPPH˙ radical-scavenging activity

The antioxidant activity of the extracts was determined in terms of hydrogen donating or radical-scavenging ability, using the stable radical DPPH˙ (2,2-diphenyl-1-picrylhydrazyl) according to the method described by López et al. (2007). The assay was carried out using 96-well plates and absorbance was recorded in the Power Wave XS microplate reader (KC Junior BioTek Program) at 25°C. A 150-µL aliquot of samples or control was mixed with 150 µL of methanol solution of DPPH˙ (4 mg/mL). The decrease in the absorbance values was measured at 517 nm at 30 min. The activity was evaluated according to the formula: radical-scavenging activity (% RSA) = (Ac − As)/Ac × 100, where As was the absorbance of control and Ac was the absorbance of sample. The percentage of inhibition was plotted versus the concentration of the samples measured and results are finally expressed as IC₅₀ values, estimated using GraphPad Prism v.4 Program. BHT (3,5-di-tert-butyl-4-hydroxytoluene) was used as positive control. All experiments were performed several times.

ABTS˙⁺ radical-scavenging activity

Assay was performed according the method described previously (García-Iñiguez de Ciriano et al., 2009). The chromogenic radical ABTS˙⁺ [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] was prepared by reaction of 7 mM aqueous ABTS˙⁺ solution and 2.45 mM potassium persulfate solution. Diluted ABTS˙⁺ radical solution (200 µL) was added to each well into 96-well microplates to react with 20 µL of stock solutions of extracts (1000–1 µg/mL in methanol) or control (methanol). Readings were recorded after 6 min at 734 nm and results are expressed as IC₅₀. All measurements were performed three times.

Superoxide anions scavenging activity

Superoxide radicals were generated by a hypoxanthine/xanthine oxidase enzymatic system and radical-scavenging activity was assayed according the method described by Valentão et al. (2001) with some modifications. In the sample wells (96-well microplate), 75 µL of 145 µM xanthine, 75 µL of 50 µM NBT (nitro blue tetrazolium chloride), and 75 µL of extracts were mixed, and all of them were dissolved in 50 mM buffer phosphate. The reaction started by adding 75 µL of xanthine oxidase (0.29 unit of enzyme/mL of 50 mM buffer phosphate) and the absorbance was measured at 560 nm after 2 min of incubation at room temperature. Results are expressed as data of IC₅₀. All measurements were performed three times.

Anti-inflammatory activity

Animals

For in vivo experiments, we used a group of ten male Wistar rats (150–200 g) and male albino Swiss mice (25–30 g) from the Center for Applied Pharmacobiology Research of the University of Navarra. Housing conditions were five animals per cage at room temperature of 22°C with free access to pellet chow and tap water ad libitum. Animals were allowed to acclimatize for 1 week before the experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Navarra.

Rat model of carrageenan-induced acute inflammation

An edema was induced on the right hind foot of rats by subplantar injection of 0.1 mL of a solution of 3% carrageenan in 1.5% (w/v) saline solution (Winter et al., 1962). The reference group was treated with indomethacin (10 mg/kg, p.o.). A control group was treated only with the vehicle. Doses of 250 and 500 mg/kg of MeOH50 extract were dissolved in carboxymethylcellulose 1% and were treated orally 30 min before carrageenan injection. The volumes of the injected and contralateral paws were measured 1, 2, 3 and 4 h after the induction of inflammation using a plethysmometer (Ugo Basile), and the edema was expressed as an increase in paw volume due to carrageenan injection. The anti-inflammatory activity was calculated at each time of observation, as percent inhibition of edema in the animals treated with the substances under testing in comparison to control animals.

Mouse model of TPA-induced acute inflammation

Inflammation was induced through topical application of 2.5 µg of TPA (12-O-tetradecanoylphorbol-13-acetate) dissolved in 20 µL of acetone on the right ear (Tonelli et al., 1965). This was applied by an automatic pipette in 10 µL volumes to both anterior and posterior surfaces of the right ear. The left ear (control) received the same volume of acetone. Indomethacin was used as a reference drug (0.5 mg/ear). MeOH50 extract of C. spinosa was applied in three doses of 2.5 mg/ear each every 30 min; the first dose was applied at the beginning of the experiment. The system of successive doses has been used in order to work in a similar way to the folk medicine. Inflammation was allowed to develop for 4 h, after which the animals were killed by cervical dislocation and a section of the central portion of both ears was obtained and weighed. The swelling was assessed in terms of the mean weight increase of each ear, while inhibition of the swelling was expressed as weight reduction in comparison to the control group treated with TPA only (Tubaro et al., 1986). Details of the protocol have been previously described by Calvo et al. (1998).

Antifungal activity

Direct bioautography with C. cucumerinum and C. albicans

Geometric dilutions of extracts were prepared in an appropriate solvent, and 10 µL of these solutions was applied on the TLC plates. It was evaluated that a range of concentrations from 100 to 1 µg of each extract and nystatin was used as a reference compound.

Direct bioautography with C. cucumerinum was as follows. After application of the samples on silica gel 60 F₂₅₄ Albacked sheets (Merck), the TLC plates were developed using appropriate solvent systems and thoroughly dried for complete removal of solvents. The plate was sprayed with a suspension of spores of C. cucumerinum in a nutritive medium and incubated for 2–3 days in polystyrene boxes with a moist atmosphere. Clear inhibition zones appeared against a dark grey background.

Direct bioautography with C. albicans was as follows. After application of the samples on a silica gel 60 F₂₅₄ glass-backed plate (Merck), the TLC plate was developed using appropriate solvent systems and thoroughly dried for complete removal of solvents. An inoculum of yeast in malt agar was prepared and spread over the TLC plate. The plates were incubated overnight at 30°C and then sprayed with methylthiazolyltetrazolium chloride. Active compounds appeared as clear spos against a purple-colored background.

Fungal cultures of A. alternata, Botrytis cinerea, P. expansum and R. stolonifer.

Fungi were obtained from “Colección Española de Cultivos Tipo” (Department of Microbiology, University of Valencia). In order to obtain microorganism growth, potato dextrose agar (PDA) was used, and the details of this method have been previously described by Casanova et al. (2008).

Statistical analysis

The relationship between total polyphenolic content of the extracts and their antioxidant activity was determined by Pearson’s Correlation Coefficient. Statistical analysis of results of anti-inflammatory activity was performed by one-way analysis of variance test followed by Dunnett’s test to compare data of the extract-treated groups and control group. Differences in all tests were considered significant if the probability value was p ≤ 0.05 and high significance if p ≤ 0.001.

Results

Preliminary phytochemical screening of aerial parts of C. spinosa showed the presence of flavonoids and phenolic acid derivatives, while alkaloids, saponins, tannins, cardiac glycosides, cyanogenic glycosides, terpenoids and anthraquinones were absent. Reversed-phase column C18 and mobile phase used by high-performance liquid chromatography diode-array detection (HPLC-DAD) were appropriate to the characterization of MeOH, MeOH50 and aqueous extracts. Figure 1 shows chromatograms at 254 nm of these extracts, which contain flavonoids and phenolic acid derivatives in different proportions. Chromatographic analysis showed that aqueous extract had the highest number of phenolic acids derivatives while MeOH extract presented the greater diversity of flavonoids. The composition of MeOH50 extract was intermediate one between both extracts. MeOH extract showed the highest content of total phenolics compounds (Table 1).

Table 1.  Total polyphenols content and free radical-scavenging activity of the extracts of Chuquiraga spinosa.

Extract/antioxidant	Total polyphenols content*	DPPH˙ radical-scavenging activity^†	ABTS˙^+ radical-scavenging activity^†	Superoxide radical-scavenging activity^†
MeOH	12.59 ± 4.06	36.51 ± 1.93	40.91 ± 2.83	10.91 ± 0.95
MeOH50	6.25 ± 1.24	21.67 ± 1.47	20.04 ± 1.28	8.54 ± 0.48
Water	5.37 ± 0.56	29.35 ± 1.22	30.49 ± 1.59	9.58 ± 0.95
BHT	—	5.05 ± 0.63	5.22 ± 0.75	2.25 ± 0.43

*Total polyphenols content is expressed as mean ± SEM (n = 12) of milligrams of gallic acid per 100 mg of extract.

^†Radical-scavenging activity is expressed as IC₅₀ (µg/mL).

Figure 1.  Chromatograms of the extracts of Chuquiraga spinosa by HPLC-DAD registered at 254 nm. (A) phenolic acid; (B) flavonoid.

The three extracts showed high antioxidant activity (lower data of IC₅₀), and the most active was MeOH50 extract (Table 1). Significant correlation was observed between total phenolic content and DPPH˙ radical-scavenging activity (r² = −0.885), ABTS˙⁺ radical-scavenging activity (r² = −0.848) and superoxide anion inhibition activity (r² = −0.855).

Only MeOH50 extract was chosen to evaluate the anti-inflammatory activity because the chemical composition of this extract is intermediate between the other extracts. The effect on carrageenan-induced edema is shown in Table 2. In control group, the subplantar injection of carrageenan produced a local edema in the following 30 min that increased progressively to reach a maximal intensity 3 or 4 h after injection of the phlogistic agent. A pre-treatment with MeOH50 extract showed different results according to the dose used. MeOH50 extract in a dose of 500 mg/kg significantly reduced the edema 2 h after carrageenan injection, the maximal inhibition (52.5%). In the third hour, the inhibition was 45.45% in comparison with the control group. MeOH50 extract in a dose of 250 mg/kg was ineffective. Indomethacin showed a clear inhibition of the inflammation induced compared with the control group. The anti-inflammatory activity caused by topical application of MeOH50 extract in the model of TPA-induced mouse ear edema is shown in Table 3. The anti-inflammatory effect of MeOH50 extract (88.07%) was higher than the effect of indomethacin (74.83%) 30 min post-dosing at dose level tested.

Table 2.  Anti-inflammatory effect of MeOH50 extract of Chuquiraga spinosa on rat paw edema.

Sample	Dose (mg/kg)	Increase in paw volume^† (percentage of inhibition of edema)
		1 h	2 h	3 h	4 h
Control group	-	0.28 ± 0.08	0.40 ± 0.04	0.66 ± 0.08	0.74 ± 0.05
Indomethacin	10	0.27 ± 0.02 (3.57%)	0.22 ± 0.09* (45%)	0.35 ± 0.02** (46.97%)	0.29 ± 0.05** (60.81%)
MeOH50 extract	250	0.28 ± 0.18 (0%)	0.38 ± 0.11 (5.0%)	0.52 ± 0.07 (21.11%)	0.69 ± 0.12 (7.02%)
MeOH50 extract	500	0.22 ± 0.08 (21.42%)	0.19 ± 0.05** (52.5%)	0.36 ± 0.08** (45.45%)	0.50 ± 0.08* (32.43%)

^†Increase of paw volume is expressed in milliliters as mean ± SEM (n = 10); statistical differences were considered significant between treated group and control group *p ≤ 0.05, **p < 0.01.

Table 3.  Anti-inflammatory effect of MeOH50 extract of Chuquiraga spinosa on mouse ear edema.

Sample	Dose (mg/ear)	Weight of edema^† (mg)	Percentage of inhibition
Control	—	27.97 ± 3.54	—
MeOH50 extract	2.5 (×3)*	3.33 ± 1.00**	88.07
Indomethacin	0.5	7.04 ± 2.97**	74.83

*Three doses of 2.5 mg/ear in intervals of 30 min.

^†Values of weight of edema are expressed as mean ± SEM (n = 10); differences were considered significant between treated group and control group **p < 0.01.

In this study, extracts of C. spinosa were screened for their antifungal activity against C. albicans using the bioautographic method (Table 4). The aqueous and MeOH50 extracts were active with a minimal inhibitory amount of 2.5 and 6.25 µg, respectively.

Table 4.  Antifungal activity of the extracts of Chuquiraga spinosa.

Extract/antifungal compound	Candida albicans*	Cladosporium cucumerinum*	Rhizopus stolonifer^†	Penicillium expansum^†	Botrytis cinerea^†	Alternaria alternata^†
MeOH extract	>>10	>10	18.49	0	19.76	0
MeOH50 extract	6.25	>10	13.52	n.d.	n.d.	n.d.
Aqueous extract	2.5	2.5	4.58	0	0	0
Nystatin	1	0.2	n.d.	n.d.	n.d.	n.d.

*Minimum inhibitory concentration of extract (µg) to inhibit fungi growth on silica gel TLC plate.

^†Percentage of inhibition at dose of 250 ppm.

n.d., not determined.

The result of the antifungal assay against C. cucumerinum showed that aqueous extract of C. spinosa was the only active with a minimal amount of 2.5 µg. The mycelia growth assays in PDA plates demonstrated that aqueous extract was the less active against the phytopathogen fungi tested. None of the extracts showed activity against A. alternata and P. expansum (percentage of inhibition lower than 10%), whereas MeOH extract showed inhibition against R. stolonifer and B. cinerea (20.81 and 19.76%, respectively).

Discussion

In the present work, three different methods were used successfully for the evaluation of the antioxidant activity of the extracts: DPPH˙ radical-scavenging assay, ABTS˙⁺ assay and superoxide radical-scavenging activity. Several publications have reported the relationship between a high phenolic content and antioxidant activity (Ismail et al., 2010), and this correlation was confirmed in this study.

In general, the topical administration of an anti-inflammatory agent at the site of inflammation is the most effective treatment. In general, extracts are more active when they are administered topically than when they are given orally (Recio et al., 1994). The reason can be related to the ability of the extracts to penetrate the skin of the ear. This finding suggests that MeOH50 extract of C. spinosa may be a therapeutic agent for the treatment of inflammation in skin diseases. It has been reported that flavonoids and phenolic acids have anti-inflammatory effects. Flavonoids may interact directly with prostaglandin system and various phenolic acid derivatives may exert their anti-inflammatory action through inhibition of superoxide radical generation (Panthong et al., 1989; Lee et al., 2005). For these reasons, flavonoids and phenolic acids present in MeOH50 extract of C. spinosa may be responsible of anti-inflammatory activity.

C. albicans is one of the most commonly encountered human pathogens and causes variety of infections in mucosal skin and systemic infections in individuals with impaired immunity (Nielsen & Heitman, 2007). Few classes of drugs are effective against these fungal infections, and they have limitations with regard to efficacy and side-effects. As there are few really effective antifungal preparations currently available for the treatment of systemic mycoses and as the efficacy of existing drugs is rather limited, it is important to find new sources of antifungal agents. Plant-derived natural products may offer potential leads for novel agents which act against these mycoses. In this paper, it has been demonstrated that is possible to find anti-inflammatory and antifungal activities in the same extract. MeOH50 extract of C. spinosa can be an important candidate for treatment of Candida infections.

C. cucumerinum has been known as an important phytopathogen that causes scab disease in cucumber all over the world (Lee et al., 1997). R. stolonifer, A. alternata, P. expansum and B. cinerea cause decay in stoned fruits, particularly peaches, but also in strawberries, raspberries and grapes (Northover & Zhou, 2002). One of the main problems of modern agriculture is the postharvest fruit losses due to pathogen’s attack and natural senescence during storage. Storage under controlled conditions and the use of synthetic pesticides are not free from problems due to human health risks and environmental effects. New strategies to solve these problems consists of developing methods to improve the natural plant resistance by using the plant’s own defense molecules to control the pathogen’s attack and applying new natural pesticides (Gonzalez et al., 2003). The antifungal activity of the extracts obtained from aerial parts of C. spinosa has been investigated here for the first time.

Conclusions

Extracts of C. spinosa aerial parts have shown high radical-scavenging activity. Furthermore, it has been demonstrated an anti-inflammatory effect using 50% methanol extract of this plant in models of acute inflammation in vivo. These results give the first step in order to confirm the validity of the traditional use of this plant. Further studies need to be performed to determine the mechanism of action and to identify the bioactive compounds. Antioxidant and antifungal activity can be interesting for applications in food conservation, storage products and pharmaceutical and cosmetic industry.

Declaration of interest

University of Navarra Foundation is thanked for the financial support. We also thank Government of Navarra and Alumni Navarrenses Association for the fellowships.