Introduction

Deterioration of food quality occurs during processing and storage and it is related to oxidative processes and microorganisms. Oxidative degradation affects lipids mainly, but also carbohydrates, proteins and nucleic acids. Many additives have long been used by food industry to preserve their products from oxidation, usually synthetic antioxidants such as butylhydroxianisole (BHA) and butylhydroxitoluene (BHT), with no clear effects on human health after chronic consumption [1, 2].

Many plants are sources of compounds with antioxidant activity that might be used as natural preservatives [3–5]. In addition, antioxidants play a special role in human healthcare and in last years prevention of cardiovascular diseases and cancer has been associated with the intake of fruits and vegetables, rich in natural antioxidants [6, 7]. Reactive oxygen species (ROS) are a class of highly reactive molecules derived from the oxygen and generated by metabolic processes in human beings and some external factors such as pollution, radiation or some dietary habits. ROS can cause DNA mutation, protein oxidation and lipid peroxidation, contributing to the development of atherosclerosis [8–9], inflammation [10], neurodegenerative diseases [11, 12], cataracts [13, 14], cancer [15] and aging.

On the other hand, fruits and vegetables suffer from infections during harvesting and packing. Rhizopus stolonifer, Alternaria alternata, Penicillium expansum and Botrytis cinerea are reported to cause decay in stoned fruits, particularly peaches, but also in strawberries, raspberries and grapes [16]. The disease normally develops after harvest, during transportation and as the fruit ripen prior to consumption. The post-harvest losses are fastly increased by the rapid spread of the fungus to adjacent fruits during ripening because the pathogen is not efficiently controlled by registered fungicides and treatments [17].

In the attempt to reduce the use of chemicals due to the concern about human health and environmental pollution, new alternative control approaches are being developed as the use of medicinal and aromatic plant extracts. Plants contain secondary metabolites, some of them with antimicrobial properties [18, 19].

Verbena officinalis, commonly called verveine, grow widespread in all temperature regions of the globe and finds use in folk medicine as diuretic, expectorant and anti-rheumatic. It is listed in the Chinese Pharmacopoeia and the British Herbal Pharmacopoeia. In Navarra (Spain), it is used extensively in traditional medicine mainly because of its anti-inflammatory properties [20]. Preliminary pharmacological studies indicate anti-inflammatory activities of the chloroform and methanolic extract of the plant in the carrageenan paw oedema model [21]. In a previous study we reported that 50% methanolic extract from the leaves of V. officinalis L. showed significant topical anti-inflammatory and analgesic properties in experimental models [22, 23]. The main components of V. officinalis L. are triterpenic acids and sterols [21], iridoids [24, 25], caffeoyl derivatives [26], and flavonoids [27].

The aim of this work was to analyze the antioxidant and antifungal activity of V. officinalis by in vitro approaches. The antioxidant capacity was measured by DPPH method and the antifungal activity by inhibition of in vitro cell cultures of A. alternata, B. cinerea, P. expansum and R. stolonifer.

Materials and Methods

Plant Material

V. officinalis L. (Verbenacea), leaves were collected in June 2001, at Ilundain (Navarra, Spain). A voucher is deposited in the Botany Department’s Herbarium, at the Faculty of Pharmacy, University of Navarra, Pamplona, Spain, and was authenticated by Dra. M.L. López.

Tested Material

Crude chloroform, ethyl acetate and 50% methanolic extracts of V. officinalis L. were obtained by 24-h maceration at 4 °C. Fifty percent of methanolic extract was fractionated by Sephadex® LH-20 to afford five fractions. The analysis of them by HPLC [27] showed the presence of iridoids in fraction I, flavonoids in fraction II and caffeoyl derivatives in fraction IV. Further purifications of fractions II and IV with Sephadex® G-25 afforded three flavonoids (luteolin-7-glucoside (1), luteolin 7-diglucuronide (2), apigenin 7-glucuronide (3)), and two phenolic acids (chlorogenic acid (4) and verbascoside (5); Fig. 1). All crude extracts, fractions I, II, IV and pure compounds (1–5) were tested for antioxidant and antifungal activity.

Fig. 1
figure 1

Chemical structure of the active constituents of Verbena officinalis leaves. Glc Glucuronide, Glu glucose

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Antioxidant Activity

Among the radical scavenging assays, one based on the utilization of DPPH was chosen due to its simplicity and worldwide acceptance for comparative purpose. DPPH method is independent of the substrate polarity. This method is based on the reduction of alcoholic DPPH solutions at 517 nm in the presence of an antioxidant compound that donates hydrogen or electron. Non-radical form DPPH-H is formed. The procedure of Brand-Williams et al. [28] has been adapted for evaluation of the free radical scavenging capacity of the tested material. One milliliter of several concentrations of each crude extract and pure compounds was added to 4 ml of 0.004% methanolic solution of DPPH. After 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. Inhibition of free radical by DPPH in (%) was calculated:

$${\text{Inhibition}}\left( {I\% } \right) = \left( {A_{{\text{blank}}} - {{A_{{\text{sample}}} } \mathord{\left/{\vphantom {{A_{{\text{sample}}} } {A_{{\text{blank}}} }}} \right.\kern-\nulldelimiterspace} {A_{{\text{blank}}} }}} \right) \times 100$$

Where A blank is the absorbance of the control reaction (containing all reagents except the test compound), and A sample is the absorbance of the test compound. IC50 was calculated using nonlinear regression module of Statistica 5.0 as a concentration exposing 50% of maximum activity. The antioxidant activity was compared with the activity of natural rutin, l-ascorbic acid and butylated hydroxyanisole (BHA). The results are given as a mean ± standard deviation (SD) of experiments done in triplicate.

Antifungal Activity

The fungal microorganisms were A. alternata, B. cinerea, P. expansum and R. stolonifer. These fungi cause different infections in many plant crops and stored products. The fungi were obtained from “Colección Española de Cultivos Tipo” edited by Department of Microbiology (University of Valencia).

In order to obtain microorganism growth, potato dextrose agar (PDA) was used (200 g potato juice, 20 g d(+)-glucose, 15 g agar, and 1,000 ml distilled water). Czapex Dox Agar was used for the determination of antifungal effect (30 g sucrose, 3 g sodium nitrate, 0.5 g magnesium sulfate, 1 g potassium hydrogen phosphate, 13 g agar, and 1,000 ml distilled water). One hundred fifty milliliter culture medium (Czapex Dox Agar) was prepared for each sample. Prepared agars were sterilized at 121 °C for 20 min in autoclave. Two hundred fifty parts per million of each tested material was added into sterilized agar medium. These mediums were poured into Petri dishes after being shaken. After keeping the agars in room conditions for one night fungi colonies with a 0.5 cm diameters corkpore were taken from the disks. Petri dishes were incubated at 24–25 °C. Assays were carried out in triplicates and with control samples. Benzoic and gallic acids were used as positive controls at the same concentration.

In order to evaluate the inhibition of mycelia growth of fungi by samples, the fungi colonies diameters were measured since the day of incubation with Leica Q Win Program (v.2.2.) and converted into percentage of inhibition (I%) according to the following formula: [(Control − Treatment / Control)] × 100.

Results and Discussion

Antioxidant Activity

The DPPH radical scavenging activity decreased in order 50% methanol > ethyl acetate > chloroform (Fig. 2). Phytochemical investigations of 50% methanolic extract showed the presence of iridoids, caffeoyl derivatives as well as flavonoid compounds [22].

Fig. 2
figure 2

Free radical scavenging capacities of the Verbena officinalis extracts measured in DPPH assay

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After fractionation of 50% methanolic extract, DPPH assay clearly indicated that the highest shown by the fraction which contained flavonoids and caffeoyl derivatives (Table 1), and the results (2.20 and 7.23 μg/ml, respectively) were comparable to those of rutin (4.8 μg/ml). Therefore, flavonoids and caffeoyl derivatives might have been the active principles responsible for the antioxidant activity of V. officinalis.

Table 1 Free radical scavenging activity (expressed as EC50) of Verbena officinalis
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The isolated compounds from V. officinalis showed a similar DPPH antiradical activity when compared with rutin, but much higher activity than ascorbic acid and BHA. In that it concerns to the flavonoids, luteolin 7-diglucuronide is the compound with more activity of the three-isolated (5.86 ± 1.8). The antioxidant activity decreased when the substitution is a glucose molecule (13.12 ± 2.1). Apigenin 7-glucuronide presents an intermediate antioxidant activity (10.95 ± 2.3). In a first approach with these results it is possible to establish that the substitution with one or several glucuronic acid molecules is related to the antioxidant activity of the flavonoids. Both caffeoyl derivatives (chlorogenic acid and verbascoside) presented high antioxidant activity in DPPH assay.

Antifungal Activity

The effect of three different extracts of V. officinalis on the mycelial growth of four plant pathogen fungi is presented in Fig. 3. None of the extracts showed significant inhibition against A. alternata and B. cinerea (<10%). The 50% methanolic crude extract was the most active against P. expansum and R. stolonifer (32.55% and 28.98%, respectively). The inhibition was more pronounced in the caffeoyl derivative fraction against P. expansum (87.45%) and R. stolonifer (79.11%), being even higher than pure compounds (Table 2). These results suggest a possible synergism between the caffeoyl derivatives present in the extract.

Fig. 3
figure 3

Antifungal effects of extracts against phytopathogenic fungi

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Table 2 Antifungal activity of three fractions of 50% methanolic extract and pure compounds (1–5)
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Although most of the natural products in agricultural area are used against insects, there are several reports on the effects of essential oils against food and cereal storage fungi, leaf pathogens, and soil-borne fungi [29, 30]. However, there are few studies on the effects of plant extract and pure natural products, so the antioxidant and antifungal abilities of caffeoyl derivatives are of particular interest for applications within the food, storage products and cosmetic industries.