Experimental material

The leaves and whole fruits of the wild and cultivated plants were dehydrated at 45 °C for 48 h, in a forced air oven (Novatech HS35-EA) and milled to a particle size of 0.5 mm in diameter. With 0.5 g of each sample of leaves and fruits separately, a mixture was prepared with 50 mL of methanol / water (80/20) in an ultrasonic bath (Branson B-200) for 20 min; of the extract, 2 mL were separated, portion that was filtered and centrifuged at 14 000 rpm for 5 min in a Labogen MINI centrifuge (^{Atanassova et al., 2011}).

Determination of phenols and total flavonoids

The content of total phenols was determined with the Folin-Cilocalteu method. One mL of each extract in triplicate was mixed with 9 mL of HPLC grade water and one mL of the Folin-Ciocalteu reagent. After 5 min, 10 mL of sodium carbonate (Na₂CO₃) was added at 7 % (w / v) and at the end the mixture was adjusted to 25 mL with HPLC grade water. They were incubated in the dark for 90 min at room temperature and the absorbance was read at 750 nm in a JENWAY model 6320D spectrophotometer. Different concentrations of gallic acid (0, 20, 40, 60, 80 and 100 mg L^-1) were used as standard to calculate the content of total phenols, expressed as mg of gallic acid equivalents (EAG) 100^-1 g of sample in dry weight (mg EAG 100 g^-1 PS) (^{Atanassova et al., 2011}).

The total content of total flavonoids was analyzed by the aluminum chloride (AlCl₃) method, for which an aliquot was taken in triplicate of one mL of the extract and mixed with 4 mL of distilled water and 0.3 mL of 5 % NaNO₂. (p / v). After 5 min, 0.3 mL of 10 % AlCl₃ and six minutes later, 2 mL of a 1M of sodium hydroxide (NaOH) solution were added and complemented to a total volume of 10 mL with water. It was mixed on a vortex shaker (OHAUS OHS-30392115) and the absorbance was read at 510 nm in a JENWAY 6320D model spectrophotometer. Different concentrations of (+) catechin (0, 20, 40, 60, 80 and 100 mg L^-1) were used for the realization of a standard curve to calculate the total flavonoid content expressed as mg equivalents of catechin per 100^-1 g of dry weight (mg EC 100 g^-1 PS) (^{Atanassova et al., 2011}).

Determination of antioxidant capacity

The antioxidant activity was evaluated using two different methods. The activity of inhibiting free radicals with DPPH, according to ^{Atanassova et al. (2011)} using DPPH (1,1-diphenyl-2-picrylhydrazyl), 0.004% (in methanol, w / v), the extracts (n = 3) were diluted at different concentrations (100, 200 and 500 μmL mL^-1) and mixed with 4 mL of DPPH, in the dark. After 60 min of incubation in the dark and at room temperature, the absorbance was read against a blank (only reagents without sample with methanol) at 517 nm in a JENWAY 6320D UV-VIS spectrophotometer. The antioxidant activity was expressed as a percentage (%) of inhibition of free radicals by DPPH, according to the following equation:

The evaluation of the capacity of donation of hydrogen atoms was determined by the ORAC method (^{Ou et al., 2001}) with some modifications. It is one of the most accepted techniques for the measurement of antioxidant capacity, since it measures the oxidation inhibition induced by peroxide radicals, generated by the thermal decomposition of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which reacts with a fluorescent probe (fluorescein), to form a colorless non-fluorescent product. A Trolox calibration curve was designed as a reference standard in concentrations of 0.02 to 3 μM of Trolox ((M ETx mL^-1). The antioxidant activity was calculated from the following equation:

Obtained from the Trolox calibration curve.

The assay was carried out in a 96-well microplate reader (Tecan infinite M200PRO Fluorometer) where 20 μL of the extracts or standard were placed (Trololox 5 mM), 120 μL of fluorescin (120 nM) were added. ) and AAPH (2,2-azobis (2-methylpropionamidine) dihydrochloride) 40mM, were incubated in a kiln (Novatech HS35-EA) at 37 °C for 30 min and the readings were recorded with excitation 485 nm and λ emission 538 nm respectively with 70 cycles each 5 minutes. The protective effect of the antioxidant was calculated using the differences of areas under the decay curve of the fluorescein between the blank and the sample. The area under the curve (AUC) was calculated as follows:

Where:

f₀ = Reading of the initial fluorescence at minute 0

f₁ = Fluorescence reading at time i.

The net AUC was obtained by subtracting the AUC from the AUC blank of the sample:

Results are expressed in μM equivalent of Trolox in 100 g of dry weight (μM ETx 100 g^-1 PS).

Statistical analysis

The results were expressed as the mean ± standard deviation; in addition, with the Microsoft Excel 2016 for Mac program, the polyphenol content data were compared with the antioxidant activity to know if there was a correlation between these values.

Content of phenols and total flavonoids

In Table 1 it can be observed that the wild plants had higher content of phenolic compounds (196.46 (0.02 mg EAG 100 g^-1 of PS) both in leaves and in fruits, in comparison with the cultivated ones (23.58 (0.12 EAG 100 g^{- 1} of PS). In fruits, the values obtained were 9.44 in wild and 6.21 in cultivated plants. The same happens with the total polyphenols; the flavonoids were obtained in greater quantity in the leaves and fruits of wild plants (148.52 (0.1 and 23.1 ± 0.01 mg EC 100 g^-1 of PS, respectively) than in those cultivated (6.01 and 3.79 respectively) (Table 1).

Antioxidant activity by DPPH

By this method, a higher percentage of free radical blocking was observed in leaves of cultivated plants than in wild plants, despite the fact that the content of polyphenols and flavonoids was higher in leaves of the latter (Table 2). The antioxidant activity improved with increasing concentrations, both in leaves and in fruits. The highest value corresponded to 500 μL mL^-1 with 11.5 ± 0.4 % and 8.28 ± 0.3 %, for cultivated and wild plants, respectively. The other concentrations (200 and 100 μL mL^-1) were 6.45 ± 0.3 and 4.19 ± 0.2 for the cultivated plants and 3.91 ± 0.1 and 2.14 ± 0.1 in wild plants.

In the fruits, the free radical blockage percentages were similar in both types of plants (Table 3), and, as in the leaves, the higher concentration of the samples increased the antioxidant activity; at 500 μL mL^-1 the highest of 61.78 ± 1.2 and 62.69 ± 1.5 %, respectively, were recorded; in 200 μL mL^-1 were 20.7 ± 0.9 and 17.42 ± 0.6, and 100 (μL mL^-1), the lowest with 9.86 ± 0.4 and 8.22 ± 0.3 %.

Antioxidant activity with the ORAC-Fluorescein assay (ORAC-FL

The highest values were found in leaves and fruits of the wild plants of 1 396 ± 15.5 and 342 ± 10 μM ETx 100 g^-1, respectively, in comparison with the cultivated plants ±156 ± 5 and 297 ( 8 μM ETx 100 g^-1 for leaves and fruits, respectively (Figure 1).

Hojas de plantas silvestres = Leaves of wild plants; Frutos de plantas silvestres = Fruits of wild plants; Hojas de plantas cultivadas = Leaves of cultivated plants; Frutos de plantas cultivadas = Fruits of cultivated plants.

Figure 1 Oxygen radical absorption capacity (ORAC) μM equivalents of Trolox 100 g^-1 PS in leaf and fruit of Physalis chenopodifolia Lam. in wild and cultivated plants. 

Phenols and flavonoids

The values of total phenols in the leaves of the wild plants of Physalis chenopodifolia (196.46 mg of EAG 100 g^-1 PS) are higher than those of the leaves of species with medicinal use in Poland (0.15 to 15.15 mg of EAG 100 g^{- 1} PS) (^{Wojdylo et al., 2007}); of Bulgaria, with values of 27.94 to 48.9 mg de EAG 100 g^-1 PS (Attanasova et al., 2011), and of some plants of traditional Chinese medicine such as Pinellia ternata (Thunb.) Makino, with 12 mg of EAG 100 g^-1 PS, up to 166 mg of EAG 100 g^-1 PS in Trichosanthes kirilowii Maxim (^{Song et al., 2010}), with the same Folin-Ciocalteu test and gallic acid as standard. Likewise, of edible vegetables, considered as important sources of phenols such as asparagus, cabbage, carrot, celery, squash, garlic, lettuce, onion, radish, spinach, chili, tomato and beet with values between 13.5 to 154.1 mg 100 g^-1 PS (^{Ninfali et al., 2005}).

However, they are inferior to other commercial medicinal plants reported by ^{Yoo et al. (2008)}, with values of 464.2 to 844.4 mg 100 g^-1 PS and by ^{Pourmorand et al. (2006)} in medicinal plants of Iran with 3 100 to 28 950 mg 100 g^-1, as well as some other popular ones in Serbia for therapeutic ethnobotanical use in diseases such as diabetes, rheumatism, urinary tract, ulcerations, hypertension, arteriosclerosis, for example, with values from 960 to 6 200 mg of EAG 100 g^-1 (^{Žugić et al., 2014}).

Compared to other Solanaceae, the species under study has a lower content than Solanum madrense Fernald, as described by ^{Fernández y Ruiz (2017)}, which reached leaf values of 202 to 377 mg EAG 100 g^-1. In other Physalis species, such as P. patula Mill, P. subulata L., P. solanacea L. and P. hederifolia A. Gray var. hederifolia, ^{Medina-Medrano et al. (2015)} estimated a leaf content of 58.75 to 12 90.06 mg EAG g^-1 and established that the most common are phenolic acids, quercetin and glycosylated kaempferol, determined by high pressure chromatography (HPLC).

The polyphenols of Physalis chenopodifolia fruits are similar to commercial fruits such as Musa cavendish L. (7 mg EAG 100 g^-1 (^{Singh et al., 2016}) but inferior to mango fruits (78.3), guava (87.5), papaya (88.2) and dates (304.24 to 563.71) (^{Amira et al., 2012}; ^{Patthamakanokporn et al., 2008};), as well as fruits from Ecuador from 25 to 2 167) and Physalis peruviana from different localities of Peru (106 to 149.3 mg EAG 100 g^-1) (^{Jurado et al., 2016}; ^{Vasco et al., 2008}).

The content of flavonoids in the leaves of the species of interest was above those of the leaves of spices and medicinal plants according to ^{Ninfali et al. (2005)}, ^{Žugić et al. (2014)} and Attanasova et al. (2011). Likewise, the figures of the present study are higher to those corresponding to edible vegetables (7.0 to 89.1 mg EC 100 g^-1) (^{Ninfali et al., 2005}).

However, they are inferior to spices and medicinal herbs according to ^{Žugić et al. (2014)}, ^{Ninfalli et al. (2005)} and ^{Yoo et al. (2008)}, whose values are go from 600 to 1 321.2 mg EC 100 g^-1. The same thing is extensive for other Solanaceae such as Solanum madrense (389 to 531 mg EC 100 g^-1) (^{Fernández and Ruiz, 2017}) and other Mexican species of Physalis, with leaf values of 6.543 to 21.265 mg of EC g^-1 of sample (^{Medina-Medrno et al., 2015}).

The antioxidant activity of herbs, fruits, food supplements and beverages is usually analyzed with in vitro tests and generally in relation to the content of polyphenols by traditional methods, and only one technique is applied. The most common methods to measure this capacity are DPPH and ORAC, because they are very easy to reproduce, but they also show significant differences in their response to antioxidants (^{Roy et al., 2010}).

Antioxidant activity, DPPH

In general, samples with a higher polyphenol or flavonoid content have greater antioxidant activity (^{Žugić et al., 2014}), which was not reflected in the samples of the present study; however, other compounds with antioxidant activity have been reported, such as the phyisalines considered to be the main metabolite in this genus. ^{Hernández (2015)} refers to antioxidant activity with DPPH in leaves of Physalis peruviana, so it would be interesting to evaluate the content of phyisalines in these samples.

The values obtained in the leaves are superior to other Solanaceae such as Solanum ferrugineum Jacq., on which 10 % is calculated at a higher concentration (1 000 mL^-1) (^{Medina-Medrano et al., 2016}), but less than the leaves of commercial and medicinal herbs such as Eucalyptus globulus Labill. and Chamaemelum nobilis L. with values of 60.1 and 91 % at a concentration of 100 μL mL^-1 also analyzed with DPPH (^{Yoo et al., 2008}). In leaves of Solanum madrense, ^{Fernández and Ruiz (2017)} estimated 90 % at 500 μL mL^-1, by means of DPPH.

At 500 μL mL^-1 in the samples of the studied fruits, the numbers are higher than those recorded in other Solanaceae such as Solanum ferrugineum (with 15 % at a concentration of 1 000 μg mL^-1) (^{Medina-Medrano et al., 2016}) and Physalis peruviana (42.22 % a 800μg mL^-1) (^{Jurado et al., 2016}).

Antioxidant activity, ORAC-Fluorescein

Unlike the obtained with the DPPH technique, the leaves of cultivated plants showed greater antioxidant capacity; this can be explained by the content of total polyphenols and flavonoids, since there is significant evidence that ORAC values are strictly dependent on the content of phenols and flavonoids; furthermore, while ORAC techniques measure the capacity to reduce oxygen radicals induced by peroxide radicals, DPPH quantifies the percentage of free radical uptake in general (^{Ninfali et al., 2005}).

The ORAC values of the leaves of wild plants of Physalis chenopodifolia are higher than those of some medicinal plants such as Petroselium hortensis L. (13 018 μmol ET 100 g^-1) and Rosmarinus officinalis L. (29 032 μmol ET 100 g^-1). The same happens with some edible vegetables such as chili, tomato, onion squash, carrot, lettuce and radish with values of 252 to 1 240 μmol ET 100 g^-1 (^{Ninfali et al., 2005}), but inferior to other medicinal ones also according to ^{Ninfali et al., 2005}) with values that reach more than 32 000 μmol ET 100 g^-1.