Streszczenie

Wprowadzenie: Stres oksydacyjny jest zaangażowany w patogenezę wielu chorób przewlekłych, toteż wzra­sta zainteresowanie naturalnymi antyoksydantami pochodzącymi z roślin w odniesieniu do ich potencjalnych korzyści zdrowotnych. Rośliny z rodzaju Physalis są bogate w metabolity wtór­ne i wykazują znaczny potencjał antyoksydacyjny. Postęp w badaniach nad transformacją ge­netyczną otworzył nowe możliwości w zwiększaniu produkcji metabolitów wtórnych w kultu­rach komórek i organów. Zregenerowane z korzeni transformowanych rośliny Physalis ixocarpa porównano z roślinami nietransformowanymi hodowanymi in vitro i ex vitro pod kątem poten­cjału antyoksydacyjnego.
Materiał/Metody: Całkowity potencjał antyoksydacyjny (TAC), całkowitą zawartość fenoli i askorbinianu mierzo­no w owocach, kwiatach, liściach i korzeniach P. ixocarpa odpowiednio, na zasadzie redukcji jonów żelaza (FRAP), z odczynnikiem Folin-Ciocalteu i metodą z 2,2′-dipyrydylem.
Wyniki/Dyskusja: Profil antyoksydacyjny wyznaczony na podstawie TAC, arkorbinian i fenole zależał od organu i warunków hodowli. Wydaje się, że całkowita zawartość fenoli, a nie poziom askorbinianu okre­ślają TAC w badanych ekstraktach. Wodne ekstrakty wykazywały mniejszą aktywność antyoksy­dacyjną w porównaniu do ekstraktów acetonowych, co wskazuje, że lipofilne antyoksydanty mają większy udział w całkowitym potencjale antyoksydacyjnym w tkankach roślin. Transformacja za pośrednictwem Agrobacterium zmieniała status antyoksydacyjny w odniesieniu do TAC, fe­noli i askorbinianu, co było widoczne zarówno w roślinach hodowanych in vitro, jak i ex vitro.

Słowa kluczowe:Physalis ixocarpa • całkowity potencjał antyoksydacyjny • askorbinian • fenole • transformacja za pośrednictwem Agrobacterium

Summary

Introduction: Oxidative stress is involved in pathogenesis of a number of chronic diseases hence is an incre­asing interest in plant-derived natural antioxidants with respect to their potential health benefits. Plants from the genus Physalis are particularly rich in secondary metabolites and show signifi­cant antioxidant potential. Recent development in transgenic research has opened new possibi­lities for enhanced production of secondary metabolites with plant cell and organ cultures. The hairy root-regenerated Physalis ixocarpa plants grown in vitro and ex vitro were compared to the non-transformed plants with respect to their antioxidant potential.
Material/Methods: The total antioxidant capacity (TAC), the contents of total phenols and ascorbate were evaluated in fruits, flowers, leaves and roots of P. ixocarpa using the ferric reducing antioxidant power as­say (FRAP), the Folin-Ciocalteu method and the 2,2′-dipyridyl method, respectively.
Results/Discussion: The antioxidant profiles, in terms of TAC, ascorbate and phenols were organ-specific and depen­ded on the culture conditions. Neither the total phenol content nor the ascorbate level appeared to determine the TAC of the studied plant extracts. The aqueous extracts exhibited lower antioxi­dant activities than the acetone ones indicating that lipophilic antioxidants made a major contri­bution to TAC of the plant tissues. Agrobacterium rhizogenes-mediated transformation changed the antioxidant status with respect to TAC, phenols and ascorbate and this effect was observed in the plants grown in vitro and ex vitro.

Key words:Physalis ixocarpa • całkowity potencjał antyoksydacyjny • askorbinian • fenole • transformacja za pośrednictwem Agrobacterium

Introduction

Plants from the genus Physalis (family Solanaceae), nati­ve to warm and subtropical regions of Middle and South Americas, are particularly rich in secondary metabolites. Phytochemical analyses showed that they contain witano­lides, physalins, calystegines, tropane and nortropane al­kaloids [32,22]. Physalis species have also shown signifi­cant antioxidant potential [6,24]. Due to the large biological activities of these compounds, Physalis plants were used for centuries as medicinal herbs in the treatment of uri­nary and skin diseases, gonorrhea, ulcers, sores and as a vermicidal drug, and recent studies have confirmed their therapeutic properties [31,15]. Physalis spp. extracts have been also reported to possess anticancer activities [16,6].

Out of the secondary metabolites found in the genus Physalis, the physalins have been most extensively screened for their biological effects [2,16,8] whereas antioxidants have been assessed less intensely. However, as oxidative stress is invo­lved in pathogenesis of a number of chronic diseases inclu­ding cancer, cardiovascular and neurodegenerative disorders [23,26], there is an increasing interest in plant-derived natu­ral antioxidants with respect to their potential health bene­fits. In plants, ascorbate and phenolic compounds contribu­te mainly to the total antioxidant potential of plant extracts and may be the basis for their biological activity.

Recent development in transgenic research has opened new possibilities for enhanced production of secondary metabo­lites with plant cell and organ cultures. It has been found that Agrobacterium-mediated transformation affected the content of secondary metabolites as rol genes act as potent activators of secondary metabolism [5]. Thus the use of transformed root cultures (hairy roots) which are establi­shed by transformation with A. rhizogenes is a promising approach to intensify the production of secondary metabo­lites. Furthermore, differentiated plants regenerated from the hairy roots can be also used for the production of bio­active metabolites in good yields. However, there are only a few reports on secondary metabolite production in hairy root-regenerated medicinal plants [27]. Additionally, most of the data did not refer to the plant growth stage and or­gan specificity of the metabolite profiles.

The aim of this study was to compare the hairy root-re­generated Physalis ixocarpa plants grown in vitro and ex vitro to the non-transformed plants with respect to their antioxidant potential in terms of total antioxidant capaci­ty (TAC) and the contents of total phenols and ascorbate.

Materials and Methods

Plant material

Non-transformed Physalis ixocarpa Brot. ex Hornem (syn. Physalis philadelphica Lam.) plants were obtained from in vitro germinated seeds and cultured in vitro on solidified Murashige and Skoog [19] basal medium with 0.7% (w/v) agar and 3% sucrose (w/v), supplemented with 5 µM kine­tin and 1 µM 6-benzyladenine for shoot induction and with 1 µM 1-naphthaleneacetic acid for rooting. Cultures were grown under 16 h/8 h photoperiod (40 µmol m^-2 s^-1 cool fluorescent light, Philips 40W) at 23±2°C. Transformed plants were obtained by regeneration from hairy roots trans­formed with Agrobacterium rhizogenes ATCC 15834 and cultured in vitro under the same conditions as described above. For acclimatization to ex vitro conditions at day 28 non-transformed and transformed plants were washed in sterile distilled water to remove traces of MS medium and transferred to pots containing perlite under the growth chamber conditions (23±2°C, 16 h/8 h photoperiod, 70% humidity and 40 µmol m^-2 s^-1 light intensity) and covered for 7 days with glass. After 14 days the plants were trans­ferred to pots filled with soil and grown under the control­led growth chamber conditions as described above. Plants cultivated in vitro were used for experiments at the age of 4 weeks and plants grown ex vitro were analyzed 8-10 we­eks after having been transferred to soil.

Total antioxidant capacity

For extraction of water-soluble and acetone-soluble antio­xidants 500 mg of fresh plant material (roots, leaves, flo­wers and fruits) was homogenized (1:10) in 0.1 M phosphate buffer (pH 7.4) and acetone, respectively. Then the mixtu­res were centrifuged and supernatants were used for me­asurements of the total antioxidant capacity of water-solu­ble (TACw) and acetone-soluble antioxidants (TACa). TAC was measured by using a modified FRAP (Ferric Reducing Ability of Plasma) assay [3]. This method depends upon the reduction of ferric 2,4,6-tripyridyltriazine (Fe (III)-TPTZ) complex to the ferrous 2,4,6-tripyridyltrazine (Fe (II)-TPTZ) by a reductant at low pH, monitored at 593 nm. Briefly, the FRAP reagent was prepared fresh daily from 300 mM ace­tate buffer at pH 3.6, 10 mM TPTZ (2,4,6-tripyridyltriazi­ne) solution in 40 mM HCl and 20 mM FeCl3×6 H2O solu­tion in proportion of 10:1:1 (v/v), respectively. Plant extracts (50 µl) were allowed to react with the FRAP reagent for 30 min in darkness. The absorbance of the reaction mixture was than recorded at 593 nm. The antioxidant capacities of the extracts were expressed as mg Trolox equivalents per gram of fresh plant material (mg g^-1 FW).

Determination of total phenols

For the determination of total phenols plant material (0.5 g FW) was homogenized in 5 cm³ ice-cold 80% aqueous methanol. Total phenol content was measured by a modi­fied Folin-Ciocalteu assay [29]. Briefly, methanolic extract (0.1 cm^-3) was mixed with 3.8 cm^-3 distilled water, 0.1 cm^-3 Folin-Ciocalteu reagent and 1 cm^-3 10% Na₂CO₃. The mi­xture was incubated for 1 h at room temperature and then the absorbance at 725 nm was measured. Calibration curve was prepared with chlorogenic acid and total phenol content was given as chlorogenic acid equivalents in µmol g^-1 FW.

Determination of ascorbate content

For the determination of ascorbate content plant material (0.5 g FW) was homogenized in 5 cm³ ice-cold 5% tri­chloroacetic acid. Ascorbate was determined spectropho­tometrically by the method described by Kampfenkel et al. [13]. Total ascorbate was estimated after reduction of de­hydroascorbate (DHA) to reduced ascorbate (AA) with di­thiothreitol. The concentration of ascorbate (µmol g^-1 FW) was determined using a calibration curve for AA as a stan­dard. Redox ratio for ascorbate was calculated as AA/DHA.

Statistical analysis

The results presented are the means of three independent experiments. Sample variability is given as the standard deviation of mean.

Results and Discussion

Hairy root-regenerated plants of P. ixocarpa grown in vitro showed phenotype features typical for plants transformed with A. rhizogenes, i.e. reduced apical dominance, short internodes and wrinkled leaves. The hairy root-regenera­ted plants grown ex vitro showed normal morphology, ho­wever the plant height and leaf area were reduced when compared with the non-transformed culture-derived plants (data not shown).

It is well known that in plants the profiles of biologically active compounds with potential health benefits, e.g. antio­xidants, depend on genetic, developmental and environmen­tal factors [14,34]. These aspects need to be also considered with respect to antioxidant production in plants in vitro-cultures [17]. In our study the antioxidant tests based on a FRAP assay revealed that the differentiated A. rhizogenes-transformed plants regenerated from hairy roots and cultu­red in vitro exhibited the highest TAC, with 1.753 TE g^-1 FW for roots and 7.43 TE g^-1 FW for leaves. In the non­-transformed plants grown in vitro the TAC of the root and leaf extracts were 39% and 64% lower, respectively. The lo­west TAC (0.783 TE g^-1 FW) was found in the hairy roots (Table 1). In general, the aqueous extracts exhibited lower antioxidant activities than the acetone ones indicating that under in vitro conditions the lipophilic antioxidants made a major contribution to TAC of the plant tissues. The lo­west TACw/TACa index was found in the roots of trans­formed plants (Table 1).

Table 1. Total antioxidant capacity (TAC) of water-soluble (TACw) and acetone-soluble (TACa) antioxidants in hairy root cultures and in roots and leaves of non-transformed and Agrobacterium rhizogenes-transformed Physalis ixocarpa plants cultured in vitro

The phenolic content of plants greatly contributes to their antioxidant potential and many studies have demonstrated the radical scavenging properties of plant phenolic com­pounds and confirmed the relationship between phenolic compounds and antioxidant activity [35]. However, some authors could not found such a relationship [18]. In this study no strong correlation between TAC and total phe­nol concentration was observed in cultures in vitro. The rank of total phenol content was as follows: leaves of non­-transformed plants > leaves of transformed plants > roots of non-transformed plants > hairy roots > roots of trans­formed plants (Table 2) and it was not similar to the TAC rank (Table 1). It is likely that not all phenols are reducing agents active in the FRAP assay. Moreover, this correla­tion does not consider the qualitative and quantitative dif­ferences in the phenolic profiles among plant tissues. It has been proposed that the specific antioxidant capacity (TAC expressed on phenolics basis), could be a reliable indica­tor of the effectiveness of neutralization of free radicals by the specific mixture of phenolic compounds present in the tissue [11]. In our study transformation increased the specific antioxidant capacity in plants grown in vitro indi­cating the presence of phenolics with higher antioxidant capacity to stabilize free radicals when compared to the phenolic profiles present in the tissues of non-transformed plants (Fig. 1). The phenol content was organ-dependent, as its concentration in leaves was markedly higher than in roots and hairy roots (Table 2). A similar relationship was also found for ascorbate (Table 2). The latter correspon­ded to the fact that the highest level of ascorbate synthe­sis takes place in the leaves [30], and ascorbate levels in roots are usually low compared to leaves [9]. In our study, under in vitro conditions, the highest ascorbate content of 7.629 µmol g^-1 FW was assayed for the leaves of transfor­med plants while their roots had the lowest ascorbate con­centration of 1.166 µmol g^-1 FW (Table 2).

Table 2. Total phenol, reduced (AA), oxidized (DHA) and total ascorbate contents and ascorbate redox ratio (AA/DHA) in hairy root cultures and in roots and leaves of non-transformed and Agrobacterium rhizogenes-transformed Physalis ixocarpa plants cultured in vitro

Figure 1. Specific antioxidant capacity in organs of non-transformed and Agrobacterium rhizogenes-transformed Physalis ixocarpa plants cultured in vitro and grown ex vitro. Data are expressed as the mean ± standard deviation (n=3). The specific antioxidant capacity was defined as the ratio of total antioxidant capacity over total phenolics (Trolox mass equivalents per mass of chlorogenic acid)

Figure 2. Total antioxidant capacity (TAC) of water-soluble (TACw) and acetone-soluble (TACa) antioxidants in organs of non-transformed and Agrobacterium rhizogenes-transformed Physalis ixocarpa plants grown ex vitro. Data are expressed as the mean ± standard deviation (n=3)

Ascorbic acid is one of the most abundant and effective an­tioxidants in plant tissues. It is involved directly in elimi­nating reactive oxygen species, regenerating vitamin E in plants and it also participates in cell metabolism and signa­ling [20]. The antioxidant potential of ascorbate depends not only on its content but also on the redox status of its pool. Our results indicated that the ascorbate-dependent redox environment was kept reduced as the total ascorba­te pool was dominated by AA (Table 2). However, the in­creased AA content and diminished DHA concentration in the leaves and roots of the transformed plants as compared with the non-transformed ones resulted in higher ascorba­te redox state expressed as the AA/DHA ratio (Table 2). In the leaves of transformed plants the high ascorbate level corresponded to the increased activity of the water-solu­ble antioxidants, whereas in the non-transformed plants it did not (Tables 1, 2). These results confirmed that under in vitro conditions leaves of the transformed and non-transfor­med plants differed in their antioxidant profiles. As gene­tic transformation of different Physalis species for the pro­duction of secondary metabolites has been reported [1,2] these data might be important for the in vitro biosynthe­sis of antioxidants from Physalis cultures.

In Physalis grown ex vitro the antioxidant characteristics varied between the transformed and non-transformed plants, but mostly in relation to organs. Leaves had the hi­ghest TAC of 5.314 and 3.764 TE g^-1 FW for non-trans­formed and transformed plants grown ex vitro, respecti­vely (Fig. 2). The lowest values of TAC, 1.289 and 0.654 TE g^-1 FW, were determined in flowers of the non-trans­formed plants and fruit extracts of the transformed ones. In flowers and roots, however the TAC values were higher in the transformed plants (Fig. 2). Similar relationships be­tween the transformed and non-transformed plants were found with respect to the antioxidant capacities of water- and acetone-extractable antioxidants, except the TACw of flower extracts which was higher in the non-transfor­med plants (Fig. 2). The lower TAC of the leaf and fruit extracts in A. rhizogenes-transformed plants could be at­tributed to the transformation-induced changes in the an­tioxidant profile. Similarly to the proportions described for in vitro cultures, the acetone extracts exhibited higher an­tioxidant capacity than the aqueous ones and the lowest ra­tio of TACw to TACa was found in the roots (Fig. 2). The highest concentration of phenolic compounds was found in the flowers and the lowest in the fruits (Fig. 3). In the generative organs (fruits, flowers) the levels of phenolics were higher in the transformed plants whereas in the vege­tative organs (leaves, roots) their contents were similar to those in the non-transformed ones (Fig. 3). As it has been found that high rates of biomass production had negative effects on the respective production of secondary metabo­lites [7], the lower content of phenols in the fruits and flo­wers in the non-transformed plants could be attributed to more intensive growth of these organs when compared to the transformed ones (data not shown).

Figure 3. Total phenols content in organs of non-transformed and Agrobacterium rhizogenes-transformed Physalis ixocarpa plants grown ex vitro. Data are expressed as the mean ± standard deviation (n=3)

Phenols are often recognized as the compounds with major relevance in the TAC of fruits and vegetables [21,35]. In P. peruviana, elagic acid was suggested to be the major com­ponent contributing to the antioxidant activity of the who­le-plant aqueous extracts [6]. Taking into account that the fruits of P. ixocarpa (tomatillos) are popular dietary pro­ducts traditionally used as ingredients in sauces in some countries in Latin America [4,32], it is worth noting that in fruits from A. rhizogenes-transformed plants the content of total phenols was 4.5-fold higher than in the respective organs of non-transformed plants (Fig. 3). However, the in­creased level of phenols did not correspond to TAC (Fig. 2) and to specific antioxidant capacity (Fig. 1) indicating the importance of other antioxidant constituents in the fruits of transformed plants, or the negative effects of changes in the phenolic pool on its antioxidant activity. The structure­-activity relationship studies have revealed that the degree of glycosylation and hydroxylation affects the antioxidant properties of phenolic compounds [10]. Thus the antioxi­dant capacity of a given plant extract cannot be predicted on the basis of the total phenolic content, as was observed also in the present study. Similar results have been repor­ted for a large number of plant material extracts of Finnish origin evaluated with respect to their total phenolic con­tent and antioxidant activity [12].

In Physalis plants grown ex vitro the content and redox ratio of ascorbate was organ-specific. The leaves of both trans­formed and non-transformed plants showed high AA con­tent (4.84 µmol g^-1 FW and 4.24 µmol g^-1 FW, respectively) and the highest AA/DHA redox ratio (Fig. 4). The general­ly high level of ascorbate accumulation in plant leaves can be explained by its constitutive role in photosynthesis [20]. Intriguingly, we observed that the flowers of non-transfor­med plants contained approximately 2 times the levels of AA and total ascorbate found in the leaves and the flowers of transformed plants maintained approximately the same content of ascorbate as the leaves (Fig. 4). Moreover, the flowers of transformed and non-transformed plants showed differential accumulation of DHA (Fig. 4) and thus diffe­red significantly with respect to their redox ratio of ascor­bate (17.8 versus 2.1). Ascorbate was the least abundant in the fruits followed by roots (Fig. 4). Transformation cau­sed marginal difference in the ascorbate level in the roots. In the fruits, however the contents of AA and DHA were markedly higher in the transformed than in non-transfor­med plants. As an opposite relationship was found with re­spect to TACw, these data could indicate that in the fruits of transformed plants ascorbate did not account for the acti­vity of water-soluble antioxidants. The contribution of AA to total antioxidant power in aqueous extracts of popular fruits ranged from 1 to 50% [33]. Taking into account the literature data revealing high variability of AA content in fruits [25,33], its level determined in Physalis fruits appe­ared to fall within the lower range. Thus, tomatillos can­not be recommended as a rich dietary source of vitamin C.

Figure 4. Concentration of reduced (AA), oxidized (DHA) and total ascorbate contents and ascorbate redox ratio (AA/DHA) in organs of non-transformed and Agrobacterium rhizogenes-transformed Physalis ixocarpa plants grown ex vitro. Data are expressed as the mean ± standard deviation (n=3)

Conclusions

In conclusion, our study provided the first indication of differential TAC and accumulation of phenols and ascor­bate in the organs of A. rhizogenes-transformed and non­-transformed Physalis plants grown in vitro and ex vitro. The antioxidant profiles, as far as TAC, AA and phenols were concerned, were organ-specific and depended on the in vitro and ex vitro culture conditions. Neither the total phenol content nor the ascorbate level appeared to deter­mine the TAC of the studied plant extracts. Transformation changed the antioxidant status in terms of TAC, phenols and ascorbate and this effect was observed in the plants grown in vitro and ex vitro. As phytochemicals of medicinal plants have been receiving increased interest, this prelimi­nary study contributes to the characteristics of secondary metabolites with antioxidant activity in the genus Physalis.

Total antioxidant capacity

For extraction of water-soluble and acetone-soluble antio­xidants 500 mg of fresh plant material (roots, leaves, flo­wers and fruits) was homogenized (1:10) in 0.1 M phosphate buffer (pH 7.4) and acetone, respectively. Then the mixtu­res were centrifuged and supernatants were used for me­asurements of the total antioxidant capacity of water-solu­ble (TACw) and acetone-soluble antioxidants (TACa). TAC was measured by using a modified FRAP (Ferric Reducing Ability of Plasma) assay [3]. This method depends upon the reduction of ferric 2,4,6-tripyridyltriazine (Fe (III)-TPTZ) complex to the ferrous 2,4,6-tripyridyltrazine (Fe (II)-TPTZ) by a reductant at low pH, monitored at 593 nm. Briefly, the FRAP reagent was prepared fresh daily from 300 mM ace­tate buffer at pH 3.6, 10 mM TPTZ (2,4,6-tripyridyltriazi­ne) solution in 40 mM HCl and 20 mM FeCl₃×6 H₂O solu­tion in proportion of 10:1:1 (v/v), respectively. Plant extracts (50 µl) were allowed to react with the FRAP reagent for 30 min in darkness. The absorbance of the reaction mixture was than recorded at 593 nm. The antioxidant capacities of the extracts were expressed as mg Trolox equivalents per gram of fresh plant material (mg g^-1 FW).

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The authors have no potential conflicts of interest to declare.

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