Dynamic of Phenolic Compounds, Antioxidant Activity, and Yield of Rhubarb under Chemical, Organic and Biological Fertilization

In recent years, rhubarb is being increasingly cultivated, as it provides early yields when the vegetables supply to market is deficient and shows high levels of both polyphenols content and antioxidant capacity in edible parts. In 2017, we investigated crops of the rhubarb cultivar Victoria to the fifth year of production. Comparisons were performed between three root phase fertilizations—chemical (NPK 16-16-16®), organic (Orgevit®), and biological (Micoseeds MB®)—plus an unfertilized control. The determinations of polyphenols, the antioxidant capacity, and the yield indicators from the stalks (petioles) of rhubarb were made at each out of the 10 harvests carried out. The highest yield (59.16 t·ha−1) was recorded under the chemical fertilization. The total polyphenols content and antioxidant capacity varied widely from 533.86 mg GAE·g−1 d.w. and 136.86 mmol Trolox·g−1 d.w., respectively in the unfertilized control at the last harvest, up to 3966.56 mg GAE·g−1 d.w. and 1953.97 mmol Trolox·g−1 d.w. respectively under the organic fertilization at the four harvest. From the results of our investigation, it can be inferred that the chemical fertilization was the most effective in terms of yield, whereas the sustainable nutritional management based on organic fertilizer supply led to higher antioxidant compounds and activity.


Introduction
Rhubarb (Rheum rhabarbarum L.) is a perennial plant [1] that is characterized by high antioxidant properties and is cultivated only for its petiole [2], as its leaves are toxic due to their content of oxalic acid [3]. In Europe, it is cultivated mainly in Germany, France, and England [4]. Stalks are used for the preparation of various culinary preparations (soup, pie, compote, sweetness), but they are also used in traditional medicine as laxatives, gastrointestinal hemorrhage, and the treatment of constipation jaundice and ulcer [5]. A study has shown promising anti-cancer properties and the broad therapeutic potential of anthraquinines in the petioles of rhubarb [6]. The consumption of rhubarb in large quantities has an adverse effect on the accumulation of calcium in the body [3].
The technology and the nutritional regime used to cultivate crops influence the petioles production and its composition [7]. Important benefits from crop production have been observed by applying radicular fertilizers especially when soil conditions are limiting root uptake [8]. This way, much lower

Results and Discussion
The total polyphenol content and antioxidant capacity were positively influenced by both the type of fertilization applied and the date of harvest compared to the unfertilized control. For the total polyphenol content, the highest statistically significant increase was recorded in the fertilized treatment with Mo (61.68% compared to Ct). The organic fertilization (Og) provided 1477.95 mg GAE·g −1 d.w., followed by chemical fertilization with a total polyphenol content of 1320.62 mg GAE·g −1 d.w. (Table 1). The role of arbuscular mychorrhizal fungi AMF should be taken into consideration in the Mo treatment as an abiotic stress according to environmental conditions [36].
The highest polyphenol content was recorded at the fourth harvesting period (R 4 ), with a value of 2450.86 mg GAE·g −1 d.w., followed by fifth harvest (R 5 ) with a total polyphenol content of 1819.34 mg GAE·g −1 d.w. The lowest polyphenol content was recorded at the last harvest (R 10 ), i.e., 670.92 mg GAE·g −1 d.w. Statistically, lower values were recorded at the eighth harvest time (R 8 ) with a total polyphenol content of 1015.14 mg GAE·g −1 d.w.
The content of TP increased from R 1 to R 4 and R 5 , after which it decreased toward the end of the production period, due to the reduction of the metabolism of the resent plants. Similar studies were also conducted in salad [37], where the total phenol content (TPC) was higher but dependent on the climatic conditions.
Regarding the influence of fertilization on antioxidant activity (AC), the highest values were recorded when fertilizing with Mo, with the value of 877.07 mmol Trolox·g −1 d.w. (280.06% against C), followed by the Og treatment, with the value of 728.05 mmol Trolox·g −1 d.w. (232.47% compared to C). Ch fertilization resulted in an antioxidant activity of 478.48 mmol Trolox·g −1 d.w. (152.52% compared to variant C). From the data presented in Table 1, a positive correlation was observed between the values of the total polyphenols content and the antioxidant capacity. Higher levels of antioxidant activity were recorded in the middle of the harvest period (R 4 and R 5 ).
Data obtained from this study are in agreement with those reported for other Polygonaceae familly species such as English spinach [38] and sorrel [39].
Regarding the influence of fertilization and harvesting time on the total polyphenols content, statistically significant differences were recorded (Table 2).
Indeed, the total polyphenols broadly varied from 433.86 mg GAE·g −1 d.w., in the case of R 10 , unfertilized, up to 3966.56 mg GAE·g −1 d.w., in the case of R 10 , Og fertilization. (Table 2). In the case of Ch, the total polyphenols content ranged from 829. 51 [5]. Higher polyphenol content can be attributed to the effect of mycorrhizal associations, which induce a decrease in carbohydrate content in cells [40].
Regarding the influence of the fertilizer on the antioxidant capacity, this varied in wide limits from 136.86 mmol Trolox·g −1 d.w., in the case of C, from R 10 , to 1953.97 mmol Trolox·g −1 d.w., in the case of Og fertilization, from R 4 ( Table 3).  Solfanelli et al. justify the higher content of polyphenolic compounds by the fact that in warmer periods, with less precipitation, the sugar content increases in plants and thus the activity of AMF is higher [41].
Studies on the influence of different fertilizers on the antioxidant capacity of the petioles of rhubarb are scant. Zhou et al. determined the antioxidant activity of commonly consumed vegetables in Colorado and found significant variation among individual samples of each vegetable tested. They ranked the antioxidant activity of the vegetables as follows: rhubarb > green bean; tomato > potato; kale > spinach > broccoli [4].
Takeoka et al. analyzed the antioxidant capacity of the 29 species of the genus Rheum. The antioxidant capacity ranged from 463 µmol Trolox·g −1 d.w., in the case of Rheum officinale, up to 1242 µmol Trolox·g −1 d.w., in the case of Valentine cv, of the species Rheum rhabarbarum L. [5].
In perennial species, as in Populus sp., high TPC and AC are also accounted for by water stress and higher temperatures [42].
In general, polyphenols are secondary metabolism compounds that are produced by plants for defensive purposes under increased biotic or abiotic stress.
The phenolic compounds content and composition were positively influenced by the application of fertilizers and the harvesting season, and in this respect, p-coumaric acid, ferulic acid, isoquercitrin, rutoside, and quercetrol were analyzed (Table 4). Table 4. Main effects of the experimental factors on the polyphenol compounds (n = 3). Out of the phenolic compounds, rutozide showed the highest content. Rutoside ranged in a wide interval from 372.57 µg·g −1 , in case C, to 490.97 µg·g −1 , in the case of Og. Under fertilization with Ch and Mo, 27.01% increases were obtained, respectively 25.58%, compared to the unfertilized control.

Treatment P-Coumaric
Quercetrol varied from 11.57 µg·g −1 in the control to 20.20 µg·g −1 in the case of the fertilization with Mo. In the case of Og and Ch fertilization, 48.16% and 42.70% increases were obtained, compared to C.
The data from Table 4 regarding the influence of the treatment on the phenols quality highlight the favorable effect of the biofertilizers application on the crop, compared with Ch and C.
The harvesting time significantly influenced (p ≤ 0.05) phenol content as follows: the highest p-coumaric acid content was obtained at R 7 , ferulic acid at R 8 , isoquercitrin at R 6 , rutozide at R 4 , and quercetrol in R 9 . The lowest content of p-coumaric acid was recorded at first harvest (R 1 ), while the lowest content of ferulic acid was recorded at R 5 , the lowest content of isoquercitrin and rutoside were recorded at the last harvest (R 10 ), and the lowest content of quercetrol was recorded at R 9 .
P-coumaric acid varied within wide limits from 6.43 µg·g −1 in the case of R 1 to 23.57 µg·g −1 in the case of R 7 . Statistically higher values of the p-coumaric acid content in the petioles of rhubarb were obtained also at the R 4 and R 9 harvest times, with increases of 286.96% and 268.3% respectively compared to R 1 , which indicates that this acid accumulates when the plants have overcome the early development stages, being also influenced by the climatic conditions [43].
Regarding the content of ferulic acid in the petioles of resin influenced by the harvesting season, it ranged from 15.69 µg·g −1 in the case of R 10 , to 39.96 µg·g −1 , in the case of R 8 . Remarkable results were also obtained at the R 7 and R 6 times, with increases of 244.99% and 199.9% respectively compared to R 10 . Ferulic acid showed statistically low values in the case of R 9 and R 8 , with increases of 4.90% and 6.8% respectively compared to R 10 . These results indicate that ferulic acid also accumulates in the second half of the harvesting period.
The values of isoquercitrin from the petioles of resin influenced by the harvesting time varied widely from 32.43 µg·g −1 , in the case of R 10 , up to 84.83 µg·g −1 , in the case of R 6 , which suggests that isoquercitrin accumulates in the middle of the harvesting period. Increases of 247.33% and 244.96% were obtained at R 9 and R 8 , compared to R 10 . The low values of isoquercitrin were recorded at R 1 and R 2 , with increases of 45.7% and 50% respectively compared to R 10 .
The content of rutoside in the petioles of resale varied in wide limits of 196.02 µg·g −1 , in the case of R 10 , up to 904.12 µg·g −1 , in the case of R 4 . A higher content of rutoside was obtained by R 5 and R 2 , with increases of 340.1% and 254% respectively compared to the 10th harvest (R 10 ). Lower values of rutoside were achieved by R 8 and R 9 , with increases of 43.54% and 59.4% respectively compared to R 10 .
The content of quercetrol in the rhubarb petioles ranged from 11.22 µg·g −1 , in the case of R 1 , to 21.64 µg·g −1 , in the case of R 9 . Higher values were obtained by R 10 and R 4 , with increases of 90.5% and 88% respectively compared to R 1 . Quercetrol achieved increases of 1.42% and 4.54% respectively in the R 2 and R 3 harvest periods.
Fertilization with Mo had a significant positive influence on p-coumaric acid, ferulic, isoquercitin, and quercetrol.
The positive influence of biological fertilizers on the content of polyphenols has also been demonstrated in species such as tomatoes [44,45] or peppers [46][47][48].
The type of fertilization and the harvesting season significantly influenced the content of the five polyphenols (p-coumaric acid, ferulic acid, isoquercitrin, rutoside, and quercetrol) ( Table 5). P-coumaric acid content widely varied from 4.00 µg·g −1 , in the case of the Ch treatment, from the eighth period (R 8 ), to 46.14 µg·g −1 , in the case of fertilization with Mo from R 7 .
Ferulic acid fluctuated from 12.16 µg·g −1 , in the case of Ch fertilization from R 5 , to 51.58 µg·g −1 , in the case of Mo from R 7 .
In addition, isoquercitrin was also significantly influenced by the fertilizer and harvesting period. It ranged in wide limits from 25.50 µg·g −1 , in the case of the Og fertilization, from R 6 , to 148.78 µg·g −1 , in the case of the control, which was also from R 6 .
The values of rutoside fluctuated from 121.06 µg·g −1 in the case of the control from R 7 to 1442.24 µg·g −1 in the case of Og fertilization from the R 4 harvest time.
The content of quercetrol ranged from 6.78 µg·g −1 in the case of the control of R 7 to 33.20 µg·g −1 in the case of the Ch treatment of R 7 and Og from R 10 .
The content of biologically active compounds depends mainly on the cultivation method as well as cultivar and harvest time [49]. Many studies indicate that organic farming systems have a significant impact on the quality of strawberries produced, such as the use of organic and biological fertilization [50,51].
The fertilization treatments significantly influenced the number of petioles rhubarb per plant, their average weight per plant, the average weight per plant, and the yield dynamics (Table 6). The number of petioles per plant ranged from 9.2 2 , in the case of the control plant, to 11.14, in the case of Ch.
The average weight of the petioles per plant was not significantly influenced by fertilization. However, this ranged from 39.11 g·plant −1 , in the case of the control plant, to 42.92 g·plant −1 , in the case of Mo.
The total yield ranged from 47.27 t·ha −1 , in the case of the unfermented treatment, to 59.16 t·ha −1 , in the case of Ch, although the differences between treatments were not significant.
The influence of different types of fertilizers on production indicators has also been demonstrated in vegetable species such as Cynara cardunculus L. [52,53].
The total quantity of stalks ranged from 1199.75 kg·ha −1 , in the case of R 4 , up to 8044.75 kg·ha −1 , in the case of the first harvesting season (R 1 ) (Figure 1). The highest yields were recorded in the first three harvests and in the last four, whereas the lowest values were recorded in the first three eras, and respectively in the last four. The lowest values were recorded in the middle of the harvesting period (R 4 , R 5 , and R 6 ). Mouna et al. conducted a study on Salvia officinalis L., where phenophases led to higher production at the beginning of the harvesting period [54].
Fertilizer and harvesting times significantly influenced the dynamic yield of rhubarb petioles. Thus, the dynamic yield for the 40 experimental treatments varied widely from 560 kg·ha −1 , in the case of the control at R4, up to 11,997 kg·ha −1 , in the case of chemical fertilization (Ch) from the ninth harvest (R9). Highest values were recorded under Ch fertilization at R7 and that with Og from R9, which showed positive increases, compared to the control from the fourth harvest time (R4) ( Table 7). Corroborating the dynamics of the production with the climatic conditions, we can say that the production at the perennial plants has its peak at the beginning of the vegetation period when the average temperature increases above 5 °C and the soil has sufficient accumulated humidity from the winter season. The vegetative buds bloom in greater numbers, but they grow slower, they are more succulent, the petioles reach the normal size in a longer period, and they accumulate water in greater quantity, when the harvest is bigger. After harvesting and in more arid onditions, the plants grow Yield (kg·ha −1 Mouna et al. conducted a study on Salvia officinalis L., where phenophases led to higher production at the beginning of the harvesting period [54]. Fertilizer and harvesting times significantly influenced the dynamic yield of rhubarb petioles. Thus, the dynamic yield for the 40 experimental treatments varied widely from 560 kg·ha −1 , in the case of the control at R 4 , up to 11,997 kg·ha −1 , in the case of chemical fertilization (Ch) from the ninth harvest (R 9 ). Highest values were recorded under Ch fertilization at R 7 and that with Og from R 9 , which showed positive increases, compared to the control from the fourth harvest time (R 4 ) ( Table 7). Corroborating the dynamics of the production with the climatic conditions, we can say that the production at the perennial plants has its peak at the beginning of the vegetation period when the average temperature increases above 5 • C and the soil has sufficient accumulated humidity from the winter season. The vegetative buds bloom in greater numbers, but they grow slower, they are more succulent, the petioles reach the normal size in a longer period, and they accumulate water in greater quantity, when the harvest is bigger. After harvesting and in more arid onditions, the plants grow slower; they recover in a longer period, because they start from dormant buds or those that appear in the respective year of culture.
The application of chemical fertilizers, which have high solubility, made the production from the second part of the vegetation period bigger, and between the control and the types of fertilization at the beginning of the year, there were not very big differences, because the first petioles initially grow from the accumulated reserves in rhizomes in the previous year.

Plant Material and Growth Conditions
A research study was carried out in 2017 at "V. Adamachi" Experimental Station of the Agronomic University of Iasi (47 • 19 25"N, 27 • 54 99"E, 150 m a.s.l.) on rhubarb (Rheum rhabarbarum L.) using root cuttings of the Victoria cv. The cambic chernozem soil is characterized by a medium fertility, with 3.1% organic matter, 32% of clay and pH = 6.6. During the experimental period, the average temperature was 18.54 • C, the precipitation was 293.5 mm, and the relative humidity of the air was 66.8% (Table 8). The planting was practiced in 2012, with plants spaced 0.75 m along the rows, which were 1.00 m apart (density of 1.33 plants·m −2 ). Due to the dry agricultural year, three irrigations were practiced, each with 250 m 3 ·ha −1 , when the soil available water decreased below 80%. Flowering stems were removed 4 to 5 days after planting in order to favor the development of the edible part [55]. During the vegetation period, two manual hoeings were performed between rows and plants. No phytosanitary treatments were performed for protecting plants against diseases and pests. The biometric and biochemical determinations were made in 2017 on a rhubarb crop set up by seedlings to the fifth year of harvest.

Experimental Protocol
Three types of fertilization were compared in a split plot design experiment with three replications. Chemical (NPK = 16-16-16), Mycorrhizal-based formulate (Micoseeds MB ® ), and Organic (Orgevit ® ) treatments were compared to an unfertilized control (C). Chemical NPK 16-16-16 was produced by Ameropa Company ® (Romania), Micoseeds MB ® was from MsBiotech (Italy), and Orgevit ® was from MeMon BV (Netherlands). The biological product consists of an arbuscular mycorrhizal fungus (Glomus spp.), PGPR (Pseudomonas sp., Bacillus spp., Streptomyces sp.), and a fungus (Trichoderma sp.) in different proportions. Organic fertilizer is chicken manure formulate with pH 7, 4% N, 2.5% P2O5, 2.3% K2O, 1% MgO, 0.02% Fe, 0.01% Mn, 0.01% B, 0.01% Zn, 0.001% Cu, and 0.001% Mo. All fertilizers were supplied to the soil. Thus, the Ch fertilizer was applied in an amount of 425 kg ha −1 . The product beneficial microorganisms-based Micoseed MB (Mo) was used in the dose of 60 kg ha −1 . Og was applied at the dose of 2400 kg ha −1 . Ch and Og fertilizers were supplied after the first petiole harvest (03.04) in five stages (04.04, 28.04, 12.05, 26.05, and 09.06); Micoseeds MB ® was applied in two stages, the first before planting (16.03) and the second one day after the first harvest (04.04) according to producer recommendations. For the determination of the doses of the fertilizers Og and Ch, the chemical composition of each product was taken into account, and for the Og, it was considered that 70% of the active substance of the product is assimilated in the first year after application.
Biometric and chemical determinations were performed at 10 harvest times:

Biometric Measurements
From the field of research were collected stalks from each plant from the four variants taken in the experiment. For each variant, we determined the average quantities obtained by measuring the weight with the electronic scale. Petioles with a minimum diameter of 10-12 mm and a length of 25-30 cm were harvested. In the laboratory, weighing was performed on the analytical balance to determine the average weight of petioles per plant. To determine the length of the stalks, the measurements were made with the graduated ruler, with the unit of measure in centimeters, and the thickness of the petioles was determined with the electronic chisel (mm).

Samples Preparation
In order to prepare the material for the laboratory analyses, 10 stalks from each repetition were selected randomly for a total of 40 pieces. The petioles were cut into 1 cm fragments for drying under normal weather conditions. The drying was carried out on a Sanyo oven, type MOV-112F, at a temperature of 70 • C up to the total loss of water from the dry material in order to determine the quantity of water and the dry substance. The samples were crushed into small fragments of 0.1-1 mm. The last step consisted in the packaging and labeling of variants in order to carry out laboratory analyses to determine the polyphenol content and antioxidant capacity in plants.

HPLC-MS Analysis of Phenolic Compounds
In this application, the presence and content of different phenolic compounds in 70% ethanolic extracts were studied using an HPLC-MS, method which allows the simultaneous detection of several phenolic compounds with a single column pass [56].
The identification and quantification of polyphenolic compounds was carried out using an Agilent Technologies 1100 HPLC Series system (Agilent, Santa Clara, CA, USA) equipped with a G13311A binary gradient pump, G1322A degasser, column thermostat, G1316A UV detector, and G1313A autosampler. The HPLC system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap SL). For the separation, a reverse-phase analytical column was employed (Zorbax SB-C18 100 × 3.0 mm i.d., 3.5 µm particle) and the work temperature was set at 48 • C. The detection of the compounds was performed in both UV and MS mode [57][58][59]. The UV detector was set at 330 nm until 17.5 min, and then at 370 nm. The MS system was operated using an electrospray ion source in the negative mode. ChemStation and DataAnalysis software from Agilent were used for processing the chromatographic data. The mobile phase was a binary gradient: methanol and acetic acid 0.1% (v/v). The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol, for 35 min; then, it had 42% methanol for the next 3 min. The flow rate was 1 mL·min −1 , and the injection volume was 5 µL [60,61].

Antioxidant Activity Test
The rhubarb stalks' antioxidant capacity was assessed in terms of radical scavenging activity, following the procedure described by Re et al. [62] with slight modifications [63]. In this respect, 500 µL aliquot of the extract or Trolox standard was added to 1 mL of DPPH methanol solution (74 mg·L −1 ). A daily prepared solution of 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) showed a final absorption at 520 nm of 1.8 AU. The mixture was shaken and allowed to stand for 1 h at room temperature; then, the absorption was measured at 520 nm in a Lambda 25 spectrophotometer. The antiradical activity of the sample is inversely correlated with its purple color intensity. Aqueous solutions of Trolox at various concentrations were used for calibration (0.15-1.15 mmol·L −1 ). The results were expressed as µmol equivalents of Trolox (an analog of vitamin E) per g of sample (TEAC-Trolox Equivalent Antioxidant Capacity) [64].

Statistical Analysis
Data statistical processing was carried out by one-way and two-way ANOVA, and mean separations were performed through Tukey's test using a SPSS version 21, referring to p ≤ 0.05 probability level.

Conclusions
The present study confirms that fertilizer management method may have a significant impact on the phenolic composition of rhubarb. The fact that there are qualitative variations between polyphenols at the same harvest date, under same environmental conditions, on the same cultivar indicates that they are influenced by the treatment used.
However, in addition, harvesting time under climatic conditions has an important effect on the quality and quantity of bioactive compounds.
The research shows that the biological fertilizer had a positive effect on the total phenol content, directly influencing the antioxidant capacity from rhubarb, compared to the chemical and organic fertilizers, which are very important in the human diet.
Biological fertilization provides satisfactory nutritional conditions for the accumulation of ferulic acid, p-coumaric acid, isoquercitrin, and quercetrol compared with rutoside, which accumulates in the highest quantities under organic fertilization. Polyphenolic compounds accumulate in petiols of rhubarb as follows: rutozide > isoquercitrin > ferulic acid > quercetrol > p-coumaric acid.
Chemical fertilization provided the highest yields, regardless of the harvesting period, but the differences with respect to organic and biological treatments were not, which means that there are favorable conditions for using the two fertilizers in sustainable agriculture, without restriction.
The treatments carried out and the harvesting period in accordance with the climatic conditions emphasize that there are the premises of promoting organic and biological treatments for a type of non-polluting, sustainable agriculture.