Effect of Semiarid Environment on Some Nutritional and Antinutritional Attributes of Calendula ( Calendula officinalis )

Stressful environments have been shown to affect themetabolism in some plants. In the present study, we assessedwhether semiarid environment (saline and saline alkaline soil) could affect the nutritional (total proteins, phenolics and riboflavin contents, and catalase activity) and antinutritional (hydrogen peroxide and malondialdehyde contents) properties differently in different plant parts (leaves, flowers, and roots) of calendula. Although salinity decreased plant biomass, it did not affect total protein and phenolics contents in the calendula. All plant parts were rich in riboflavin contents. However, plants grown under saline-alkali soil had relatively more riboflavin contents in the flowers. Salinity increased hydrogen peroxide (H 2 O 2 ) concentration in the flowers and roots, whereas saline-alkali soil did not affect it. Plants exposed to both saline and saline-alkali soil had greater catalase activity in the flowers and leaves. Plants exposed to salinity had higher malondialdehyde (MDA) contents in the flowers compared with nonsaline and saline-alkali conditions. Nonetheless, the possibility of safely using different parts of calendula as nutraceutical was in the order flower > root > leaf. Overall, the results suggested that plant can be grown in mild saline-alkali (EC ≤ 7; pH = 8.5) soils without affecting its nutraceutical properties.


Introduction
Calendula officinalis L. commonly known as marigold is an annual plant with yellow to orange flowers and is grown for medicinal [1][2][3] and ornamental uses [4][5][6].All plant parts can be used either dried or fresh.The buds, leaves, and blossoms are used to formulate herbals and homeopathic medicines.Fresh flowers of C. officinalis and dried petals are used in salad and in tea, respectively.The flowers are also used in food industry to flavor cakes, cookies, and puddings as well as coloring of several culinary products [7,8].
Plant based dietary supplements and natural botanicals are better sources of antioxidants and anti-inflammatory compounds [9].Approximately 80% of the world population depend exclusively on plants for their health and healing.Pharmacological studies have shown that calendula exhibited a broad range of biological effects such as antimicrobial, anti-inflammatory [10,11], antioxidant [12], wound healing [13], antiviral, and antitumoral [14].It primarily contains two classes of pigments, the flavonoids and carotenoids.Plant based phenolics constituents retard oxidative degradation of lipids and thereby improve quality and nutritional value of food [15,16].Flavonoids have antioxidant activities which play an important role in the food preservation and human health [17].Calendula has a high number of carotenoids that are important antioxidants and are precursors of Vitamin A and retinoids [18].
Various environmental factors can affect growth, essential oil production, and other secondary products in plants [19,20].Although the natural products are gaining a revitalized attention in medical community and their therapeutic uses are gradually increasing, there is a lack of information about the effects of environmental conditions on the nutritional (functional food) and antinutritional (nonfood) properties of different medicinal plants.Different stresses have been shown to produce reactive oxygen species that result in the accumulation of toxic substances [21][22][23][24].The most mutagenic lipid oxidation product is MDA [25].It modifies both proteins and DNA, and its reaction with proteins is associated with the development of pathological conditions such as atherosclerosis, diabetes, preeclampsia, Parkinson disease, and colon cancer [26].A better strategy, therefore, is to look for the variation of natural substances like oxidants and antioxidants in the medicinal plants when grown under diverse environments.Accordingly, the primary objective of the study was to assess up to what extent semiarid environment could affect the nutritional and antinutritional properties of calendula.

Plant Materials and Growing Conditions.
The experiment was conducted in the field under semiarid environmental conditions at University of Agriculture Faisalabad (UAF), Ayub Agricultural Research Institute, Faisalabad (AARI), and Government College University, Faisalabad (GCUF).The healthy calendula seeds were sown at the three sites and the germination percentage was over 75%.The meteorological data during the growing season is shown in Figure 1.The pH and electrical conductivity (EC e ) of the soil (from where the plants were uprooted) was tested before and after conduction of the experiment, and the average soil properties are shown in Figure 2. The plants were irrigated with tap water as and when required.All other agronomic practices were kept as normal and uniform at the three growing sites.At flowering stage (about 50 days after planting), the whole plants of calendula were uprooted, washed with distilled water so as to remove all unwanted material, and blotted and fresh weights were recorded.After drying at 65 ∘ C for 72 h, shoot dry weights were recorded.The fresh, washed, and blotted plants were separated in to leaves, flowers, and roots for different analyses.However, for total phenolics determination, plant parts dried at 40 ∘ C for 10 days were used.

Total Soluble Proteins.
The concentration of total soluble protein was determined by using the method as described earlier [27].Briefly, plant sample (0.5 g) was taken and grounded in 10 mL phosphate buffer (pH 7.8).After centrifugation at 16,000 g for 15 min at 4 ∘ C, the supernatant was treated with 2 mL Bradford reagent and incubated for 15 min.The absorbance was measured at 595 nm.Total soluble proteins were computed using standard graph using bovine serum albumin (BSA) as standard.

Riboflavin Concentration.
The concentration of riboflavin was determined by using the method of Okwu and Josiah [28].Plant sample (1 g) was extracted with 20 mL of 50% ethanol for 1 h.To the supernatant (5 mL), 5 mL of 5% potassium permanganate and 4.5 mL of 30% H 2 O 2 were added and allowed to stand over a hot water bath for 30 min.Then, 1 mL of 40% sodium sulphate was added and the volume was made up to 25 mL.The absorbance was measured at 510 nm with a spectrophotometer.

Total Phenolics.
Total phenolics were determined by using Folin-Ciocalteu method with some modifications [29].Briefly, the extracts were mixed with 5 mL of diluted (1 : 10 v/v) Folin-Ciocalteu reagent and 4 mL of sodium carbonate solution (75 g/L).The tubes were vortexed for 15 s and then incubated at 40 ∘ C for 30 min.The absorbance was measured at 765 nm using the UV-VIS spectrophotometer (Hitachi U-2910).Total phenolic contents were expressed as mg g −1 tannic acid equivalent (TAE).

Catalase (CAT) Activity.
The CAT activity was determined as previously described [30].Briefly, the reaction solution (3 mL) for CAT was comprised of 50 mM phosphate buffer (pH 7.0), 15 mM H 2 O 2 , and 50 L enzyme extract.Reaction was initiated by the addition of H 2 O 2 to the reaction solution.The CAT activity was determined by following the decomposition of H 2 O 2 and measuring the absorbance decrease at 240 nm for 1 min.The CAT activity was expressed as kat mg −1 protein min −1 .
2.6.MDA Contents.The MDA was determined as described earlier [31].Briefly, in 2 mL of supernatant, 2 mL of 0.6% thiobarbituric acid was added and incubated at 100 ∘ C for 20 min in water bath.After heating, the samples were immediately cooled in ice-bath and centrifuged at 20,000 g for 10 min.The absorbance of the supernatant was recorded at 450 nm, 532 nm, and 600 nm on UV-VIS spectrophotometer (Hitachi U-2910).The MDA contents (mmol g −1 FW) were calculated according to the given formula: (1) The H 2 O 2 concentration was determined following Velikova et al. [32].Briefly, fresh leaves (0.1 g) were homogenized in 5 mL of 0.1% trichloroacetic acid (TCA).After centrifugation at 12,000 rpm for 15 min, the supernatant (0.5 mL) was then mixed with 0.5 mL of phosphate buffer (pH 7) and 1 mL of 1 M KI.After incubation, the absorbance was taken at 390 nm.The content of H 2 O 2 was determined using a calibration curve constructed using a series (1-50 M) of analytic grade H 2 O 2 .

Statistical Analysis.
The data collected was subjected to analysis of variance technique (ANOVA) by using a computer software CoStat version 6.2, CoHort Software, 2003, Monterey, CA, USA.When the difference among means was significant ( ≤ 0.05), the mean values were compared using the least significant difference (LSD).

Plant Biomass and Nutritional
Properties.Calendula exhibited more growth in terms of fresh and dry masses under nonsaline conditions (EC ≤ 4dS m −1 ) (Figure 3).However, the minimum fresh and dry masses of calendula were observed under saline and saline-alkali environment.Arid environment nonsignificantly affected total protein contents in all calendula parts (leaf, flower, and root) (Figure 4(a)).However, the environmental conditions significantly affected riboflavin (Vitamin B2) concentration in all tissues of calendula (Figure 4(b)).Interestingly, plants growing in saline-alkali environment had maximum riboflavin concentration in the flowers and the minimum riboflavin concentration in the roots.Irrespective of the growing conditions, all plant parts (flower, leaf, and root) were rich in riboflavin contents.
In the present study, changes in environmental conditions had nonsignificant effect on the total phenolics contents in different plant parts (Figure 5(a)).Nonetheless, growing conditions significantly affected CAT activity in all calendula parts (leaves, flowers, and roots) (Figure 5 collected from saline and saline-alkali environment had more CAT activity in the flowers as compared with plants grown in the nonsaline environment.Overall, growing conditions did not affect CAT activity in the roots of calendula.The minimum CAT activity was observed in the leaves of plants grown in nonsaline environment.

Antinutritional Properties.
Changes in environmental conditions significantly affected H 2 O 2 concentration in all plant parts (Figure 6(a)).Nonetheless, flowers had more H 2 O 2 concentration followed by leaves and roots under different growing conditions.Salinity caused greater H 2 O 2 accumulation in the calendula roots when compared with nonsaline and saline-alkali regimes.Arid environment significantly affected MDA concentration in all parts of calendula (Figure 6(b)).Salinity increased MDA concentration in the flowers while nonsaline and saline-alkali regime increased MDA concentration in the leaves.In contrast, saline-alkali environment increased MDA concentration in the roots when compared with nonsaline and saline environments.

Discussion
Changes in environmental conditions (abiotic stresses) have been shown to affect the physiochemical attributes in different plants [33][34][35].In the present study, soil salinity and alkalinity decreased plant biomass.However, different environments did not affect total proteins contents in calendula.Earlier studies have shown variable effects of different environments on total proteins contents in different plants.For instance, salinity did not affect total soluble protein content in narrow-leafed lupine [36] and wheat [37].In contrast, salinity significantly decreased protein contents in some plants [38,39].Khedr et al. [40] found an increase in protein content at low and decline at high NaCl levels in both shoot and root of Pancratium maritimum.Our results suggested that calendula could be grown under mild saline or saline-alkali environments without affecting its nutritional value in terms of soluble protein contents.Riboflavin is used for human nutrition and therapy and also as an animal feed additive.Riboflavin produced by plants acts as a coenzyme in many physiological reactions in plants and animals [41,42].In this work, saline-alkali environment increased riboflavin concentration in the flowers whereas it was decreased in the roots.This showed that different calendula tissues faced stress differently.Nonetheless, calendula accumulated higher riboflavin concentration in the flowers under different environments.Riboflavin, being an antioxidant [43], affected H 2 O 2 accumulation in Arabidopsis thaliana [44].Jaleel et al. [45] stated that higher riboflavin contents could decrease the membrane degradation under stressful environments possibly by preventing lipid peroxidation [46].Accordingly, our results suggested that higher concentration of riboflavin in the flowers could increase membrane stability and thus increase longevity of flowers.Furthermore, flowers being rich in riboflavin contents could be used as functional food.
Phenolics are the most abundant secondary metabolites of plant origin which form an important part of both human and animal diets.Phenolics are more potent antioxidants than Vitamins C and E and carotenoids because phenolics could act as hydrogen donators, reducing agents and quenchers of singlet oxygen [47,48], and thus it prevents the stress-induced oxidative stress [49].Both saline and saline-alkali environments did not affect total phenolics in different calendula parts.The results suggested that calendula plants might have faced less stress under different regimes.Furthermore, plants might have used another antioxidative pathway/kind of antioxidants to cope with the stressful environment.Nonetheless, phenolics played an important role in the abiotic stress tolerance in some plants [50], where the synthesis of soluble phenolics directly correlated with the stress tolerance [51].All tissues of calendula accumulated phenolics irrespective of the growing environment.
The favourable activity of the calendula in a number of health related matters has also been associated with the accumulation of total phenolics [9].Catalases are used to decrease oxidants like H 2 O 2 [52].In the present study, more CAT activity was observed in the flowers of calendula grown under both saline and salinealkali conditions.A contrasting response of CAT activity to different abiotic stresses has been shown in the literature.For instance, salinity stress enhanced CAT activity in rice plants [53,54], whereas it was downregulated in some other plants [55].This could be either due to salt stress-induced degradation of CAT by endogenous proteases or inhibition of CAT activity by the accumulation of salicylic acid [56].Earlier studies have shown that calendula extract efficiently scavenged different radicals in vitro [57].The results suggested that extract of calendula flowers particularly collected from stressful environments could be effectively used to scavenge different radicals.
Plants grown under salt stress are prone to oxidative damage induced by the elevated levels of reactive oxygen species (ROS) such as hydrogen peroxide [58][59][60].In the present investigations, both flowers and roots of calendula accumulated higher levels of H 2 O 2 under saline regimes compared with nonsaline and saline-alkali conditions.Relatively, leaves of plants growing in nonsaline environment had slightly more H 2 O 2 contents compared with those growing in saline and saline-alkali environments.This could be due to either less synthesis or activity of different antioxidative enzymes that altered H 2 O 2 concentration in leaves under nonsaline conditions.Higher cellular H 2 O 2 levels have been shown to adversely affect anthocyanins and ascorbic acid contents in different plants [61,62].Hydrogen peroxide mediated cell damage was associated with the initiation and progression of many diseases [63].In vivo, although H 2 O 2 is short-lived [64], it is highly toxic and induces cell death in vitro [65].The present results suggested that roots of calendula plants should be preferred over other parts having low H 2 O 2 concentrations when collected from saline environments.
Lipid peroxidation is a major problem in the food industry.It leads to quality deterioration, rancidity, and accumulation of potentially toxic compounds in foods [66].One of the major and most toxic by-products of lipid peroxidation is MDA [24,67].Several deleterious effects of MDA have been reported such as induction of intracellular oxidative stress [24] and formation of highly mutagenic DNA adducts in human cells [68].When ingested, MDA increases frequencies of tumour and atherosclerosis [66] and modifies both proteins and DNA [67].In the present study, salinity increased MDA contents in the flowers while nonsaline and saline-alkali environment increased MDA contents in the leaves.Salinity mediated increased production of MDA has been shown in many plants [37,65,68].Interestingly, plants grown under saline-alkali conditions had less MDA contents in the flowers and more in the roots.This could be due to the reason that flowers accumulated antioxidants such as riboflavin and phenolics and had higher CAT activity that scavenged H 2 O 2 and possibly prevented lipid peroxidation.
In conclusion, flowers collected from nonsaline and saline-alkali environments (ECe ≤ 7 dS/m) and roots from saline environments (ECe > 7 dS/m) should be preferred over other plant parts being more safe having relatively low antinutritive (H 2 O 2 and MDA) contents.Overall, based on higher nutritive and low antinutritive contents, the possibility of using different parts of calendula as food additive/nutraceutical was in the order flower > root > leaf.

MonthsFigure 1 :
Figure 1: Meteorological data showing variations in temperature, relative humidity, and precipitation levels during the growing season of calendula.Sampling was done in the last week of February.

Figure 3 :
Figure 3: Shoot biomass (fresh and dry weights) of calendula plants collected from semiarid zone.Values ( = 9 ± SE) with different superscripts (a-d) are significantly different at  < 0.05.

Figure 4 :Figure 5 :
Figure 4: Total protein concentration (a) and riboflavin concentration (b) in different tissues of calendula plants collected from semiarid zone.Values ( = 9 ± SE) with different superscripts (a-f) are significantly different at  < 0.05.