Nutritional, Phytochemical Characteristics and In Vitro Effect on α-Amylase, α-Glucosidase, Lipase, and Cholinesterase Activities of 12 Coloured Carrot Varieties

Twelve carrot varieties with different colours (purple, orange, yellow, and white) and sizes (normal, mini, and micro) were analysed for prospective health benefits (activities against diabetes-, obesity-, and aging- related enzymes—α-amylase, α-glucosidase, lipase, acetylocholinesterase, and butyrylocholinesterase, respectively) and nutritional contents (polyphenols, carotenoids, and chlorophylls). The conducted studies showed that the highest content of total polyphenols was observed in different sizes of purple carrots. The normal yellow and mini orange carrots demonstrated the highest content of carotenoids. According to the study results, the mini purple carrot showed the highest activities against diabetes-related enzyme (α-glucosidase); furthermore, the highest activities of cholinesterase inhibitors were observed for micro purple carrot. Nevertheless, normal orange carrot exhibited the highest activity against lipase. The results of the present study showed that purple-coloured carrot samples of different sizes (normal, mini, and micro) exhibited attractive nutritional contents. However, their pro-health effects (anti-diabetic, anti-obesity, anti-aging) should not be seen in the inhibition of amylase, glucosidase, lipase, and cholinesterase. Probably the mechanisms of their action are more complex, and the possible health-promoting effect results from the synergy of many compounds, including fibre, phytochemicals, vitamins, and minerals. Therefore, it would be worth continuing research on different varieties of carrots.


Introduction
Daucus carota L. is an Apiaceae member and grows in Europe, Asia, Africa, and Macaronesia. The cultivated carrot has evolved from the crossing of D. carota ssp. carota and D. carota ssp. maximus [1]. Interestingly, the first cultivated carrots were purple and yellow, followed by white carrots, and currently, orange carrots are the most popular in the world. Carrot is one of the top 10 consumable vegetables around the world, with high market share and nutrient values. One hundred grams of carrot provides approximately 41 kcal energy, 0.93 g protein, 9.58 g carbohydrates, and 2.8 g fibre [2,3].
Cultivated carrots are grouped by root colour, sugar-carotenoid content, and root shape, which are affected by development period, temperature during this time, and fertilizers [4,5]. Another reason for colour change in carrots is carotenoids, which in young carrots begin to accumulate after their first month of growth and is maintained about until the secondary growth is concluded [6].

Plant Material and Sample Preparation
Normal and mini-sized carrots were purchased from Fusion Gusto (Dąbrowa, Poland). Micro carrots were purchased from Cato Produce (Johannesburg, South Africa) in June 2020.
Carrot samples were grouped according to their sizes and colours. "Normal size" carrots in diameter (d) were between 20 mm and 45 mm and they weighed (m) from 50 g to 150 g; "mini size" carrots-20 mm > d > 10 mm and 50 g > m > 8 g; "micro size" d < 10 mm and m < 8 g.

Identification and Quantification of Polyphenols
The powder of roots (∼1 g) was mixed with 9 mL of the mixture containing HPLCgrade methanol: H 2 O (30:70%, v/v), ascorbic acid (2%), and acetic acid (1%) of the reagent. The extraction was performed twice by incubation for 20 min under sonication (Sonic 6D, Polsonic, Warsaw, Poland) and with periodic shaking [23]. Following, the slurry was centrifuged at 19,000× g for 10 min. The supernatant was filtered through a hydrophilic PTFE (polytetrafluoroethylene) 0.20 µm membrane (Millex Simplicity Filter, Merck, Darmstadt, Germany) and used for analysis. The extraction was performed in triplicate. Qualitative (liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-MS-Q/TOF)) and quantitative (ultra performance liquid chromatography with photodiode array detec- tor (UPLC-PDA)) analyses of polyphenols (phenolic acids at 320 nm, and anthocyanins at 520 nm) were performed according to Wojdyło et al. [24]. Polyphenols were separated by ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters Corporation, Milford, USA) at 30 • C. The injection and elution of the samples (5 µL) were concluded in 15 min with a sequence of linear gradients and a flow rate of 0.42 mL/min. The solvent A (2.0% formic acid, v/v) and solvent B (100% acetonitrile) comprised the mobile phase. The procedure operated through gradient elution with 99-65% solvent A (0-12 min), solvent A was later lowered to 0% for condition column (12.5-13.5 min), and the gradient returned to the initial composition (99% A) for 15 min to re-equilibrate the column. In addition, in different varieties of carrot, the content of polymeric procyanidins was determined and performed according to Kennedy and Jones [25]. All estimations were performed in triplicate. The results were expressed as mg per kg of dry matter (dm).

Identification and Quantification of Carotenoids and Chlorophylls
To obtain the samples for the determination of carotenoids and chlorophylls, the lyophilized carrot powders (~0.20 g) containing 10% MgCO 3 and 1% butylhydroxytoluene (BHT) were shaken with 5 mL of a ternary mixture of methanol/acetone/hexane (1:1:2, by vol.) at 300 rpm (DOS-10L Digital Orbital Shaker, Elmi Ltd., Riga, Latvia) for 30 min in the dark to prevent oxidation. The samples were centrifugated (4 • C, 7 min at 19,000× g; MPW-350, Warsaw, Poland), and recovered supernatants were acquired after the 4 times re-extracted from solid residue. Combined fractions were evaporated. The pellet was solubilized using methanol and filtered through a hydrophilic polytetrafluoroethylene (PTFE) 0.20 µm membrane (Millex Samplicity ® Filter, Merck, Darmstadt, Germany). Then carotenoids and chlorophylls were analysed by LC-MS-Q/TOF (identification) and UPLC-PDA (quantification) on an ACQUITY UPLC BEH RP C18 column protected by a guard column of the same materials (1.7 mm, 2.1 mm × 100 mm, Waters Corp., Milford, MA, USA) was performed at 30 • C. The elution solvents were a linear gradient of acetonitrile:methanol (70:30%, v/v) (A) and 0.1% formic acid (B) at flow rates of 0.42 mL/min. The analysis was performed at 450 nm (carotenoids) and 660 nm (chlorophylls). The obtained spectra and retention times were compared with the authentic standards and in this way the bioactive compounds were determined. The tests were implemented in triplicate, and the results are presented as mg per kg of dm.

Determination of Biological Activities of Carrot Varieties
Roots were investigated for α-amylase, α-glucosidase [26], and lipase inhibitory effects [27]. The acarbose was used as a positive control in the case of α-amylase and αglucosidase, whereas orlistat was used as a positive control for pancreatic lipase. Additionally, the activities of cholinesterase inhibitors were evaluated with the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChe) methods according to Ferreres et al. [28].

Statistical Analysis
The two-way analysis of variance (ANOVA, p ≤ 0.05) and Duncan's test were performed by Statistica version 13.3 (Stat-Soft, Cracow, Poland). The results are demonstrated as the mean value (n = 3).

Identification and Quantification of Phenolic Compounds in Coloured Carrot Varieties
Polyphenolic compounds are the secondary plant metabolites and are crucial for the colour, nutritional, antioxidant, and sensory features of foods. In the present study, the polyphenolic compositions were determined by UPLC-PDA-Q/TOF-MS, and the results are shown in Table 1. Twenty-four phenolic compounds (15 phenolic acids and nine anthocyanins) were detected. The identified phenolic acids showed similarities for purple, yellow, orange, and white carrot varieties to those reported in the literature [29]. The determined phenolic acids were derivatives of caffeic, caffeoylquinic, coumaroylquinic, ferulic, and feruloylquinic acid. In the present study, 3-O-caffeoylquinic acid, 3-O-feruloylquinic acid, O-q-coumaroylquinic acid, dicaffeoylquinic acid derivative, and di-ferulic acid derivative were detected in white and orange carrots for the first time. 4-O-caffeoylquinic acid, 3-O-feruloylquinic acid, di-ferulic acid derivative, and diferuoylquinic acid derivative were found in yellow carrots. 4-O-caffeoylquinic acid, 3-O-feruloylquinic acid, Oq-coumaroylquinic acid, dicaffeoylquinic acid derivative, di-ferulic acid derivative, and diferuoylquinic acid derivative were detected in purple carrots for the first time.  Table 2 presents the contents of phenolic compounds in carrot samples. The carrot samples showed significant differences in polyphenolic compositions (p ≤ 0.05). In the present study, di-ferulic acid derivative and 5-O-caffeoylquinic acid (5-CQA) were found in all coloured carrot samples. In the literature, 5-CQA (chlorogenic acid) was also detected in sea fennel, bean, and spinach, and it is important because of health benefits such as anti-carcinogenic, anti-inflammatory, anti-diabetic, and anti-obesity properties [30,31]. Other phenolic acids were found in different amounts in all carrot varieties. The highest total phenolic acid contents were observed in NPC (945.00 mg/100 g dm), MiOC (503.93 mg/100 g dm), and MiPC (479.33 mg/100 g dm). According to the obtained results, mini-sized and purple carrot samples had abundant phenolic acid content. Thus, the differences in phenolic acid profiles of carrots might be related to the root size, colour, and chemical compositions of the carrot varieties. Orange, purple, yellow, and white carrots are results of phenolic and carotenoid contents during the maturation period as well as storage temperature [32,33].
Procyanidins are subclasses of proanthocyanidins and are known for their antihypertensive, anti-allergic, antioxidant, and anti-microbial activities. Procyanidins have not been quantified in carrot varieties thus far. The present study is the first to quantify procyanidin contents in carrots. In the present study, the highest procyanidin contents were found in MiWC, NOC, and NWC. Thus, normal-sized and white carrot samples were rich in polymeric procyanidins. The structures of procyanidins consist of stereochemistry, hydroxylation pattern, flavan-3-ol constitutive units, and degree of polymerisation (DP, number of flavanol units) [38]. In the present study, the highest DP values were detected in MiWC (2.06), NYC (1.99), and MiYC (1.72). According to the DP values noted in the present study, mini-sized and yellow carrot samples were rich in flavanol units. Therefore, MiWC showed high procyanidin contents and DP values.
To summarise the polyphenolic contents of coloured carrot varieties, the highest total phenolic contents were found in NPC, MiPC, and MiOC. Thus, purple carrot has the highest total content of polyphenols, which was also confirmed by other authors [29,36,[39][40][41]. Moreover, phenolic contents may change depending on genes, environment, and climate factors [42]. Table 3 shows the content of carotenoids and chlorophylls of the 12 carrot varieties with significant differences (p ≤ 0.05). In the present study, violaxanthin, astaxanthin, lutein, zeaxathin, α-cryptoxanthin, β-cryptoxanthin, (6R)-δ-carotene, α-carotene, γ-carotene, εcarotene, β-carotene, and trans-apo-carotenal were identified as carotenoids. NPC exhibited the highest ratio of different carotenoid types (lutein, zeaxathin, α-cryptoxanthin, β-cryptoxanthin, γ-carotene, and trans-apo-carotenal). Trans-apo-carotenal was detected only in NPC. Carotenoids, which cannot be synthesised by animals and humans, have to be obtained from the diet as crucial precursors for healthy body functions [43]. According to Alasalvar et al. [39], β-carotene and α-carotene are the most abundant carotenoids in carrots. In the present study, β-carotene was detected in NYC > MYC > MiYC > MPC > NWC, whereas α-carotene was observed in NYC > MiOC > NOC > MiYC = NWC > MiWC. The highest content of β-carotene was found in NYC and MYC, whereas the highest α-carotene content was observed in NYC and MiOC. Thus, NYC showed the highest presence of both α and β-carotenes. Reif et al. [44] showed a different trend in their study and indicated a low amount of β-carotene in yellow carrots. The contents of β-carotene and α-carotene change with temperature, which may have caused discrepancies in the observed results.

Identification and Quantification of Carotenoids in Carrot Varieties
Carotenoids are unstable pigments, which can create some problems while working with them. For instance, orange carrot is known to be a rich source of β-carotene and α-carotene [44]. However, in the present study, α-carotene was not detected in MOC; moreover, according to the applied method, and retention time differences in β-carotene were not found in NOC, MiOC, or MOC. This was a surprising observation but was confirmed by the standard of β-carotene. Maybe the content was very low, and therefore no peak of this compound was observed. Moreover, αand β-carotene can be pressed by chlorophylls and xanthophylls as well [45]. In addition, storage period and temperature during the storage are other important factors for pigmentation.
High levels of δ-carotene and ε-carotenes were found in orange carrot samples of various sizes and have not been reported before.
Six xanthophyll pigments were identified and quantified in carrot samples (Table 3). In the present study, lutein was found solely in NPC and NYC. Similar results have been reported in the literature [44,46].
Chlorophyll pigments are essential to prevent chronic diseases [47]. In the present study, pheophorbide a, chlorophylls a, and b were found as chlorophylls pigments in the carrot samples. Pheophorbide a was solely determined in NOC and NPC, in turn chlorophyll a was identified in NOC, NYC, NWC, MiYC, MWC, and MYC. Except for MiPC, all carrot varieties showed the content of chlorophyll b. Moreover, chlorophyll a and b contents were high in NYC. Thus, chlorophyll contents were found to change within cultivars [48].
To summarise, the total carotenoid contents were the highest in NYC, MiOC, NPC, and MPC. Normal-sized and purple carrot samples showed greater total carotenoid contents. The total chlorophyll contents were the highest in NYC, MiYC, NPC, and MYC. Therefore, normal-sized and yellow carrot samples were rich in chlorophyll contents. The highest total carotenoid and chlorophyll results were observed in NYC, MiOC, NPC, and MPC. Normal-sized and purple carrot samples were rich in carotenoid and chlorophyll contents. The carotenoid contents of carrots may change depending on variety, maturity, growth conditions, growing season, soil, and genetic factors [49].

Enzyme Inhibitory Activities by Coloured Carrot Roots
The enzyme inhibitory activities of 12 carrot varieties against α-amylase, α-glucosidase, pancreatic lipase, acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were evaluated by in vitro assays and expressed as IC 50 -the amount of sample can reduce enzyme activity by 50%. The results are shown in Table 4. The carrots showed significant differences in all five inhibition activities (p ≤ 0.05). It should be noted that enzyme inhibition activities of carrot varieties have not been investigated thus far. The 12 carrot varieties were assayed for their anti-diabetic properties (inhibition of α-amylase and α-glucosidase enzymes). The enzymes α-amylase and α-glucosidase are responsible for carbohydrate digestion, and the inhibition of these enzymes may decrease postprandial blood glucose levels by reducing the breakdown of polysaccharides into glucose. The bioactivities of many plant species have been shown to decrease earlystage diabetes-related enzyme activities [50]. In the present study, the IC 50 values of the carrot samples for inhibiting α-amylase ranged from 107.85 to 807.92 mg/mL. MiWC and MOC showed the highest activities against α-amylase, whereas the lowest activity was shown by NOC. Most carrot extracts had higher IC 50 for α-amylase than sour cherry, red grapefruit, pineapple, orange, and kiwi [27]. To clarify, α-amylase inhibitory activity is a result of bioactive compounds of plants such as glycosides, polysaccharides, steroids, and terpenoids [51]. Overall, mini carrots showed the highest inhibitory effect against α-amylase.
The IC 50 values of the carrot samples for inhibiting α-glucosidase ranged from 97.02 to 897.79 mg/mL. MiPC and MYC showed the highest inhibitory activities against αglucosidase, whereas the lowest result was shown by MiWC. The IC 50 values for αglucosidase were higher than chokeberry, apple, pear, and blackberry, a group of fruits recognized as important in the prevention of diabetes [27]. A previous study reported that the high anti-diabetic activity of onion is associated with its content of phenolic acids, flavonoids, and anthocyanins [52]. Polyphenol and carotenoid contents were shown to increase the anti-diabetic activities of fruits and vegetables [53,54]. A comparison of αamylase and α-glucosidase inhibitory activities of carrot samples revealed that MiWC had the highest α-amylase inhibitory activity but the lowest α-glucosidase inhibitory activity. Micro-sized samples showed the highest α-glucosidase activities. Moreover, α-amylase inhibitory activity demonstrated non-significant correlations with total carotenoid and chlorophyll contents (R 2 = 0.05), phenolic acid content (R 2 = 0.04), and procyanidin content (R 2 = 0.01). However, chemical contents of carrot varieties showed correlations with the α-glucosidase inhibitory activity that were different from those observed for α-amylase inhibitory activity; the following R 2 values were noted for procyanidin, and total carotenoid and chlorophyll contents: 0.50 and 0.01, respectively, and no correlation was observed with phenolic acid content (R 2 = 0.00). Therefore, phenolics are correlated with diabetes-related enzymes [55]; however, in the present study high positive correlation in carrot varieties was not determined.
Pancreatic lipase hydrolyses triacylglycerols into free fatty acids, bile salts, and fatsoluble vitamins [56]. In the present study, the IC 50 values for pancreatic lipase inhibitory activities ranged from 5.29 to 12.25 mg/mL. The highest IC 50 values were observed for NOC (5.29), MiYC (5.69), MYC (6.05), and NPC (6.12); however, NWC (12.25) and MWC (11.36) showed the lowest activities against pancreatic lipase. According to Fabroni [57], total anthocyanin content correlates with pancreatic lipase activity; however, in the present study, similar results were not observed, because normal-sized orange carrot exhibited elevated activities against pancreatic lipase. Moreover, in the literature lentil cultivars have shown potent activity (IC 50 from 6.26 to 9.26 mg/mL) against pancreatic lipase [58]. Hence, phenolics, ascorbic acid, and carotenoids are responsible for pancreatic lipase inhibition [59].
More importantly, the carrot samples exhibited higher inhibitory activities against pancreatic lipase than against diabetes-related enzymes. The pancreatic lipase inhibitory activity showed a positive correlation with the total carotenoid and chlorophyll contents (R 2 = 0.29), phenolic acid content (R 2 = 0.17), and to a low extent with procyanidin content (R 2 = 0.06).
The inhibitions of AChE and BuChE are thought to be important for the diagnosis and treatment of diseases such as bladder distention, glaucoma, myasthenia gravis, and Alzheimer's disease [60]. In the present study, carrot samples were investigated for potential inhibitor activities against AChE and BuChE. The IC 50 values of the carrot samples for AChE inhibition ranged from 10.14 to 18.96 mg/mL. The highest AChE inhibitory activities were observed for MPC (10.14 mg/mL), MWC (12.05 mg/mL), and MiWC (12.31 mg/mL), whereas the lowest result was observed for NPC (18.96 mg/mL). Moreover, in the literature white ginger exhibited more potent activities for AChE inhibition than red ginger variety [61]. In addition, a previous study showed that the IC 50 value of Hippophaë cultivars for inhibiting AChE ranged from 20.16 to 40.60 mg/mL [62]. Dipsacus root showed higher activity against AChE than the other parts of the plant [63]. The content of polyphenols and carotenoids in carrots was shown to correlate with activities of cholinesterase inhibitors [64,65]. In the present study, micro-sized and white carrot varieties showed elevated activities against AChE. Furthermore, the IC 50 values of the carrot samples for inhibiting BuChE ranged from 7.83 to 19.02 mg/mL. MPC (7.83 mg/mL), NPC (7.85 mg/mL), and NYC (8.01 mg/mL) showed the highest BuChE inhibitory activities, whereas the lowest activity was shown by NWC (19.02 mg/mL). Thus, normal-sized and purple carrot samples showed superior activities against BuChE. On the other hand, AChE inhibitory activity was correlated with the content of total carotenoid and chlorophyll (R 2 = 0.27), procyanidins (R 2 = 0.17), and phenolic acids (R 2 = 0.02), whereas BuChE inhibitory activity was correlated with the content of procyanidins (R 2 = 0.17), total carotenoid and chlorophyll (R 2 = 0.07), and phenolic acids (R 2 = 0.03). Therefore, phenolics are thought to be related to cholinesterase inhibition [66].
Overall, MiPC showed the highest activities against α-glucosidase, whereas MPC exhibited the highest activity against AChE and BuChE. NOC showed the highest inhibition activity against pancreatic lipase. Thus, purple carrot samples were more functional for enzyme inhibition than other coloured carrots of different sizes.

Conclusions
The present study evaluated 12 carrot varieties of different sizes and colours for bioactive compounds and their biological activities. The study results showed that purple carrot samples had the highest values for the content of polyphenolics and carotenoids, with the highest activities against cholinesterase. Normal yellow carrot showed the lowest values for the content of polyphenols. Micro white carrot demonstrated the lowest results for total phenolic acid, total carotenoid, and chlorophyll content. Moreover, the activities against diabetes-, obesity-and aging-related enzymes of the carrot varieties were found to be correlated with the content of phenolics and carotenoids.
Overall, the present study attempted to find the best carrot variety with high nutrients for food processing. Normal purple carrot showed the highest health-promoting activities in all tests, followed by mini purple carrot. Thus, different-sized purple carrot varieties can provide high contents of bioactive compounds to combat oxidative stress-related diseases.
However, the mechanisms of the pro-health action of carrots were not confirmed in this study, and their activities as α-amylase, α-glucosidase, lipase, acetylocholinesterase, and butyrylocholinesterase inhibitors was rather poor. Probably the mechanisms of their action are more complex, and the possible health-promoting effect results from the synergy of many compounds, including fibre, phytochemicals, vitamins, and minerals. Therefore, it would be worth continuing research on different varieties of carrots.
Author Contributions: Conceptualization, P.N. and A.W.; methodology, E.Y., A.W., J.O., and P.N.; formal analysis, P.N., J.O., and E.Y. writing-original draft preparation, E.Y. and P.N.; project administration and supervisor, P.N. All authors have read and agreed to the published version of the manuscript.