A Review Article of an Antioxidant Activity of Daucus Carota Root (Carrot)

Fruits and vegetables are rich sources of nutrients that contain phytochemicals (also known as bioactive compounds), which are recognised for their nutraceutical effects and health benefits [1]. The cultivated carrot (Daucus carota L.) is one of the most important vegetable plants in the world because of its high yield potential and use as fresh or processed product. With an annual world production (carrots and turnips) of >428 million tons and a total growing area of about 11.5 million hectares [2]. Carrots rank among the top 10 vegetable crops in the world [3]. They play a major role in human nutrition, because of their high dietary value and good storage attributes [4,5]. Phytochemicals contribute to the dietary value of carrots and comprise mainly four types; namely, phenolic compounds, carotenoids, polyacetylenes, and ascorbic acid. This review article comprehensively describes the occurrence, biosynthesis, factors affecting concentration, and health benefits of phytochemicals found in Daucus carota.

2. Methods:

The literature for this review paper was retrieved from Google Scholar by using the following key words: occurrence of phenolics or phenols or phenolic acids, carotenoids, polyacetylenes, and ascorbic acid or vitamin C in carrot; biosynthesis of phenolics, or phenols or phenolic acids or hydroxycinnamic acids or chlorogenic acids, carotenoids, polyacetylenes, and ascorbic acid or vitamin C, in carrot; factors affecting the concentration of phenolics or phenols or phenolic acids, carotenoids, polyacetylenes, and ascorbic acid or vitamin C in carrot; nutritional importance or nutritional benefits of phenolics or phenols or phenolic acids, carotenoids, polyacetylenes, and ascorbic acid or vitamin C in carrot; health effects of carrot phenolics, carotenoids, polyacetylenes, and ascorbic acid/vitamin C after carrot consumption. The key words: structures of phenols or phenolic acids, carotenoids, polyacetylenes, and ascorbic acid or vitamin C in carrot, were searched in the NCBI website and redrawn in MS word using PNG format. Two hundred and fifty-five (255) articles including original research papers, books, and book chapters were downloaded, of which one hundred and thirty articles (130) most relevant to the topic were selected for writing the review article. The rejected research papers were too old or irrelevant. Literature was summarised according to the four types of phytochemicals found in carrots; namely, phenolics, carotenoids, polyacetylenes, and ascorbic acid. Under each phytochemical, the literature was summarised according to occurrence, biosynthesis, factors affecting concentrations, and resulting health benefits.

Reagent and chemicals:

Folin Ciocalteu’s Phenol reagent, sodium carbonate, gallic acid, quercitin, 10%Aluminium chloride, ethanol, 2, 2 Diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, methanol, 2,2’-Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), potassium persulfate, beta carotene, chloroform, linoleic acid, butylated hydroxytoluene (BHT) were purchased from Sigma chemical.

Preparation of plant extracts:

Dry powder of Daucus carota were taken from local market.

Phytochemical screening:

Total phenolic content

The total phenolic contents in baby carrot and carrot crude extracts were determined by using the Folin-Ciocalteu method [6]. 50 µl of extracts (1 mg/ml) or standard solution of gallic acid (6.25, 12.5, 25, 50, 100 µg/ml) in distilled water were added to 50 µl of distilled water. Distilled water was used as blank. 50 µl of 10% Follin Cicocalteu’s phenol reagent and 50 µl of 1 M sodium carbonate solution were added to the mixture in a 96-well plate. Reactions were incubated for 60 minutes at room temperature and protected from light. The absorbance was measured at 750 nm with a Microplate Reader [7]. Total phenolic contents in baby carrot and carrot were expressed as mg Gallic Acid Equivalents per gram of dry plant material [8]. All samples were analyzed in triplicate.

Total flavonoid content:

Total flavonoid content in baby carrots and carrots determined by the aluminium chloride colorimetric assay. 50 µl of extracts (1 mg/ml) or standard solution of quercetin (6.25, 12.5, 25, 50, 100 µg/ml) in 80% ethanol was added to 10 µl of 10% the aluminium chloride solution and followed by 150 µl of 95% ethanol. 80% ethanol was used as reagent blank. 10 µl of 1 M sodium acetate was added to the mixture in a 96 well plate. All reagents were mixed and incubated for 40 minutes at room temperature protected from light. The absorbance was measured at 415nm. Total flavonoid contents in baby carrots and carrots were expressed as mg Quercitin Equivalents (QE) per gram of dry plant material. All samples were analyzed in triplicates.

Free radical scavenging activity:

DPPH assay:

The 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity was performed [8]. Briefly 20 µl of carrot extracts (1 mg/ml) or standard solution of ascorbic acid (3.125, 6.25, 12.5, 25, 50 µg/ml) in absolute methanol was added to 180 µl of DPPH reagent in 96 well plate. Absolute methanol was used for reagent blank. All reagents were mixed and incubated for 30 minutes at room temperature, protected from light. The absorbance was measured at 517 nm with a Microplate Reader [9]. Experiments were done in triplicates. The percentages of the DPPH free radical scavenging activity were calculated as follows:

% Scavenging activity = [(Abs control–Abs sample) Abs control] ×100

The percentages of the DPPH free radical scavenging activity were determined by comparing with free radical scavenging activity of ascorbic acid and expressed as mg vitamin C Equivalent Antioxidant Capacity (VCEAC) per gram of dry plant material.

ABTS assay:

The ABTS free radical scavenging activity was performed [9]. The 2, 2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) or ABTS•+ formation was generated by oxidation of ABTS reagent and potassium sulphate. 20 µl of extracts (1 mg/ml) or ascorbic acid standard (3.125, 6.25, 12.5, 25, 50 µg/ml) in absolute ethanol was added to 180 µl of ABTS•+ working reagent in a 96-well plate. Absolute ethanol was used as reagent blank. The plate was incubated for 45 minutes at room temperature in a dark condition. The absorbance was measured at 734 nm with a Microplate Reader [10]. Experiments were all done in triplicates.

% Scavenging activity = [(Abs control–Abs sample) Abs control] ×100

The percentages of the ABTS free radical scavenging activity were determined by comparing with calibration curve of ascorbic acid and expressed as mg vitamin C Equivalent Antioxidant Capacity (VCEAC)/g dry plant material.

MATERIALS AND METHODS:

No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.

Composition of Carrot Powder:

The chemical composition of carrot powder samples is shown in Table 1. The moisture content of carrot powder was 4.5%, whereas ash, fat, TP, and fiber con tents were 4.80, 2.75, 8.25, and 11.94% on a dry basis, respectively. It was found that ash, fat, TP, and fibre in carrot powder were approximately 6.80, 2.90, 9.20, and 7.20%, respectively, on a dry basis [11]. Another study stated that carrot samples have moisture content of 11.70%, whereas ash, fat, TP, and fibre contents were approximately 13.40, 5.40, 10.40, and 13.70% on a dry basis, respectively [12]. The total carbohydrate content of carrot powder in current research recorded was around 72.30%, while the caloric value was 346.8 kcal/100 g on a dry basis. Our results were in agreement with those reported as they observed that carbohydrate and caloric values in carrot powder were 68.3% and 336.0 kcal/100 g on a dry basis, respectively [13]. Potassium, phosphorus, calcium, sodium, magnesium, iron, and zinc contents in carrot powder are illustrated in Table 1. In the present study, potassium, phosphorus, calcium, sodium, magnesium, iron, and zinc were approximately 532.7, 199.3, 880.0, 615.0, 171.7, 20.7, and 3.4 mg/100 g on a dry basis, respectively. Similar findings were exemplified, as they found that zinc content in carrots was 3.2 mg/100 g on a dry basis [14]. Moreover, it was reported that calcium, phosphorus, and iron contents in carrot pulp waste were 314.95, 317.77, and 12.65 mg/100 g on a dry basis, respectively [15].

Invitro process in carrot Powder:

Vitamin C, β-carotene, vitamin A, total phenolics, and antioxidant activity of carrot powder samples are presented in Table. Data showed that vitamin C content was 50.0 mg/100 g on a dry basis. It was reported that ascorbic acid was 8.0 mg/100 g of fresh orange-coloured carrots [16].

in the current study, β-carotene content was 123.0 mg/100 g on a dry basis. However, another study observed lower content of β-carotene in carrot powder at a rate of 1,154.20 µg/g or 115.42 mg/100 g [17]. Whereas another reported higher β-carotene (254.0 mg/100 g) on a dry basis when compared with our findings [18]. Such differences could be attributed to the variety and environmental factors. Furthermore, The retinol equivalent of carrot powder was about 20,500 (RE/100g), which was obtained using the standard conversion formula. Likewise, others studies examined 5 groups of fresh carrots and found that the β-carotene and vitamin A contents were 12,300 µg/100 g and 2,054.1 RE/100 g, respectively [19].The total phenolics content  in carrot pow der was 203.6 mg/100 g on a dry basis sample, whereas it was reported that the range of total phenolic content in 15 varieties of orange carrot ranged from 18.7 to 33.8 mg/100 g as gallic acid equivalents on a fresh weight basis [20]. The antioxidant activity of carrot powder (determined by DPPH scavenging activity) was 64.45%. Our value is higher than the values reported in the study conducted by which ranged from 7.45 to 34.9% [21]. Another study by estimated radical scavenging activity using a stable DPPH radical. They found that the DPPH ranged from 3.5 to 13.7% in 15 varieties of orange carrot extracts (on a fresh weight basis) [22]. Moreover, the importance of carotenoids (which are located in orange carrots) was referred to as a valuable antioxidant component that can neutralize the effect of free radicals [23].

Cultivar:

A seven to eleven-fold difference in-carotene concentration was observed in cultivars with different genetic makeups [24]. There is controversy in the literature about the highest number of carotenoids present in different carrot genotypes. In previous studies, it was found 2.3 times more-carotenes in purple carrots than orange varieties. Similar results were presented [25,26]. However, according to recent studies, higher contents of and-carotene are present in orange carrots, lutein in yellow carrots, lycopene in red carrots, anthocyanin in the roots of purple carrots, and phenolic compounds in black carrots [27]. studied the effect of genetic variability on carotenoids in carrots of different colours (genotypes) and found that the range of carotenoids in yellow and purple carrots is 469 to 605 µg/100 g, while 10 times more carotenoids are present in orange carrots. The highest carotenoids’ content, particularly-carotene (170 mg/kg), is present in dark orange carrots, whereas purple carrots have the lowest-carotene content (3.2 mg/kg). Suggestions for retaining carotenoid concentrations include the usage of cultivars known to have higher ranges of the useful compounds, and those which might be more suited to the local weather and geographical location. Gene expression partly describes the differences in carotenoids’ accumulation in the secondary phloem and xylem of the fleshy roots of carrots [28].

Environment:

Environmental conditions during growth and packaging alter the level of carotenoids, sugars, and volatiles [29]. However, results may vary when research is conducted under different conditions. Other researchers and emphasise that crops grown in sandy soil tend to build up fewer provitamin A carotenoids than those grown in clay soils [30].

Storage Conditions and Temperature:

Retail storage of carrots often takes place at a temperature range of 18 to 22 C. Carrots can be subjected to these temperatures for a few days. Storage’s effect on carrot-carotene is inconsistent based on different temperature levels [31]. And-carotene concentrations increased up to 35% and 25%after three days of storage and up to 42% and 34% after ten days of storage in Nantes carrots stored at 2 C and 90% relative humidity. Significant increases in-carotene were observed in both Nevis and Kingston cultivars stored at 20 C for seven days. Longer storage periods of 21 days at 20 C have a negative effect on and-carotene [32]. After four weeks of storage, beta-carotene was enhanced from 8% to 23% at 4 C, compared to the levels at harvesting time reported that-carotene contents were reduced after eight days of storage at different temperatures, by 46% (7.5 to 8.5 C), 51% (17 to 21 C), and 70% (22 to 37.5 C) [33]. Some studies also evidenced slight variations in ↵ or-carotene when carrots were stored at 0 C, even for six months [34].

Health Benefits of Carotenoids:

The dietary intake of carotenoids, especially vitamin A, has been related to the protection of DNA, proteins, and lipids from oxidative damage; as well as to the maintenance of the normal function of the immune system, normal skin, normal mucosal membranes, and normal vision [35]. Digested purple carrot extract when passed through the colon mucosal cells, decreases oxidative DNA damage by 20.7%, protecting colon cells against reactive oxygen species stress [36]. Beta-carotene, which is present in purple and orange carrots, is the most widely studied carotenoid so far, due to its significance in medical science. Dietary provitamin A carotenoids derived from plants are a major source of our vitamin A needs, and bioconversion to retinol may account for one-third of total retinol intake in developed countries. Vitamin A is essential for normal organogenesis, immune functions, tissue differentiation, and eyesight [37]. Alpha-carotene, beta-carotene, and-cryptoxanthin obtained from carrot consumption are the carotenes that are converted into retinol in the human body. Lutein from yellow carrot and its isomer, zeaxanthin, both accumulate in the centre of the retina (also known as the macula) of the eye. These are the only carotenoids that pass through the retinal barrier and form the macula in the eye. The macula enhances eyesight through its light-filtering characteristics. They are also powerful antioxidants and essential for healthy eyes. They protect eyes from diseases by absorbing harmful blue light that enters the eye. Lutein is also the most dominant carotenoid in brain tissue and the predominant carotenoid in the developing primate brain and retina. The amount of lutein is twice as much in paediatric brains than in adult brains, indicating its role in neural growth, and it may play a role in biological functions, including anti-oxidation, anti-inflammation, and in structural activity. It shields neural tissue, especially during infancy when the retina and brain are continuously in a state of change after birth. In adults, it is linked to cognitive health, and its supplementation enhances cognition. High ingestion (near 6 mg per day) of lutein is associated with low risk of muscular degeneration during old age, although actual intake of lutein varies between 1 and 2 mg per day in adults. It can also prevent the production of harmful free radicals, such as reactive oxygen species, via physical or chemical quenching of singlet oxygen.

Ascorbic Acid:

l-ascorbic acid or vitamin C is one of the most abundant water-soluble low molecular weight antioxidants found throughout the kingdom Plantae. It is known to play a central role in regulating the cellular redox potential in cells [38]. As humans and some other primates lack the ability to synthesize and store vitamin C, they depend on fresh fruits and vegetables to cover their daily requirements (75–90 mg RDA). All recent studies point toward a diet rich in vitamin C for improving human health [39]. Suggest that vitamin C should be a clear target for the nutritional enhancement of horticultural crops. The accumulation of vitamin C within the same species may vary between different cultivars tissue types and developmental stages. Regardless of this variability, vitamin C is tightly regulated through net biosynthesis, recycling, degradation/oxidation, and/or intercellular and intracellular transport [40].

Occurrence of Ascorbic Acid:

There are many authors reporting on differences between carrot cultivars regarding the content of vitamin C [41]. According to [42]. Vitamin C content in six carrot cultivars ranged from 54 mg/kg to 132 mg/kg, while concentrations as low as 21 mg/kg [43]. And high as 775 mg/kg of Vitamin C may accumulate at up to 20 mM in chloroplasts, and occurs in almost all parts of the cell [44]. Dark orange carrots contain 4 times more vitamin C than yellow, purple and orange carrots [45].

Biosynthesis of Ascorbic Acid:

In plants, four alternative pathways for ascorbic acid biosynthesis have been reported; namely, the d-mannose/l-galactose (d-Man/l-Gal) pathway, myoinositol pathway, galacturonate pathway, and l-glucose pathway. The ten-step d-Man/l-Gal pathway is the most acceptable for ascorbic acid biosynthesis in carrots (Figure). d-Glucose-6-phosphate (d-glucose-6-P), obtained from the hexokinase of d-glucose, is converted into its furanose derivative d-fructose-6-phosphate (d-fructose-6-P in the presence of phosphor glucose isomerase (PGI). Phosphomannose isomerase (PMI) convertsd-fructose-6-P into d-mannose-6-phosphate (d-mannose-6-P). d-Mannose-6-P subsequently rearranges (phosphate moves from C6 to C1) due to the catalytic action of phosphomannose mutase (PMM) to yield d-mannose-1-phosphate (d-Mannose-1-P).

Figure: Schematic representation of the biosynthetic path way of ascorbic acid in carrot: (1) hexokinase; (2) phosphoglucose isomerase; (3) phosphomannose isomerase; (4) phosphomannose mutase; (5) guanosine diphosphate (GDP)-mannose pyro phosphorylase; (6) GDP-mannose epimerase; (7) GDP l-galactose phosphorylase; (8) l-galactose-phosphatase; (9) l-galactose-dehydrogenase; (10) l-galactose 1,4-lactone dehydrogenase.