Abstract
The chemical composition of the different plant parts of green (Hibiscus cannabinus L. cv. Jangdae) and purple (H. cannabinus L. cv. Jeokbong) kenaf cultivars were investigated using high-performance liquid chromatography. Ten carotenoid and phenolic compounds were quantified in the different parts of green and purple kenaf. The accumulation of carotenoids and phenolic compounds in the different parts of both cultivars was slightly different. The total carotenoid content in all parts of purple kenaf was higher than that in green kenaf. In particular, lutein, β-carotene, and 9Z-β-carotene were found in all the plant parts of both purple and green kenaf. Most levels of these 3 carotenoids were higher in all parts of the purple cultivar than those found in the green cultivar. According to the detected phenolic compound content, the leaves of the green cultivar contained a higher level of total phenolics, even though the most levels of the individual phenolic compounds were higher in the purple cultivar due to the level of kaempferitrin, a major compound found in kenaf, being much higher than the levels observed for the other phenolic compounds. However, the flowers and stems of the purple cultivar had a higher level of total phenolics. Among the 10 different phenolic compounds, 7 (4-hydroxybenzoic acid, chlorogenic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin, and kaempferol) were present in all the plant parts of both kenaf cultivars. Purple kenaf leaves contain higher levels of 4-hydroxybenzoic acid, chlorogenic acid, and rutin, whereas the purple cultivar flowers have higher levels of p-coumaric acid, ferulic acid, and benzoic acid. This study provides valuable information on the chemical composition of different plant parts of green and purple kenaf cultivars.
Kenaf (Hibiscus cannabinus L.), a member of the Malvaceae family, is an annual plant originating from Africa, which is a fiber crop commercially cultivated for many industrial applications, such as the production of insulation mats, pulp, paper, fabrics, biocomposites, textiles, and absorption materials. 1 It has been used as an important medicinal crop to treat fever, bruises, and bilious conditions, and as a food additive to improve the fiber and calcium content. 1,2 According to previous studies, kenaf contains many bioactive compounds, such as phenolics, saponins, tocopherols, phytosterols, fatty acids, alkaloids, and phospholipids. 3 Furthermore, it has been reported that kenaf contains a high amount of vitamin C, phosphorus, iron, calcium, and nitrogen. 4 In particular, kenaf seed oils are good for human consumption because of their unique fatty acid composition and antioxidant properties. 5
Plant carotenoids belonging to the tetraterpenoids are natural pigments that exhibit yellow, orange, and red colors, 6 and can accumulate in the plastids. 7 Carotenoids are involved in various physiological processes observed in plants. These pigments act as antioxidants that protect chlorophyll from photo-oxidative damage and as light absorbers in photosynthetic membranes. 8
Furthermore, some carotenoids are important nutrients and others exhibit protective effects against several diseases. For example, provitamin A carotenoids (α- and β-carotene) are the precursors of vitamin A, which plays a role in the prevention of blindness, xerophthalmia, and premature death. 9,10
Plant phenolics are secondary metabolites found in most higher plant species, which exhibit protective effects toward a variety of stresses, including biotic stress (insect and pathogen), temperature (heat and cold), salinity (salt), water (drought and flooding), radiation (light and ultraviolet [UV]), and mechanical stress (wind, soil movement, and submergence). 11 -13 Moreover, the intake of plant products containing diverse phenolic compounds can be useful for human health because previous papers have reported that dietary phenolic compounds exhibit cardioprotective, 14 antioxidant, 15 anti-inflammatory, 16 anticancer, 17 and anticarcinogenic 18 properties.
It is necessary to find new ways to improve food taste and quality, and add health benefits for future consumers in the food industry. To determine whether a chosen plant is suitable to be added to food in the future, its qualitative and quantitative composition must be determined. Nevertheless, there are no reports on the analysis of the composition of the secondary metabolites (phenolics and carotenoids) found in the different plant parts of kenaf or on the phytochemical comparison of different kenaf cultivars. Therefore, the present study aims to describe the phytochemical variation among the different plant parts (roots, stems, leaves, and flowers) of green kenaf (H. cannabinus L. cv. “Jangdae”) and purple kenaf (H. cannabinus L. cv. “Jeokbong”) using high-performance liquid chromatography (HPLC).
Results
Carotenoids Found in the Different Plant Parts of Green and Purple Kenaf
Analysis of the different parts (roots, stems, leaves, and flowers) of green and purple kenaf revealed 10 carotenoids: violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, 13Z-β-carotene, α-carotene, β-carotene, 9Z-β-carotene, 15Z-phytoene, and all-E-phytoene (Table 1), which were present in the leaves of both cultivars. However, only 3 carotenoids (lutein, β-carotene, and 9Z-β-carotene) were found in the roots of both cultivars. The flowers of green kenaf contain 7 carotenoids (lutein, zeaxanthin, β-cryptoxanthin, 13Z-β-carotene, α-carotene, β-carotene, and 9Z-β-carotene). However, purple kenaf flowers contain 8 carotenoids (violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, 13Z-β-carotene, α-carotene, β-carotene, and 9Z-β-carotene). Four carotenoids (lutein, 13Z-β-carotene, β-carotene, and 9Z-β-carotene) were found in the stems of the green cultivar, but, in contrast, 6 (lutein, zeaxanthin, 13Z-β-carotene, α-carotene, β-carotene, and 9Z-β-carotene) were detected in the stems of the purple cultivar. The total carotenoid content in all parts of purple kenaf was higher than that of green kenaf. The total carotenoid content was 1928.7, 334.8, 96.2, and 0.8 µg/g dry wt. in the leaves, flowers, stems, and roots of purple kenaf, respectively, whereas the total carotenoid content was 1759.3, 266.9, 19.2, and 0.7 µg/g dry wt. in the leaves, flowers, stems, and roots of green kenaf, respectively. Among the 10 different carotenoids, lutein, β-carotene, and 9Z-β-carotene were found in all the plant parts (leaf, stem, root, and flower) of both purple and green kenaf. The levels of these 3 carotenoids were higher in all the parts of purple kenaf when compared with those of green kenaf. The lutein content was 1.32, 3.01, and 1.21 times higher in the leaf, stem, and root of purple kenaf, respectively, when compared with those of green kenaf. In the flowers of green kenaf, a 1.33-fold higher lutein content was detected when compared with that found in the purple kenaf flowers. The leaves, stems, and flowers of the purple cultivar contained 1.09-, 11.48-, and 1.36-fold higher levels of β-carotene than those of green kenaf, respectively. The levels of β-carotene were comparable in the roots of both cultivars. The 9Z-β-carotene content observed in the stems and flowers was 6.09- and 1.57-fold higher in the purple cultivar than that found in the green cultivar, respectively. The 9Z-β-carotene content was not significantly different between the leaves and roots of the 2 cultivars. 15Z-phytoene and all-E-phytoene were only detected in the leaves and β-cryptoxanthin was only found in the flowers of both kenaf cultivars. The 15Z-phytoene content was 1.41-fold higher in the leaves of purple kenaf and the β-cryptoxanthin content was 1.43-fold higher in the flowers of purple kenaf. However, the all-E-phytoene content was not significantly different in the leaves of both cultivars. Zeaxanthin and α-carotene were only detected in the leaves and flowers of green kenaf, whereas they were found in the leaves, stems, and flowers of purple kenaf. The zeaxanthin content was higher in the leaves and flowers of purple kenaf. The leaves of purple kenaf contained higher levels of α-carotene, but a higher α-carotene content was detected in the flowers of green kenaf. 13Z-β-carotenoid was absent in the roots of both types of kenaf. The stems and flowers of purple kenaf have a higher 13Z-β-carotenoid content, while the values were not significantly different in the leaves of both cultivars.
The Accumulation of Carotenoids (mg/g Dry Wt.) in Hibiscus cannabinus L. Cv. “Jangdae” and H. cannabinus L. Cv. “Jeokbong.”
Abbreviation: ND, not detected.
1Mean values with different letters were significantly different (P < 0.05, analysis of variance, Duncan’s multiple-range test).
Phenolic Compounds Found in the Different Plant Parts of Green and Purple Kenaf
HPLC analysis of the phenolic compounds found in the different plant parts of both purple and green kenaf revealed the presence of 10 different phenolic compounds (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin, kaempferitrin, quercetin, and kaempferol) (Table 2). All the plant parts of both kenaf cultivars contain most of these phenolic compounds, except caffeic acid, kaempferitrin, and quercetin. Specifically, caffeic acid and quercetin were not detected in the roots of both purple and green kenaf, and kaempferitrin was not found in the stems and roots of both kenaf cultivars. The total amount of phenolic compounds accumulated in the leaves, stems, roots, and flowers of green kenaf were 24409.8, 404.4, 169.3, and 6469.9 µg/g dry wt., accounting for 77.6%, 1.29%, 0.54%, and 20.6% of the total amount of all the plant parts, respectively. On the other hand, the total amount of phenolic acid found in the different plant parts of purple kenaf were 21787.3, 575.0, 159.5, and 6983.3 µg/g dry wt. accounting for 73.8%, 1.95%, 0.54%, and 23.7%, of the total amount of all the plant parts, respectively. Among the phenolic compounds accumulated in different parts of purple and green kenaf, the level of kaempferitrin was much higher than that of the other compounds. The amount of kaempferitrin in the leaves and flowers of green kenaf was 20890.1 and 2270.1 µg/g dry wt., respectively, whereas the amount of kaempferitrin in the leaves and flowers of purple kenaf was 17080.1 and 1320.0 µg/g dry wt., respectively. After kaempferitrin, the second and third highest phenolic compounds were chlorogenic acid and rutin. The chlorogenic acid contents observed in the flowers of green and purple kenaf were 3756.9 and 5008.0 µg/g dry wt., respectively, and the rutin contents in the leaves of both kenaf cultivars were 974.1 and 1102.1 µg/g dry wt., respectively. Furthermore, the amounts of 4- hydroxybenzoic acid, caffeic acid, quercetin, and kaempferol were higher in the leaves of both kenaf cultivars and the amounts of chlorogenic acid, p-coumaric acid, ferulic acid, and benzoic acid were higher in the flowers of both cultivars. Most phenolic compounds (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, and rutin) were found in higher amounts in all the plant parts of purple kenaf when compared to green kenaf. However, the levels of quercetin, kaempferitrin, and kaempferol were higher in most of the plant parts of the green cultivar.
Accumulation of Phenolic Compounds (mg/g Dry Wt.) in Hibiscus cannabinus L. Cv. “Jangdae” and H. cannabinus L. Cv. “Jeokbong.”.
Abbreviation: ND, not detected.
1Mean values with different letters were significantly different (P < 0.05, analysis of variance, Duncan’s multiple-range test).
The principal component analysis (PCA) results obtained for the carotenoid and phenolic compound profiles clearly show the marked variance among the roots, stems, leaves, and flowers of H. cannabinus L. cv. “Jangdae” and H. cannabinus L. cv. “Jeokbong,”, respectively. Two components of the score plot accounted for 92% of the total variance (principal component 1 [PC1], 67.7%, and principal component 2 [PC2], 24.3%). PC1 shows the separation of the purple and green kenaf leaves from the other plant parts. Moreover, the metabolomes of the purple and green kenaf flowers were separated by PC2 above and below. Comparing the metabolic loadings in PC1 and PC2, the significant compounds in PC1 observed in the loading plot were 4-hydroxybenzoic acid, rutin, chlorogenic acid, kaempferitrin, caffeic acid, and quercetin, for which the eigenvector values were >0.20 (Figure 1). The most important metabolites of PC1 in the loading plot were β-carotene, 9Z-β-carotene, 13Z-β-carotene, β-cryptoxanthin, and p-coumaric acid with eigenvector values of 0.27027, 0.26918, 0.26893, −0.043857, and –0.043556, respectively. The significant metabolites of PC2 were kaempferol and ferulic acid, in which the eigenvector values were 0.19259 and –0.44958, respectively.
(A) Scores and (B) loading plots of principal components 1 and 2 obtained from principal component analysis of the secondary metabolite data measured for the roots, stems, leaves, and flowers of Hibiscus cannabinus L. cv. “Jangdae” and H. cannabinus L. cv. “Jeokbong” respectively. GF, green kenaf flower; GL, green kenaf leaf; GR, green kenaf root; GS, green kenaf stem; PC, principal component; PF, purple kenaf flower; PL, purple kenaf leaf; PR, purple kenaf root; PS, purple kenaf stem.
Discussion
In this study, 10 carotenoids (violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, 13Z-β-carotene, α-carotene, β-carotene, 9Z-β-carotene, 15Z-phytoene, and all-E-phytoene) and 10 phenolics (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin, kaempferitrin, quercetin, and kaempferol) were detected in the roots, stems, leaves, and flowers of purple and green kenaf, respectively. These findings were consistent with previous biochemistry analytical studies using liquid chromatography (LC)-mass spectrometry analysis, which indicated the presence of chlorogenic acid, kaempferol, quercetin, and kaempferitrin. 19 In addition, HPLC analysis of kenaf seeds showed the presence of 4-hydroxybenzoic acid. 20 Ryu et al have reported the presence of caffeic acid in the leaves, bark, and flowers of kenaf, and p-coumaric acid in the seeds of kenaf using ultra performance liquid chromatography. 21 Furthermore, seed oils have been shown to contain 4-hydroxybenzoic acid, caffeic acid, p-coumaric acid, and ferulic acid using LC analysis. 22 Furthermore, Mamatha et al reported the identification of lutein, zeaxanthin, α-carotene, β-carotene, neoxanthin, and violaxanthin in kenaf 23 and Djuikwo et al(2011) reported that the leaves of kenaf cultivated in Africa contain β-carotene, α-carotene, 13-cis-β-carotene, 9-cis-β-carotene, lutein, zeaxanthin, and β-cryptoxanthin. 24 In addition, neoxanthin, lutein, and β-carotene have been detected in kenaf leaves. 25
In this study, the composition of carotenoids and phenolic compounds varied in the different plant parts of green and purple kenaf. In both cultivars, the leaves contained higher amounts of most of the phenolic compounds than those observed in the other plant parts. In particular, the levels of kaempferitrin accounted for 85.6% and 78.4% of the total phenolic content in both cultivars, respectively. This was consistent with a previous study that reported a wide variation in the production of kaempferitrin in different parts of kenaf and that young and mature leaves contain higher levels of kaempferitrin than those in the roots, stems, young flowers, and old flowers, respectively. 26 Similarly, Ryu et al (2017) reported that leaf water extracts exhibited a higher amount of total phenolic compounds than those of the flower, seed, and stem bark extracts. However, the water extracts of flowers contain a higher total flavonoid content than those of the leaf, seed, and stem bark extracts. 21 Furthermore, the composition of carotenoids varied in the different parts of green and purple kenaf. The leaves also contain higher levels of most carotenoids and the levels of β-carotene, lutein, and all-E-phytoene were higher than those of the other carotenoids. These findings are in accordance with previous studies reporting the wide variation in the accumulation of carotenoid in kenaf leaves and that lutein and β-carotene were representative carotenoids found in kenaf leaves. 23 -25
The variations in the results obtained for the roots, stems, leaves, and flowers of kenaf and the different cultivars of kenaf were not surprising and were consistent with previous studies demonstrating the variation in the chemical composition within the plant species, varieties, and different plant parts. These previous studies have described the great variation in the phenolic and carotenoid composition of different parts of H. cannabinus, 26 Agastache rugosa, 27 Momordica charantia, 28 and different cultivars of Liriope platyphylla, 29 and Lactuca sativa. 30
According to the World Health Organization, ~80% of the global population is dependent on herbal medicines for primary health care. 31,32 This current study shows that the different parts of kenaf and different cultivars of kenaf contain various phenolics and carotenoids, which show a variety of biological properties, including antioxidant, 33,34 anticancer, 35 36,37 and anti-inflammatory activities. 38,39 Furthermore, kenaf seed extracts inhibit inflammation, 40 exhibit cytotoxic effects against human cancer cell lines, 41 display high cholesterol-lowering properties, 42 and antioxidant activity. 43 These properties can be exploited for the prevention and treatment of several diseases. Thus, this study suggests the potential use of kenaf roots, stems, leaves, and flowers of different kenaf cultivars in traditional herbal medicine applications.
Conclusion
In summary, this is the first study to quantify carotenoids and phenolic compounds found in the different plant parts of green and purple kenaf. The carotenoid and phenolic contents in the different parts of green and purple kenaf varied widely. Furthermore, 10 different carotenoids and 10 phenolic compounds were detected in both kenaf cultivars. In particular, the highest total amounts of phenolics and carotenoids were found in the leaves of both cultivars as well as flowers of both cultivars contained high levels of phenolics and carotenoids. Specifically, Jeokbong cultivar had the highest amounts of phenolic compounds, except kaempferitrin, and carotenoids when compared with the Jangdae cultivar. In contrast, the levels of kaempferitrin, one of the representative phenolic compounds found in kenaf, were much higher in the different parts of the green kenaf cultivar when compared to those of the purple kenaf cultivar. Therefore, this study suggests that Jeokbong cultivar can be a potential source of carotenoids and phenolic compounds. However, Jangdae cultivar can be regarded as a better source of kaempferitrin for human consumption. Considering their nutrient value, leaves and flower of both purple and green cultivars can be used as a vegetable or tea material. The results of the present study will help enhance food quality and taste, and develop new varieties with specific metabolite compositions.
Experimental
Plant Materials
The seeds of Jangdae and Jeokbong (Figure 2) were obtained from the Advanced Radiation Technology Institute (Jeongeup, Jeonbuk, Korea). Two varieties of kenaf (Jangdae and Jeokbong) were cultivated in a greenhouse at Chungnam National University. After 3 months, different plant parts (roots, stems, leaves, and flowers) were harvested and washed using distilled water. The plant samples were then frozen using liquid nitrogen and lyophilized using a lyophilizer (Ilshin Lab Co. Ltd., Yangju, Korea). The dried samples were ground into fine powder for further HPLC analysis of the carotenoids and phenolic compounds.
A comparison of the 2 types of kenaf studied: (A) green kenaf (Jangdae) and (B) purple kenaf (Jeokbong). This picture was taken from our previous study. 43
Extraction of Phenolic Compounds From 2 Varieties of Kenaf and Their HPLC Analysis
According to a previously reported method, 44 the phenolic compounds were extracted and analyzed from the different plant parts (roots, stems, leaves, and flowers) of green and purple kenaf. Three biological replicates were used for extraction and analysis. The extraction step using 80% methyl alcohol (v/v) was performed 3 times. Specifically, fine dried powders (100 mg) of the different plant parts of kenaf were added into 15 mL tubes, respectively, followed by adding 1.5 mL of 80% methyl alcohol (v/v). Afterward, the samples were sonicated at 25 °C for 60 minutes and then centrifuged at 12,000 × g for 10 minutes. The supernatant was collected in a fresh tube. Next, the crude extracts were evaporated using nitrogen gas and redissolved in 2 mL of 80% methyl alcohol (v/v). After filtration through a 0.45 µm syringe filter, the resulting extract was placed into a 2.0-mL vial. HPLC analysis was performed using an HPLC instrument (NS-4000, Futecs Co., Daejeon, South Korea) equipped with a UV-Vis detector, autosampler (NS-6000, Futecs Co., Daejeon, South Korea), and C18 column (OptimaPak, 250 × 4.6 mm, 5 µm, RStech Co., Daejeon, South Korea) to isolate the phenolic compounds. The analysis conditions and gradient program were taken from a previous study. 44 Individual peaks were identified based on the retention times of the respective chemical standards and spiking experiments. Moreover, the calculation was conducted using the corresponding calibration curves. Standards of kaempferol (≥97%), rutin hydrate (≥94%), quercetin dihydrate (≥98%), kaempferitrin (≥97%), benzoic acid (≥99.5%), trans-ferulic acid (≥99%), p-coumaric acid (≥98%), caffeic acid (≥98%), chlorogenic acid (≥95%), and 4- hydroxybenzoic acid (99%) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA).
Extraction of Carotenoids From Varieties of Kenaf and HPLC Analysis
According to a previously reported method, 45 carotenoids were extracted and analyzed from different plant parts of green and purple kenaf. Three biological replicates were used for extraction and analysis. Fine dried powders (20 mg) of the different plant parts of kenaf were added to 15-mL tubes, followed by the addition of 3 mL of absolute ethyl alcohol containing 0.1% ascorbic acid (w/v). The samples were then incubated at 85 °C in a water bath for 5 minutes and 120 µL of 80% potassium hydroxide (w/v) was added. The crude extracts were then vortexed for 25 seconds and incubated at 85 °C in a water bath for 10 minutes. The extracts were then placed in an ice bath for 5 minutes and 0.1 mL of an internal standard (β-apo-8′-carotenal in ethyl alcohol; 25 µg/mL), 1.5 mL of distilled water, and 1.5 mL of hexane were added. After centrifugation at 1200 × g for 5 minutes at 4 °C, the upper layer was transferred to a 50-mL tube. The centrifugation procedure was repeated 2 more times using the residue. The collected supernatants were dried using nitrogen gas and then dissolved in 0.25 mL of a 50:50 (v/v) methanol–dichloromethane solution. An HPLC system (Agilent 1100, Agilent Technologies, Massy, France) equipped with a photodiode array detector and C30 column (YMC Carotenoid, 250 × 4.6 mm, 3 µm, YMC Co., Kyoto, Japan) was used to isolate the carotenoids. The analysis conditions and gradient program were taken from a previous study. 45 The identification of the individual carotenoids was conducted with the help of our previous guidelines and the combined use of the retention time and coelution of β-apo-8′-carotenal used as an internal standard. Quantification of the individual carotenoids was conducted using their corresponding calibration curves. Standards of violaxanthin (≥95%), lutein (≥96%), zeaxanthin (≥95%), β-cryptoxanthin (≥95%), 3Z-β-carotene (≥96%), β-carotene (≥96%), 9Z-β-carotene (≥99%), α-carotene (≥95%), phytoene (≥98%), and β-apo-8’-carotenal (≥97%) were purchased from CaroteNature (Lupsingen, Switzerland).
Statistical Analysis
Statistical analysis was performed using the statistical analysis system (SAS) software (version 9.4, 2013; SAS Institute, Inc., Cary, NC, USA). The data acquired from the HPLC analyses of the roots, stems, leaves, and flowers of green and purple kenaf were analyzed using analysis of variance and Duncan’s multiple range test at P <0.05. PCA consisted of score plots to visualize the contrast between the different plant parts of H. cannabinus L. cv. “Jangdae” and H. cannabinus L. cv. “Jeokbong,” respectively, and loading plots to account for cluster separation.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by grants from the Research Program of KAERI, Korea.
