Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

Jae Il Lyu; Jung Min Kim; Dong-Gun Kim; Jin-Baek Kim; Sang Hoon Kim; Joon-Woo Ahn; Si-Yong Kang; Jaihyunk Ryu; Soon-Jae Kwon

doi:10.9787/PBB.2020.8.1.1

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Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

Jae Il Lyu¹, Jung Min Kim^1,2, Dong-Gun Kim^1,3, Jin-Baek Kim¹, Sang Hoon Kim¹, Joon-Woo Ahn¹, Si-Yong Kang¹, Jaihyunk Ryu¹, Soon-Jae Kwon^1,*

Plant Breeding and Biotechnology 2020;8(1):1-10.
Published online: March 1, 2020

DOI: https://doi.org/10.9787/PBB.2020.8.1.1

¹Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 5622, Korea

²Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju 61186, Korea

³Department of Life Resources, Graduate School, Sunchon National University, Suncheon 57922, Korea

*Corresponding author Soon-Jae Kwon, soonjaekwon@kaeri.re.kr, Tel: +82-63-570-3312, Fax: +82-63-570-3813

• Received: October 15, 2019 • Revised: November 12, 2019 • Accepted: November 20, 2019

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
References

Abstract

The leaves of roselle (Hibiscus sabdariffa L.) have been used as a traditional folk medicine that has diuretic and mild laxative effects. Roselle is cultivated in many countries for medicines and food. However, studies on the variation of functional compounds in different accessions are relatively limited. In this study, we investigated the phenolic compound content of leaf extracts from 49 different roselle accessions from a worldwide collection by ultra-high performance liquid chromatography mass spectrometry. The aim was to chemically classify and assess the pharmacological properties of the phenolic compounds in the leaves. The phenolic compounds were identified as neochlorogenic acid, cryptochlorogenic acid, rutin, rutin isomer, isoquercitin, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, quercetin, quercetin isomer, and kaempferol. These compounds were present at significantly different levels among the accessions. The most abundant phenolic compound was rutin, which was found in 45 accessions where the total phenolic compound content was 18.75-46.51 mg/g (average 31.52 mg/g). The phenolic compounds were classified into four groups. The two accessions with the highest total phenolic compound content were from Ghana (PI286316) and Senegal (PI275413). The hierarchical cluster analysis of the 49 roselle accessions showed that they formed five groups according to their phenolic compound content. Our results will be useful for the selection of roselle genotypes with improved functional compounds.
Key words: Roselle, Phenolic compounds, Rutin, UPLC

INTRODUCTION

Roselle (Hibiscus sabdariffa L.), which belongs to family Malvaceae, is a valuable medicinal crop originating from West Africa that contains a variety of functional compounds (Ali et al. 2005; Wang et al. 2014; Wang et al. 2016). Roselle is cultivated widely in Africa, Asia, and America for its leaves, seeds, stem, and calyces, which are used as food (Babatunde 2003; Sayago-Ayerdi et al. 2007; Ochani and D’Mello 2009). Roselle leaves have three to five lobes and they are arranged alternately on the stem (Daudu et al. 2015). The leaves are rich in flavonoids, which may contribute to their antioxidant capacity (Mohd-Esa et al. 2010; Wang et al. 2014; Wang et al. 2016). Roselle leaves are used traditionally for their diuretic, choleretic, febrifugal, and hypotensive effects (Lin et al. 2007; Kuo et al. 2012; Guardiola and Mach 2014). Extracts from roselle leaves have a variety of biological activities, including antioxidant, antitumor, antihyperammonemic, antiatherosclerotic, antifilarial, and antihyperlipidemic activities (Fernández-Arroyo et al. 2011; Yang et al. 2011; Chen et al. 2013; Guardiola and Mach 2014). Roselle leaves are used as a vegetable in soup and salad mainly in Africa, but are disregarded as a food source in many countries (Wang et al. 2014; Daudu et al. 2015; Wang et al. 2016).

Plants are used widely as sources of food and drugs and the phytochemical content of many of them has been investigated (Wang et al. 2016; Ryu et al. 2016; Kwon et al. 2018; Ryu et al. 2019). Phytochemicals are bioactive compounds that provide abundant health benefits (Zhen et al. 2016; Ryu et al. 2017a). The classification and analyses of phytochemicals have revealed new medicinal and industrial resources among naturally available materials that could be developed into profitable crops (Obouayeba et al. 2015; Ryu et al. 2017a; Kwon et al. 2018). Flavonoids and phenolic acid are bioactive compounds that have important pharmacological effects. They are abundant in roselle leaves where the major flavonoids are rutin, quercetin, and kaempferol and its derivatives (Chen et al. 2013; Wang et al. 2016; Zhen et al. 2016).

Breeders have developed new cultivars of various medicinal crops that are more nutritious and have improved levels of bioactive compounds (Sheela and Sheena 2014; Ryu et al. 2017a). Although the flavonoid content of roselle leaves has been studied (Wang et al. 2014; Wang et al. 2016; Zhen et al. 2016), a limited number of accessions was used and the flavonoid content of roselle grown in Korea has not been investigated so far. The aim of the present study was to investigate the flavonoid content of roselle leaves from worldwide accessions that could be grown commercially in Korea.

MATERIALS AND METHODS

Plant materials

Forty-nine roselle accessions were used in this study (Table 1, Fig. 1). These accessions were obtained from the United States Department of Agriculture (USDA) in 2016 and were cultivated from 2016 to 2017 at the Korean Atomic Energy Research Institute (Jeongeup, Korea). The leaves of each accession were harvested in August 2017 for functional compound analysis. The leaves were handpicked from plants in three separate plots on the same plantation. Seeds were planted in plots (3 × 4.2 m) with alternate row spacings of 20 and 60 cm. Fertilizer (N:P:K 4:2:2 w/w/w) was applied at 600 kg/ha shortly after seedling. Manure was spread before planting, and the plants were not fertilized after planting.

Ultra-high performance liquid chromatography (UPLC) mass spectrometry (MS) analysis

Phenolic compounds were analyzed using a UPLC system (CBM-20A, Shimadzu Co., Kyoto, Japan) with two gradient pump systems (LC-30AD, Shimadzu), a UV-detector (SPD-M30A, Shimadzu), an auto sample injector (SIL-30AC, Shimadzu), and a column oven (CTO-30A, Shimadzu). Separation was achieved on an XR-ODS column (3.0 × 100 mm, 1.8 mm; Shimadzu, Japan) using a linear gradient elution program with a mobile phase containing solvent A (0.1% (v/v) trifluoroacetic acid in distilled deionized water) and solvent B (0.1% (v/v) trifluoroacetic acid in acetonitrile). Samples for UPLC analysis of phenolic compound content were ground using a grinder immediately prior to analysis. All samples were ground to achieve a particle size that passed through a 500 mL sieve. For the UPLC analysis, ground samples (1 g) were extracted in 5 mL water for 16 hours and filtrated through a 0.45 mm membrane filter. The phenolic compounds were separated using the following gradient: 0-5 minutes from 10%-15% solvent B; 5-10 minutes from 15%-20% solvent B; 10-15 minutes from 20%-30% solvent B; 15-25 minutes from 30%-50% solvent B; 25-30 minutes from 50%-75% B; 30-35 minutes from 75%-100% solvent B; 35-40 minutes from 100%-5% solvent B; and 40-45 minutes from 5%-0% solvent B. The phenolic compounds and anthocyanins were detected by MS at 280 nm and 520 nm, respectively. Chlorogenic acid (Sigma, USA), caffeic acid (Sigma, USA), delphinidin-3-sambubioside (Sigma, USA), delphinidin-3-glucoside (Sigma, USA), cyanidin-3-sambubioside (Sigma, USA) were determined by MS according to the retention times of commercial standards and UV–visible spectral characteristics.

Statistical analysis

The chemical analysis data were assessed by analysis of variance using a multiple comparisons method in the SPSS version 12 statistical software package (SPSS Inc., Chicago, IL, USA). Differences were considered to be significant at a = 0.05. When the treatment effect was significant, means were separated using Duncan’s multiple range test.

The clustering analysis of samples from the leaves of the 49 roselle accessions was performed using the complete linkage method in the SPSS software. The phenolic compounds were visualized as z-values in a heatmap.

RESULTS

Leaf morphological characteristics

The evaluation of leaf morphological characteristics is presented in Table 1 and Fig. 1. The leaf shapes of the roselle accessions were divided into five types: 24 accessions with palmatifid leaves, 10 accessions with palmatisect, 8 accessions with semi-palmatifid leaves, 6 accessions with trifid leaves, and 1 accession with entire leaves. All the roselle accessions had green leaves, except PI286312, which had purple leaves.

Phenolic compounds

The phenolic compounds in the leaves of the forty-nine accessions were analyzed by UPLC-MS. The chemical compounds identified in the roselle accessions are listed in Table 2. Fifteen compounds, delphinidin-3-O-sambubioside (D3S), neochlorogenic acid (NCA), cyanidin-3-O-sambubioside (C3S), cyanidin-3-O-glucoside (C3G), delphinidin-3-O-glucoside (D3G), cryptochlorogenic acid (CCA), rutin (RTN), rutin isomer (RTN-I), isoquercitin (IQN), kaempferol-3-O-rutinoside (K3R), kaempferol-3-O-glucoside (K3G), quercetin (QRN), quercetin isomer (QRN-I), kaempferol, and unknown compounds were detected. Four anthocyanins (D3S, C3S, C3G, D3G) were detected only in PI286312. Significant differences in phenolic compound content were detected among 11 roselle accessions (Table 3). RTN, NCA, and K3R were the most abundant phenolic compounds in roselle leaves from all the accessions. The NCA content ranged from 4.43-18.16 mg/100 g, and the highest level was in PI268100. The CCA content was highest in PI268100 (4.17 mg/100 g) and lowest in PI500724 (1.12 mg/100 g). The RTN content differed significantly among the roselle accessions and ranged from 0.66 mg/100 g (PI468409) to 19.47 mg/100 g (PI275413) (i.e., a 29.5-fold difference among accessions). The RTN-I content was highest in PI291128 and PI295592 (7.16 and 7.15 mg/100 g). The IQN content was highest in PI468409. The K3R content was highest in PI274247 (17.51 mg/100 g), and lowest in PI365477 (1.48 mg/100 g). The K3G content ranged from 0.38-3.06 mg/100 g (mean 1.23 mg/100 g) across all the accessions. The QRN content was highest in PI207920 and PI468409 (1.24 and 1.21 mg/100 g) and was relatively low in the other accessions. The QRN-I content ranged from 0-0.84 mg/100 g across all the accessions. The KFL content ranged from 0.05-0.96 mg/100 g across all the accessions and was highest in PI500734. The unknown compound content was approximately 2.9-fold higher in PI275413 than in all the other roselle accessions.

Chemical hierarchical cluster analysis

The results of the hierarchical cluster analysis are presented in Fig. 2. The 49 roselle accessions clustered into five groups, which formed two independents supergroups (Groups I and II, and Groups III, IV, and V). Group I contained 16 accessions (PI500719, PI500734, PI468413 and PI500729, PI500725, PI500731, PI500727, PI500721, PI500732, PI591549, PI365477, PI291128, PI500752, PI500723, PI638933, and PI468409). Group II contained five accessions (PI256038, PI273388, PI263224, PI500724, and PI267778). Group III contained 17 accessions (PI180026, PI207920, PI500705, PI500747, PI500740, PI591551, PI256039, PI273391, PI500699, PI500720, PI500736, PI273459, PI500701, PI500706, PI265319, PI468412, and PI669506). Group IV contained six accessions (PI274245, PI500698, PI500737, PI496938, PI286312, and PI295592). Group V contained five accessions (PI268100, PI275413, PI286316, PI500713, and PI274247).

The chemical hierarchical cluster analysis divided the nine chemical compounds into four clusters. Cluster I contained four compounds (K3G, KFL, IQN, and RTN-I). Cluster II contained three compounds (QRN-I, Unknown, and K3R) compounds; Cluster III contained two compounds (QRN and RTN) as well as total content. Cluster IV contained two compounds (CCA and NCA).

DISCUSSION

Roselle originated in West Africa and has been cultivated for about 6000 years. It was introduced to Asia and South America from the 17^th to 20^th century (Ali et al. 2005; Da-Costa-Rocha et al. 2014; Daudu et al. 2015). Roselle leaves are a promising economic product, but information about their morphological traits and chemical composition is limited, which significantly impacts their affective applications. Morphological studies of roselle have been limited to the calyx of native Nigerian species (Daudu et al. 2015), and accessions collected from various countries are considered to be optimized in each environment (Wang et al. 2016). This is the first study at the functional compound level of roselle plants in Korea. In the kenaf plant, a Hibiscus species, its leaves also divided into three types, such as non-lobed (entire), shallow-lobed, deeply lobed (palmate). Ryu et al. (2017a) reported that kenaf has presented five major phenolic compounds, these highly correlated flowering date, hypocotyl and petal color rather than type of leaves. Our study, also observed no direct relationship among phenolic compounds level and leaf shape types.

Roselle is considered an important medicinal plant in Africa and Asia (Chen et al. 2013), Its leaves are used to treat colds, toothaches, and hangovers and have long been used in traditional medicines in Africa (Ali et al. 2005; Kuo et al. 2012; Guardiola and Mach 2014). The phenolic compounds are very important biological activates (Min et al. 2015; Deng et al. 2018; Ryu et al. 2019). Previous studies concluded that variations in phenolic compound content depend on the origin, harvest time, and cultivation conditions (Sheela and Sheena 2014; Wang et al. 2016; Ryu et al. 2016). High-performance liquid chromatography analysis of roselle leaves detected eight flavonoids (RTN, RTN-I, IQN, K3R, K3G, QRN, QRN-I, and KFL), four anthocyanins (D3S, C3S, C3G, and D3G) and two phenolic acids (NCA and CCA) compounds. Wang et al. (2016) identified nine compounds in the leaves of 31 different roselle accessions, including six flavonoids (RTN, IQN, K3R, K3G, QRN, and KFL) and three phenolic acids (NCA, CCA, and chlorogenic acid). In addition, the liquid chromatography/quadrupole-time-of-flight mass spectrometry method successfully identified five compounds (NCA, CCA, chlorogenic acid, RTN, and RTN-I) in eight roselle accessions (Wang et al. 2014). In the present study, the highest total phenolic compound content was found for PI275413. The phenolic compound RTN exhibits strong antioxidant properties in roselle leaves (Ochani and D’Mello 2009; Chen et al. 2013; Wang et al. 2014). We found that the RTN content was highest for PI275413, which was from Senegal, and PI286316, which was from Ghana, compared to the other accessions. Generally, environmental conditions are important factors that affect the phytochemical content of plants. Phenolic compound content is considerably influenced by soil, temperature, light, fertilizer conditions, and genetic background (Min et al. 2015; Ryu et al. 2017a; Deng et al. 2018). In this study, the plants were cultivated under the same conditions and the leaves were collected at the same time for phytochemical analysis, so any differences in phenolic compound content are likely genotype dependent, which may have implications for selection in breeding programs.

Anthocyanins play important roles in the response of plants to oxidative stress because of their ability to scavenge reactive oxygen species (Ryu et al. 2017b; Ryu et al. 2018). In this study, the anthocyanins D3S, C3S, C3G, and D3G were found in the roselle leaves, which may prevent cell damage and could be beneficial in the treatment of various human diseases (Obouayeba et al. 2014; Ryu et al. 2016; Ryu et al. 2018). This is the first report on the anthocyanin composition of roselle leaves. The predominant anthocyanins in roselle petals are D3S and C3S (Obouayeba et al. 2014), and two anthocyanins (C3G and D3G) were identified in calyces of roselle (Obouayeba et al. 2015). The identification of Hibiscus species as a source of functional compounds may result in an increase in consumption (Sheela and Sheena 2014; Wang et al. 2016; Ryu et al. 2018). Breeding novel cultivars with high flavonoid and anthocyanin content may be possible. Our results suggest that PI286312 is the most appropriate resource for food and drug industries because it contains high levels of anthocyanins.

The hierarchical cluster analysis grouped the 49 roselle accessions according to their chemical similarities. The chemical hierarchical cluster analysis provided information that can be used to select for chemotype in breeding programs and other useful information (Sheela and Sheena 2014; Ryu et al. 2019). In this study, we found high levels of chemical diversity among the 49 roselle accessions analyzed. Roselle accessions could be divided into five major groups, Group I mainly contained Zambia accessions and high isoquercitin content, Group II included five accessions with exhibited lack of all phenolic compounds. In particular, the highest RTN content and total phenolic content were found in Groups III and V, and RTN and NCA content was the main feature highlighted in the cluster analysis. This suggests that the RTN and NCA content of roselle leaves could be used as a marker to assess chemotypes. Although Group I was apparent pattern of geographical origin, but other groups were not exhibited. These results could be used in breeding programs to develop roselle cultivars with improved functional compounds.

ACKNOWLEDGEMENTS

This work was supported by the research program of KAERI, Republic of Korea.

Fig. 1

Leaf morphological characteristics of the 49 roselle accessions used in this study. The numbers in the boxes correspond to the numbers in column 1 of Table 1.

Fig. 2

Hierarchical cluster analysis of the 49 roselle accessions according to their phenolic compound content.

Table 1

Origin and leaf morphological characteristics of the 49 roselle accessions used in this study.

Table 1
Lines No.	Accession number	Origin	Leaf shape	Leaf color
1	PI180026	India	Palmatified	Green
2	PI207920	Cuba	Palmatisect	Green
3	PI256038	Bangladesh	Palmatisect	Green
4	PI256039	Bangladesh	Palmatified	Green
5	PI263224	Zambia	Palmatisect	Green
6	PI265319	Cuba	Palmatisect	Green
7	PI267778	Sudan	Trifid	Green
8	PI268100	Nigeria	Palmatified	Green
9	PI273388	Taiwan	Palmatisect	Green
10	PI273391	Taiwan	Palmatified	Green
11	PI273459	Transvaal,	Trifid	Green
12	PI274245	Nigeria	Semi-palmatified	Green
13	PI274247	Poland	Semi-palmatified	Green
14	PI275413	Senegal	Palmatified	Green
15	PI286312	Ghana	Palmatified	Purple
16	PI286316	Ghana	Palmatisect	Green
17	PI291128	Ghana	Trifid	Green
18	PI295592	Niger	Palmatisect	Green
19	PI365477	Thailand	Trifid	Green
20	PI468409	United States	Palmatisect	Green
21	PI468412	United States	Entire	Green
22	PI468413	United States	Palmatified	Green
23	PI496938	Sudan	Palmatified	Green
24	PI500698	Zambia	Palmatified	Green
25	PI500699	Zambia	Palmatified	Green
26	PI500701	Zambia	Semi-palmatified	Green
27	PI500705	Zambia	Palmatified	Green
28	PI500706	Zambia	Palmatified	Green
29	PI500713	Zambia	Palmatified	Green
30	PI500719	Zambia	Palmatified	Green
31	PI500720	Zambia	Palmatified	Green
32	PI500721	Zambia	Semi-palmatified	Green
33	PI500723	Zambia	Trifid	Green
34	PI500724	Zambia	Palmatified	Green
35	PI500725	Zambia	Palmatified	Green
36	PI500727	Zambia	Semi-palmatified	Green
37	PI500729	Zambia	Palmatified	Green
38	PI500731	Zambia	Semi-palmatified	Green
39	PI500732	Zambia	Semi-palmatified	Green
40	PI500734	Zambia	Palmatified	Green
41	PI500736	Zambia	Palmatified	Green
42	PI500737	Zambia	Palmatified	Green
43	PI500740	Zambia	Palmatified	Green
44	PI500747	Zambia	Palmatified	Green
45	PI500752	Zambia	Trifid	Green
46	PI591549	Zambia	Semi-palmatified	Green
47	PI591551	Zambia	Palmatified	Green
48	PI638933	South Africa	Palmatisect	Green
49	PI669506	Tanzania	Palmatisect	Green

Table 2

Details of phytochemical compounds identified in leaf extracts from the 49 roselle accessions used in this study.

Table 2
No.	RT	M+H⁺	Identification
1	12.89	597	Delphinidin-3-O-sambubioside (D3S)
2	12.97	355	Neochlorogenic acid (NCA)
3	13.05	581	Cyanidin-3-O-sambubioside (C3S)
4	13.20	449	Cyanidin-3-O-glucoside (C3G)
5	13.40	465	Delphinidin-3-O-glucoside (D3G)
6	13.56	355	Cryptochlorogenic acid (CCA)
7	14.64	611	Rutin (RTN)
8	14.91	611	Rutin isomer (RTN-I)
9	15.08	465	Isoquercitin (IQN)
10	15.24	595	Kaempferol-3-O-rutinoside (K3R)
11	15.75	449	Kaempferol-3-O-glucoside (K3G)
12	16.02	303	Quercetin (QRN)
13	16.91	303	Quercetin isomer (QRN-I)
14	18.37	287	Kaempferol (KFL)
15	19.79	449	Unknown

RT: Retention time, M+H⁺: Molecular ion value.

Table 3

Phenolic compoundz) content in leaf extracts from roselle accessions.

Table 3
No.	NCA	CCA	RTN	RTN-I	IQN	K3R	K3G	QRN	QRN-I	KFL	Unknown	Total
1	8.60c	1.39bc	16.75c	0.70de	1.81ij	3.78ef	2.60ab	0.67cd	0.26d	0.17cd	0.06de	36.79bc
2	7.47de	1.81bc	17.49ab	0.45ef	2.29ij	4.18de	0.71de	1.24a	0.44c	0.10de	0.02de	36.19bc
3	5.27gh	1.42bc	9.46fg	0.41ef	1.53ij	2.97fg	0.49de	0.57de	0.25d	0.07e	0.02de	22.47fg
4	5.95gh	1.80bc	16.48c	0.75cd	1.79ij	3.60ef	0.58de	0.78cd	0.24d	0.19cd	0.05de	36.19bc
5	5.51gh	1.91bc	6.74g	0.29f	1.45ij	3.37fg	0.51de	0.24f	0.12d	0.05e	0.01e	20.21fg
6	6.20fg	1.32bc	15.26cd	1.17cd	1.84ij	3.00fg	0.54de	0.99b	0.34cd	0.31bc	0.08de	31.05cd
7	6.51fg	1.45bc	7.14g	1.73bc	1.57ij	2.89fg	0.49de	0.05f	0.02d	0.37bc	0.13cd	22.36fg
8	18.16a	4.17a	8.60fg	0.64ef	1.49ij	3.01fg	0.46de	0.61cd	0.20d	0.13de	0.01e	37.47bc
9	4.98h	1.22c	8.40fg	0.87c	1.20j	2.49fg	0.38e	0.75cd	0.24d	0.19cd	0.06de	20.78fg
10	7.27de	1.76bc	17.42ab	1.60cd	2.09ij	3.89de	0.54de	0.48de	0.15d	0.53bc	0.14cd	35.73bc
11	6.86ef	1.71bc	17.40ab	1.01cd	3.85fg	4.44de	0.99cd	0.78cd	0.30cd	0.31bc	0.09de	37.75bc
12	9.00c	2.45b	7.55fg	1.91cd	5.14ef	3.68ef	1.89bc	0.08f	0.04d	0.84ab	0.29b	32.87cd
13	6.61fg	2.25bc	5.42g	2.19b	1.71ij	17.51a	3.06a	0.35f	0.84a	0.51bc	0.85a	41.31ab
14	10.95b	2.20bc	19.47a	1.92bc	3.80fg	5.68c	0.91cd	0.60cd	0.21d	0.62ab	0.16cd	46.51a
15	8.87c	1.92bc	12.05de	0.64ef	3.54gh	4.48de	1.47cd	0.29f	0.11d	0.19cd	0.04de	33.60cd
16	9.27c	1.81bc	19.33a	1.62bc	5.30ef	4.26de	0.94cd	0.04f	0.00e	0.79ab	0.15cd	43.52ab
17	5.51gh	1.71bc	1.21h	7.16a	4.69fg	2.43fg	2.07ab	0.07f	0.15d	0.59ab	0.20cd	25.79ef
18	4.43h	1.32bc	10.42ef	7.15a	4.70fg	2.45fg	2.10ab	0.67cd	0.19d	0.20cd	0.04de	33.61cd
19	7.22de	1.96bc	0.85h	1.71bc	6.60cd	1.48g	1.25cd	0.15ef	0.17d	0.89ab	0.16cd	22.44fg
20	6.31fg	1.72bc	0.66h	1.35cd	10.91a	7.04b	1.37cd	1.21a	0.06d	0.78ab	0.10de	31.49cd
21	4.62h	1.27bc	15.29cd	0.81cd	2.09ij	2.81fg	0.55de	0.21ef	0.23d	0.30bc	0.05de	28.23de
22	6.98e	1.89bc	6.39g	1.56bc	6.67cd	1.99g	1.56cd	0.12f	0.03d	0.74ab	0.18cd	28.10de
23	8.77c	2.05bc	10.48ef	1.55bc	8.77b	2.39fg	1.52cd	0.22ef	0.78ab	0.75ab	0.13cd	37.43bc
24	7.88d	2.16bc	9.05fg	1.58bc	7.61bc	2.44fg	1.53cd	0.20ef	0.06d	0.89ab	0.18cd	33.59cd
25	6.33fg	1.91bc	17.16b	1.51bc	2.55hi	4.75cd	0.86de	0.17ef	0.07d	0.72ab	0.23bc	36.26bc
26	7.32de	2.12bc	17.22b	1.87bc	2.98hi	4.23de	0.83de	0.15ef	0.13d	0.82ab	0.22bc	37.89bc
27	7.38de	1.89bc	17.67ab	0.72de	3.03hi	4.26de	1.18cd	0.02f	0.03d	0.28bc	0.07de	36.54bc
28	7.50de	2.28bc	18.12ab	1.76bc	2.77hi	5.01cd	0.99cd	0.25ef	0.22d	0.75ab	0.23bc	39.87ab
29	10.44b	2.15bc	17.43ab	0.92cd	3.27gh	5.64c	1.15cd	0.13f	0.10d	0.21cd	0.07de	41.51ab
30	7.00ef	1.92bc	6.49g	0.98cd	6.17de	2.34fg	1.63cd	0.16ef	0.13d	0.47bc	0.12de	27.40ef
31	6.92ef	1.98bc	16.58c	0.72de	2.47hi	4.42de	0.79de	0.02f	0.11d	0.22cd	0.06de	34.29cd
32	6.92ef	2.24bc	6.10g	1.21cd	6.32de	2.74fg	2.59ab	0.04f	0.05d	0.52bc	0.15cd	28.88de
33	6.33fg	1.87bc	6.05g	1.17cd	6.21ef	2.67fg	1.89bc	0.16ef	0.14d	0.61ab	0.19bc	27.28ef
34	5.61gh	1.12c	7.11g	0.56ef	1.48ij	2.13fg	0.41de	0.09f	0.08d	0.13de	0.03de	18.75g
35	5.66gh	1.32bc	6.63g	1.42cd	6.35de	1.59g	1.08cd	0.11f	0.09d	0.77ab	0.14cd	25.15ef
36	6.77ef	1.71bc	6.94g	1.26cd	7.94bc	1.79g	1.33cd	0.11f	0.08d	0.60ab	0.11de	28.64de
37	6.50fg	1.91bc	7.13g	1.43cd	6.63cd	2.15fg	1.90bc	0.04f	0.04d	0.63ab	0.14cd	28.50de
38	6.50fg	1.64bc	6.32g	0.86cd	6.81cd	1.91g	1.83bc	0.04f	0.04d	0.39bc	0.09de	26.43ef
39	5.74gh	1.64bc	6.17g	1.41cd	6.02ef	1.90g	1.86bc	0.04f	0.04d	0.79ab	0.18cd	25.78ef
40	6.06g	1.68bc	6.37g	1.51bc	6.52cd	2.09fg	1.57cd	0.13f	0.12d	0.96a	0.24bc	27.25ef
41	7.12de	1.67bc	16.81bc	0.70de	2.58hi	3.74ef	0.61de	0.08f	0.08d	0.21cd	0.05de	33.66cd
42	6.44fg	2.07bc	7.83fg	1.32cd	8.29b	2.18fg	2.28ab	0.06f	0.04d	0.88ab	0.19bc	31.57cd
43	8.58c	1.68bc	17.58ab	0.88cd	2.67hi	4.18de	0.73de	0.13f	0.12d	0.24bc	0.06de	36.86bc
44	7.45de	2.16bc	17.19b	1.31cd	2.54hi	4.38de	0.85de	0.18ef	0.16d	0.37bc	0.10de	36.69bc
45	6.09g	1.53bc	6.16g	0.71de	6.08de	2.05fg	1.55cd	0.12f	0.09d	0.34bc	0.08de	24.78ef
46	5.50gh	1.45bc	6.23g	0.61ef	6.28de	1.88g	1.61cd	0.04f	0.03d	0.27bc	0.06de	23.97ef
47	7.09de	1.75bc	18.92ab	0.66de	3.26gh	3.25ef	0.58de	0.14f	0.12d	0.22cd	0.03de	36.03bc
48	6.60fg	1.52bc	5.49g	1.29cd	5.71ef	1.66g	1.13cd	0.10f	0.06d	0.49bc	0.10de	24.17ef
49	6.76fg	1.45bc	12.83d	1.14cd	1.73ij	2.76fg	0.57de	0.15ef	0.68b	0.37bc	0.09de	28.53de

^z)NCA: Neochlorogenic acid, CCA: Cryptochlorogenic acid, RTN: Rutin, RTN-I: Rutin isomer, IQN: Isoquercitin, K3R: Kaempferol-3-O-rutinoside, K3G: Kaempferol-3-O-glucoside, QRN: Quercetin, QRN-I: Quercetin isomer, KFL: Kaempferol. Data are shown as the mean values; n = 3.

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Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

Plant Breed. Biotech.. 2020;8(1):1-10. Published online March 1, 2020

DOI: https://doi.org/10.9787/PBB.2020.8.1.1

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Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

Plant Breed. Biotech.. 2020;8(1):1-10. Published online March 1, 2020

DOI: https://doi.org/10.9787/PBB.2020.8.1.1

Figure

Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

Fig. 1 Leaf morphological characteristics of the 49 roselle accessions used in this study. The numbers in the boxes correspond to the numbers in column 1 of Table 1.

Fig. 2 Hierarchical cluster analysis of the 49 roselle accessions according to their phenolic compound content.

Fig. 1

Fig. 2

Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

Table 1

Origin and leaf morphological characteristics of the 49 roselle accessions used in this study.

Lines No.	Accession number	Origin	Leaf shape	Leaf color
1	PI180026	India	Palmatified	Green
2	PI207920	Cuba	Palmatisect	Green
3	PI256038	Bangladesh	Palmatisect	Green
4	PI256039	Bangladesh	Palmatified	Green
5	PI263224	Zambia	Palmatisect	Green
6	PI265319	Cuba	Palmatisect	Green
7	PI267778	Sudan	Trifid	Green
8	PI268100	Nigeria	Palmatified	Green
9	PI273388	Taiwan	Palmatisect	Green
10	PI273391	Taiwan	Palmatified	Green
11	PI273459	Transvaal,	Trifid	Green
12	PI274245	Nigeria	Semi-palmatified	Green
13	PI274247	Poland	Semi-palmatified	Green
14	PI275413	Senegal	Palmatified	Green
15	PI286312	Ghana	Palmatified	Purple
16	PI286316	Ghana	Palmatisect	Green
17	PI291128	Ghana	Trifid	Green
18	PI295592	Niger	Palmatisect	Green
19	PI365477	Thailand	Trifid	Green
20	PI468409	United States	Palmatisect	Green
21	PI468412	United States	Entire	Green
22	PI468413	United States	Palmatified	Green
23	PI496938	Sudan	Palmatified	Green
24	PI500698	Zambia	Palmatified	Green
25	PI500699	Zambia	Palmatified	Green
26	PI500701	Zambia	Semi-palmatified	Green
27	PI500705	Zambia	Palmatified	Green
28	PI500706	Zambia	Palmatified	Green
29	PI500713	Zambia	Palmatified	Green
30	PI500719	Zambia	Palmatified	Green
31	PI500720	Zambia	Palmatified	Green
32	PI500721	Zambia	Semi-palmatified	Green
33	PI500723	Zambia	Trifid	Green
34	PI500724	Zambia	Palmatified	Green
35	PI500725	Zambia	Palmatified	Green
36	PI500727	Zambia	Semi-palmatified	Green
37	PI500729	Zambia	Palmatified	Green
38	PI500731	Zambia	Semi-palmatified	Green
39	PI500732	Zambia	Semi-palmatified	Green
40	PI500734	Zambia	Palmatified	Green
41	PI500736	Zambia	Palmatified	Green
42	PI500737	Zambia	Palmatified	Green
43	PI500740	Zambia	Palmatified	Green
44	PI500747	Zambia	Palmatified	Green
45	PI500752	Zambia	Trifid	Green
46	PI591549	Zambia	Semi-palmatified	Green
47	PI591551	Zambia	Palmatified	Green
48	PI638933	South Africa	Palmatisect	Green
49	PI669506	Tanzania	Palmatisect	Green

Table 2

Details of phytochemical compounds identified in leaf extracts from the 49 roselle accessions used in this study.

No.	RT	M+H⁺	Identification
1	12.89	597	Delphinidin-3-O-sambubioside (D3S)
2	12.97	355	Neochlorogenic acid (NCA)
3	13.05	581	Cyanidin-3-O-sambubioside (C3S)
4	13.20	449	Cyanidin-3-O-glucoside (C3G)
5	13.40	465	Delphinidin-3-O-glucoside (D3G)
6	13.56	355	Cryptochlorogenic acid (CCA)
7	14.64	611	Rutin (RTN)
8	14.91	611	Rutin isomer (RTN-I)
9	15.08	465	Isoquercitin (IQN)
10	15.24	595	Kaempferol-3-O-rutinoside (K3R)
11	15.75	449	Kaempferol-3-O-glucoside (K3G)
12	16.02	303	Quercetin (QRN)
13	16.91	303	Quercetin isomer (QRN-I)
14	18.37	287	Kaempferol (KFL)
15	19.79	449	Unknown

Table 3

Phenolic compoundz) content in leaf extracts from roselle accessions.

No.	NCA	CCA	RTN	RTN-I	IQN	K3R	K3G	QRN	QRN-I	KFL	Unknown	Total
1	8.60c	1.39bc	16.75c	0.70de	1.81ij	3.78ef	2.60ab	0.67cd	0.26d	0.17cd	0.06de	36.79bc
2	7.47de	1.81bc	17.49ab	0.45ef	2.29ij	4.18de	0.71de	1.24a	0.44c	0.10de	0.02de	36.19bc
3	5.27gh	1.42bc	9.46fg	0.41ef	1.53ij	2.97fg	0.49de	0.57de	0.25d	0.07e	0.02de	22.47fg
4	5.95gh	1.80bc	16.48c	0.75cd	1.79ij	3.60ef	0.58de	0.78cd	0.24d	0.19cd	0.05de	36.19bc
5	5.51gh	1.91bc	6.74g	0.29f	1.45ij	3.37fg	0.51de	0.24f	0.12d	0.05e	0.01e	20.21fg
6	6.20fg	1.32bc	15.26cd	1.17cd	1.84ij	3.00fg	0.54de	0.99b	0.34cd	0.31bc	0.08de	31.05cd
7	6.51fg	1.45bc	7.14g	1.73bc	1.57ij	2.89fg	0.49de	0.05f	0.02d	0.37bc	0.13cd	22.36fg
8	18.16a	4.17a	8.60fg	0.64ef	1.49ij	3.01fg	0.46de	0.61cd	0.20d	0.13de	0.01e	37.47bc
9	4.98h	1.22c	8.40fg	0.87c	1.20j	2.49fg	0.38e	0.75cd	0.24d	0.19cd	0.06de	20.78fg
10	7.27de	1.76bc	17.42ab	1.60cd	2.09ij	3.89de	0.54de	0.48de	0.15d	0.53bc	0.14cd	35.73bc
11	6.86ef	1.71bc	17.40ab	1.01cd	3.85fg	4.44de	0.99cd	0.78cd	0.30cd	0.31bc	0.09de	37.75bc
12	9.00c	2.45b	7.55fg	1.91cd	5.14ef	3.68ef	1.89bc	0.08f	0.04d	0.84ab	0.29b	32.87cd
13	6.61fg	2.25bc	5.42g	2.19b	1.71ij	17.51a	3.06a	0.35f	0.84a	0.51bc	0.85a	41.31ab
14	10.95b	2.20bc	19.47a	1.92bc	3.80fg	5.68c	0.91cd	0.60cd	0.21d	0.62ab	0.16cd	46.51a
15	8.87c	1.92bc	12.05de	0.64ef	3.54gh	4.48de	1.47cd	0.29f	0.11d	0.19cd	0.04de	33.60cd
16	9.27c	1.81bc	19.33a	1.62bc	5.30ef	4.26de	0.94cd	0.04f	0.00e	0.79ab	0.15cd	43.52ab
17	5.51gh	1.71bc	1.21h	7.16a	4.69fg	2.43fg	2.07ab	0.07f	0.15d	0.59ab	0.20cd	25.79ef
18	4.43h	1.32bc	10.42ef	7.15a	4.70fg	2.45fg	2.10ab	0.67cd	0.19d	0.20cd	0.04de	33.61cd
19	7.22de	1.96bc	0.85h	1.71bc	6.60cd	1.48g	1.25cd	0.15ef	0.17d	0.89ab	0.16cd	22.44fg
20	6.31fg	1.72bc	0.66h	1.35cd	10.91a	7.04b	1.37cd	1.21a	0.06d	0.78ab	0.10de	31.49cd
21	4.62h	1.27bc	15.29cd	0.81cd	2.09ij	2.81fg	0.55de	0.21ef	0.23d	0.30bc	0.05de	28.23de
22	6.98e	1.89bc	6.39g	1.56bc	6.67cd	1.99g	1.56cd	0.12f	0.03d	0.74ab	0.18cd	28.10de
23	8.77c	2.05bc	10.48ef	1.55bc	8.77b	2.39fg	1.52cd	0.22ef	0.78ab	0.75ab	0.13cd	37.43bc
24	7.88d	2.16bc	9.05fg	1.58bc	7.61bc	2.44fg	1.53cd	0.20ef	0.06d	0.89ab	0.18cd	33.59cd
25	6.33fg	1.91bc	17.16b	1.51bc	2.55hi	4.75cd	0.86de	0.17ef	0.07d	0.72ab	0.23bc	36.26bc
26	7.32de	2.12bc	17.22b	1.87bc	2.98hi	4.23de	0.83de	0.15ef	0.13d	0.82ab	0.22bc	37.89bc
27	7.38de	1.89bc	17.67ab	0.72de	3.03hi	4.26de	1.18cd	0.02f	0.03d	0.28bc	0.07de	36.54bc
28	7.50de	2.28bc	18.12ab	1.76bc	2.77hi	5.01cd	0.99cd	0.25ef	0.22d	0.75ab	0.23bc	39.87ab
29	10.44b	2.15bc	17.43ab	0.92cd	3.27gh	5.64c	1.15cd	0.13f	0.10d	0.21cd	0.07de	41.51ab
30	7.00ef	1.92bc	6.49g	0.98cd	6.17de	2.34fg	1.63cd	0.16ef	0.13d	0.47bc	0.12de	27.40ef
31	6.92ef	1.98bc	16.58c	0.72de	2.47hi	4.42de	0.79de	0.02f	0.11d	0.22cd	0.06de	34.29cd
32	6.92ef	2.24bc	6.10g	1.21cd	6.32de	2.74fg	2.59ab	0.04f	0.05d	0.52bc	0.15cd	28.88de
33	6.33fg	1.87bc	6.05g	1.17cd	6.21ef	2.67fg	1.89bc	0.16ef	0.14d	0.61ab	0.19bc	27.28ef
34	5.61gh	1.12c	7.11g	0.56ef	1.48ij	2.13fg	0.41de	0.09f	0.08d	0.13de	0.03de	18.75g
35	5.66gh	1.32bc	6.63g	1.42cd	6.35de	1.59g	1.08cd	0.11f	0.09d	0.77ab	0.14cd	25.15ef
36	6.77ef	1.71bc	6.94g	1.26cd	7.94bc	1.79g	1.33cd	0.11f	0.08d	0.60ab	0.11de	28.64de
37	6.50fg	1.91bc	7.13g	1.43cd	6.63cd	2.15fg	1.90bc	0.04f	0.04d	0.63ab	0.14cd	28.50de
38	6.50fg	1.64bc	6.32g	0.86cd	6.81cd	1.91g	1.83bc	0.04f	0.04d	0.39bc	0.09de	26.43ef
39	5.74gh	1.64bc	6.17g	1.41cd	6.02ef	1.90g	1.86bc	0.04f	0.04d	0.79ab	0.18cd	25.78ef
40	6.06g	1.68bc	6.37g	1.51bc	6.52cd	2.09fg	1.57cd	0.13f	0.12d	0.96a	0.24bc	27.25ef
41	7.12de	1.67bc	16.81bc	0.70de	2.58hi	3.74ef	0.61de	0.08f	0.08d	0.21cd	0.05de	33.66cd
42	6.44fg	2.07bc	7.83fg	1.32cd	8.29b	2.18fg	2.28ab	0.06f	0.04d	0.88ab	0.19bc	31.57cd
43	8.58c	1.68bc	17.58ab	0.88cd	2.67hi	4.18de	0.73de	0.13f	0.12d	0.24bc	0.06de	36.86bc
44	7.45de	2.16bc	17.19b	1.31cd	2.54hi	4.38de	0.85de	0.18ef	0.16d	0.37bc	0.10de	36.69bc
45	6.09g	1.53bc	6.16g	0.71de	6.08de	2.05fg	1.55cd	0.12f	0.09d	0.34bc	0.08de	24.78ef
46	5.50gh	1.45bc	6.23g	0.61ef	6.28de	1.88g	1.61cd	0.04f	0.03d	0.27bc	0.06de	23.97ef
47	7.09de	1.75bc	18.92ab	0.66de	3.26gh	3.25ef	0.58de	0.14f	0.12d	0.22cd	0.03de	36.03bc
48	6.60fg	1.52bc	5.49g	1.29cd	5.71ef	1.66g	1.13cd	0.10f	0.06d	0.49bc	0.10de	24.17ef
49	6.76fg	1.45bc	12.83d	1.14cd	1.73ij	2.76fg	0.57de	0.15ef	0.68b	0.37bc	0.09de	28.53de

Table 1 Origin and leaf morphological characteristics of the 49 roselle accessions used in this study.

Table 2 Details of phytochemical compounds identified in leaf extracts from the 49 roselle accessions used in this study.

RT: Retention time, M+H⁺: Molecular ion value.

Table 3 Phenolic compoundz) content in leaf extracts from roselle accessions.
z)

NCA: Neochlorogenic acid, CCA: Cryptochlorogenic acid, RTN: Rutin, RTN-I: Rutin isomer, IQN: Isoquercitin, K3R: Kaempferol-3-O-rutinoside, K3G: Kaempferol-3-O-glucoside, QRN: Quercetin, QRN-I: Quercetin isomer, KFL: Kaempferol. Data are shown as the mean values; n = 3.

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Phenolic Compound Content of Leaf Extracts from Different Roselle (Hibiscus sabdariffa) Accessions

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