Anthocyanin and Potential Therapeutic Traits in Clitoria, Desmodium,

Corchorus, Catharanthus and Hibiscus Species

J.B. Morris and M.L. Wang
USDA, ARS, PGRCU
1109 Experiment St., Griffin, GA 30223–1797
USA

Keywords: medicinal plants, nutraceuticals, phytochemicals, health enhancing

compounds, germplasm, phytopharmaceutical

Abstract
The USDA, ARS, Plant Genetic Resources Conservation Unit curates several
important nutraceutical and medicinal plant species. Anthocyanins are responsible
for flower, leaf and seed coat color in plants, and are antioxidants as well. However,
little is known about anthocyanin content in Clitoria ternatea, Desmodium
adscendens, Corchorus olitorius, Catharanthus roseus, Hibiscus sabdariffa and Acer
saccharum. This study was conducted to identify anthocyanin indexes and additional
health enhancing components in these species. An anthocyanin meter with an LED
diode of 520 nm was used to measure the anthocyanin index of leaves and flowers
from plants growing in the field during July, September and October 2006. We
found anthocyanin indexes ranging from 4.1 to 52.4 for leaves and 0.8 to 26.8 for
flowers in all five species. The highest leaf anthocyanin index average of 14.6 was
found in D. adscendens (PI 316623) followed by sugar maple (A. saccharum) with a
leaf anthocyanin average index of 13.0. Catharanthus roseus (PI 608581) also had the
highest flower anthocyanin index average of 17.3. These data were recorded at or
prior to 50% maturity. In addition, potential health uses for these species are
discussed. Phytochemicals identified include but are not limited to an antimicrobial
protein from C. ternatea, anthocyanin rich extracts from H. sabdariffa for potential
use as a chemopreventive agent, and antitumor promoters in C. olitorius leaves.
These anthocyanins and phytochemicals have potential value in the nutraceutical
and medical industries. These variable anthocyanin indexes among these species will
assist breeders and other scientists with valuable germplasm for development of
anthocyanin enriched cultivars.

INTRODUCTION
Several genera of economic importance to the nutraceutical as well as the
medicinal plant markets are conserved and curated at the USDA, ARS, Plant Genetic
Resources Conservation Unit (PGRCU), Griffin, GA. Anthocyanins are responsible for
flower, plant, seed coat color, and recently known to be an antioxidant as well as free
radical scavenging (Polya, 2003). Not only have several new anthocyanins been reported
in C. ternatea (Terahara et al., 1998) but an antimicrobial and insecticidal protein known
as finotin has been discovered as well (Kelemu et al., 2004). The objectives of this
research were to compare anthocyanin indexes from these field grown species and to
report some additional phytochemicals found in C. ternatea, D. adscendens, C. olitorius,
C. roseus and H. sabdariffa for potential use as a nutraceutical or medicinal plant.
MATERIALS AND METHODS
Seed from each of eight legumes including five C. ternatea, three D. adscendens,
as well as one C. olitorius, one C. roseus and one H. sabdariffa accessions were planted
in Pro-Mix HP potting soil during mid to late March inside a greenhouse. After 45–
50 days, seedlings from all species were transplanted to field plots of a clayey, kaolinitic,
thermic typic kanhapludults soil series at Griffin, GA, in a completely random design.
Each plot consisted of 1 row at 6 m x 3 m with 3 m between rows. Data were analyzed
using analysis of variance (P<0.001) (SAS Institute, Cary, NC). Mean separations were

Proc. IS on Med. and Nutraceutical Plants 381

Ed.: A.K. Yadav
Acta Hort. 756, ISHS 2007
conducted using Duncan’s multiple range test (P<0.05).
An Opti Sciences CCM-200 chlorophyll content meter was converted to an
experimental hand held anthocyanin meter. The manufacturer replaced the 655 nm light
emitting diode (LED) of the CCM with a 520 nm LED in order to measure absorbance
near the wavelength at which free anthocyanin aglycones in beans, cyanidin and
pelargonidin monoglucosides absorb (Macz-Pop et al., 2004).
Prior to and near 50% seed maturity, anthocyanin indexes were recorded from
each of three leaves using this modified anthocyanin meter on four different dates
(18 July, 25 September, and 11 or 24 October) from each accession of C. ternatea,
D. adscendens, C. olitorius, C. roseus, H. sabdariffa and sugar maple growing in the
field. Anthocyanin index data recorded on 11 and 24 October were combined because
some accessions produced unsuitable leaves due to senescence. Anthocyanin indexes
from sugar maple (Acer saccharum) leaves were included because the similar
anthocyanin, cyanidin can be detected with the 520 nm LED (van den Berg and Perkins,
2005). For the first time, additional species including C. olitorius, C. roseus and
H. sabdariffa were analyzed for their anthocyanin indexes also using this 520 nm LED.
Leaf tissue from C. ternatea (PI 226265), D. adscendens (PI 237955 and 316623),
Hibiscus sabdariffa (PI 291126) and C. olitorius (PI 404028) were prepared for HPLC
analysis for the flavonoids myricetin, daidzein, quercetin, genistein and kaempferol
following a similar procedure by Wang and Morris (2007).
RESULTS AND DISCUSSION

Anthocyanins
Leaf anthocyanin indexes ranged from 5.5 to 13.9 on 18 July 2006 (Table 1). The
species C. olitorius leaves produced the highest but not significantly average anthocyanin
index at 12.4 followed by H. sabdariffa leaves (11.6), sugar maple (11.0) and C. ternatea
PI 209591 (10.0) (Fig. 1). During 25 September 2006, sugar maple leaves produced a
significantly higher anthocyanin index (22.2) than C. roseus. Interestingly as the season
progressed on 11 October 2006, PI 316623 of D. adscendens produced a significantly
higher leaf anthocyanin index (19.6) than all other species. During 24 October 2006, both
PI 316623 and PI 237955 of D. adscendens produced significantly higher leaf
anthocyanin indexes (17.4 and 16.9, respectively) than all other species. The three
D. adscendens accessions increased by an average of 60% during October in leaf
anthocyanin index. Corchorus olitorius produced greater than 100% more leaf
anthocyanin indexes in July and September than during October. Anthocyanin indexes
were generally stable during July and September with the exception of sugar maple which
dramatically increased in leaf anthocyanin index by 100% in September.
For all months combined, anthocyanin flower and leaf indexes ranged from 1.0 to
17.3. The accession PI 608581 (C. roseus) produced the significantly highest flower (dark
salmon) anthocyanin index (17.3) followed by D. adscendens (PI 316623) leaves (14.6),
dark blue flower of C. ternatea PI 283232 (13.5), sugar maple leaves (13.0),
D. adscendens PI 237955 (12.7) and H. sabdariffa PI 291126 (11.1). The white flower of
C. ternatea PI 209591 produced the significantly lowest anthocyanin index of 1.0 while
the blue flower produced by C. ternatea (PI 226265) and leaves from C. roseus yielded
significantly lower anthocyanin indexes of 3.1 and 5.9, respectively, as well (Fig. 2).
Corchorus olitorius produced the most quercetin (578 ng/µl) followed by
H. sabdariffa (280.8 ng/µl) and C. ternatea (161.6 ng/µl). Clitorai ternatea leaves
produced the most kaempferol (658.6 ng/µl) followed by H. sabdariffa (183.4 ng/µl).
Negligible amounts of quercetin and kaempferol were found in both D. adscendens
accessions (Fig. 3). Interestingly, quercetin and kaemferol are isoflavones with similar
biochemical pathways as the anthocyanin, cyanidin and pelargonidin.

Potential Nutraceuticals and Medicinals

The African traditional medicinal plant, D. adscendens exerted analgesic effects in

382
mice (N’gouemo et al., 1996) and is used for asthma in Ghana, Africa plus ovarian
inflammation in Mato Grosso, Brazil (Barreto, 2002). A soluble dietary fiber from
C. olitorius suppressed blood gluscose elevation in rats and humans (Innami et al., 2005).
Corchorus olitorius has been found to inhibit the malaria parasite (Plasmodium
falciparum) above 96% (Sathiyamoorthy et al., 1999).
The common ornamental plant, periwinkle (Catharanthus roseus) has been shown
to be effective against leukemia, skin cancer, lymph cancer, breast cancer, and Hodgkin’s
disease (Kintzios et al., 2004). Hibiscus sabdariffa has anticancer properties (Hou et al.,
2005) as well as inhibition of LDL oxidation plus potential service as a chemopreventive
agent (Chang et al., 2006). See Table 2 for additional potential nutraceuticals and
medicinals from these species.

CONCLUSIONS
Even though some significant differences for leaf and flower anthocyanin indexes
occur among these species tested, several species decrease in anthocyanin index while
others increase as the season progresses from 18 July till 24 October. Anthocyanin
indexes remained stable for the majority of the species, however sugar maple increased
dramatically on 25 September. During plant senescence on 24 October, anthocyanin
indexes for both C. olitorius and sugar maple dropped while H. sabdariffa and
D. adscendens increased. Anthocyanins provide chemical defenses against pests and may
help protect plants during excessively cold days. Anthocyanins also increase in plants as
the season progresses and can be observed in some species. For example, D. adscendens
and H. sabdariffa produced green leaves containing large areas of purple leaf color with
progressively darker purple leaf areas as the season extended into October.
Dark flower colors in C. roseus and C. ternatea produced the greatest anthocyanin
indexes overall during the entire season while green with purple tinges in leaves of
D. adscendens and H. sabdariffa produced high anthocyanin indexes as well.
Anthocyanin indexes were very low from those species producing light colors such as
white or blue flowers and green leaves. Further testing should be considered for
environmental effects on anthocyanin indexes. However, in this first report for
anthocyanin indexes from these species, we suggest that several accessions from within
these species need testing to verify potential use in breeding programs with eventual
cultivar development for enriched anthocyanin indexes. This hand held anthocyanin meter
can be successfully used to estimate anthocyamin indexes from these species tested.
Nutraceutical and phytopharmaceutical species do exist in the USDA, ARS,
National Plant Germplasm System at the Plant Genetic Resources Conservation Unit,
Griffin, GA. Not only do some of these species provide current nutraceutical uses but
many others have potential to be used in this type of market as well as for medicinal uses.

Literature Cited
Azuma, K., Nakayama, M., Koshioka, M., Ippoushi, K., Yamaguchi, Y., Kohata, K.,
Yamauchi, Y., Ito, H. and Higashio, H. 1999. Phenolic antioxidants from the leaves of
Corchorus olitorius L. J. Agric. Food Chem. 47:3963–3966.
Barreto, G.S. 2002. Effect of butanolic fraction of Desmodium adscendens on the
anococcygeus of the rat. Braz. J. Biol. 62:223–230.
van den Berg, A.K. and Perkins, T.D. 2005. Nondestructive estimation of anthocyanin
content in Autumn sugar maple leaves. HortSci. 40:685–686.
Chang, C.C., Jeng, D.H., San, F.W., Huei, C.C., Mon, Y.Y., Erl, S.K., Yung, C.H. and
Chau, J.W. 2003. Hibiscus sabdariffa extract inhibits the development of
Atherosclerosis in cholesterol-fed rabbits. J. Agric. Food Chem. 51:5472–5477.
Chang, Y.C., Huang, H.P., Hsu, J.D., Yang, S.F. and Wang, C.J. 2005. Hibiscus
anthoxyanins rich extract-induced apoptotic cell death in human promyelocytic
leukemia cells. Toxicol. Appl. Pharmacol. 205:201–212.
Chang, Y.C., Huang, K.X., Huang, A.C., Ho, Y.C. and Wang, C.J. 2006. Hibiscus
anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages

383
 apoptosis. Food Chem. Toxicol. 44:1015–1023.
Farombi, E.O. and Fakoya, A. 2005. Free radical scavenging and antigenotoxic activities
of natural phenolic compounds in dried flowers of Hibiscus sabdariffa L. Mol. Nutr.
Food Res. 49:1120–1128.
Furumoto, T., Wang, R., Okazaki, K., Hasan, A.F.M.F., Ali, M.I., Kondo, A. and Fukui,
H. 2002. Antitumor promoters in leaves of jute (Corchorus capsularis and Corchorus
olitorius. Food Sci. and Technol. Res. 8:239–243.
Hou, D.X., Tong, X., Terahara, N., Luo, D. and Fujii, M. 2005. Delphinidin 3-
sambubioside, a Hibiscus anthocyanin, induces apoptosis in human leukemia cells
through reactive oxygen species-mediated mitochondrial pathway. Arch. Biochem.
Biophys. 440:101–109.
Innami, S., Ishida, H., Nakamura, K., Kondo, M., Tabata, K., Koguchi, T., Shimizu, J.
and Furusho, T. 2005. Jew’s mellow leaves (Corchorus olitorius) suppress elevation
of postprandial blood glucose levels in rats and humans. Int. J. Vitam. Nutr. Res.
75:39–46.
Kelemu, S., Cardona, C. and Segura, G. 2004. Antimicrobial and insecticidal protein
isolated from seeds of Clitoria ternatea, a tropical forage legume. Plant Physiol.
Biochem. 42:867–873.
Kintzios, S.E., Barberaki, M.G. and Makri, O.G. 2004. Terrestrial plant species with
anticancer activity. p.35–194. In: S.E. Kintzios and M.G. Barberaki (eds.), Plants That
Fight Cancer, CRC Press LLC, Boca Raton, Florida.
Macz-Pop, G.A., Rivas-Gonzalo, J.C., Perez-Alonso, J.J. and Gonzalez-Paramas, A.M.
2004. Natural occurrence of free anthocyanin aglycones in beans (Phaseolus vulgaris
L.). Food Chem. 94:448–456.
N’gouemo, P., Baldy-Moulinier, M. and Nguemby-Bina, C. 1996. Effects of an ethanolic
extract of Desmodium adscendens on central nervous system in rodents. J.
Ethnopharm. 52:77–83.
Polya, G. 2003. Biochemical Targets of Plant Bioactive Compounds. CRC Press, Boca
Raton, Florida.
Sathiyamoorthy, P., Lugasi-Evgi, H., Schlesinger, P., Kedar, I., Gopas, J., Pollack, Y. and
Golan-Goldhirsh, A. 1999. Screening for cytotoxic and antimalarial activities in desert
plants of the Negev and Bedouin market plant products. Pharma. Biol. 37:188- 195.
Terahara, N., Toki, K., Saito, N., Honda, T., Matsui, T. and Osajima, Y. 1998. Eight new
anthocyanins, ternatins C1-C5 and D3 and preternatins A3 and C4 from young clitoria
flowers. J. Nat. Prod. 61:1361–1367.
Wang, M.L. and Morris, J.B. 2007. Flavonoid content in seeds of guar germplasm using
HPLC. Plant Gen. Res.: Chara. and Util. (in press).
Yun-Ching, C., Kai-Xun, H., An-Chung, H., Yung, C.H. and Chau, J.W. 2006. Hibiscus
anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages
apoptosis. Food and Chem. Toxicol. 44:1015–1023.
Zakaria, Z.A., Somchit, M.N., Zaiton, H., Mat Jais, A.M., Sulaiman, M.R., Farah, W.O.,
Nazaratulmawarina, R. and Fatimah, C.A. 2006. The in vitro antibacterial activity of
Corchorus olitorius extracts. Internat. J. of Pharmacol. 2:213–215.

384
 Tables

Table 1. Range of leaf and flower anthocyanin indexes from Clitoria ternatea, Desmodium adscendens, Corchorus olitorius,
Catharanthus roseus, and Hibiscus sabdariffa accessions growing in the field at Griffin, GA during July, September and October
2006.

Anthocyanin Index Range

Taxon July 18 September 25 October 11 October 24
Leaves
Clitoria ternatea 5.5–13.4 4.1–12.7 4.9–10.1 5.3–13.6
Desmodium adscendens 7.2–12.7 4.9–14.7 6.4–23.2 8.1–22.2
Corchorus olitorius 12.0–13.2 11.6–13.1 4.7- 6.4 -
Catharanthus roseus 7.9- 9.0 4.7- 4.8 4.8- 6.1 4.3- 6.9
Hibiscus sabdariffa 10.4–13.9 7.1–14.0 - 8.1–17.6
Sugar maple 8.2–13.3 5.3–52.4 - 4.8- 7.5
Flower (color)
Clitoria ternatea (PI 209591) (white) 1.0- 1.2 0.8- 1.1 - -
C. ternatea (PI 283232) (dark blue) 7.1- 9.8 14.0–26.8 - -
C. ternatea (PI 226265) (blue) 1.0 1.1- 6.0 1.2- 1.7 -
Catharanthus roseus (dark salmon) 19.5–23.9 10.9–15.5 3.0- 3.1 -
385
386

Table 2. Potentially useful phytochemicals in Clitoria, Desmodium, Corchorus, Catharanthus and Hibiscus species.

Potential
therapeutic
Taxon phytochemical Potential use Plant part

Clitoria ternatea Anthocyanin Flower

Finotin Pesticidal (Kelemu et al., 2004) Seeds
Desmodium adscendens Ethanolic extract Analgesic (N’gouemo et al., 1996) Leaves
Ovarian inflammation (Barreto, 2002)
Corchorus olitorius Phenolic compounds Antioxidative (Azuma et al., 1999) Leaves
Soluble dietary fiber Suppress blood glucose elevation (Innami et al., 2005) Leaves
Phytol Antitumor promoter (Furumoto et al., 2002) Leaves
Inhibited malaria parasite > 90% (Sathiyamoorthy
et al., 1999)
Extract Antibacterial (Zakaria et al., 2006) Leaves
Hibiscus sabdariffa Extract Hypertension, pyrexia, liver disorders
(Chang-Che et al., 2003)
Anthocyanins Inhibit LDL oxidation, apoptosis for chemoprevention
(Yun-Ching et al., 2006) Flower (dried)
Anticancer (Hou et al., 2005; Chang et al., 2005) Calix (dried)
Phenolic compounds Antioxidant, antimutagenic, radical scavenging
(Farombi and Fakoya, 2005) Flower (dried)
Figures

25
a

20
Anthocyanin index

ab
15
a ab
abc
a
a
ab
ab ab Cor. Olitorius
10 a a
a abcd H. sabdariffa
Sugar maple
cd
c cd Clitoria ternatea
b
5 D. adscendens
Cath roseus

0
July Sept. Oct.
Month

Fig. 1. Mean anthocyanin indexes from six species tested in the field during summer and
fall.

a
Dark salmon flower
ef C.ros. Flr
Blue flower C.ter.3Flr
Leaf and flower colors

f C.ter.2Flr
White flower
C.ter.1Flr
ab C.ros.Leaf
Dark blue flower
D.ads. Leaf
Green orange leaf C.ter. Leaf
abc
S. ma. Leaf
Green purple leaf abcd H.sab. Leaf
bcd
C.ol. Leaf
Green leaf def
bcde
bcd

0 5 10 15 20
Anthocyanin index

Fig. 2. Combined mean leaf and flower anthocyanin indexes from all six species during
the entire season (July-October). Those accessions producing similar leaf and
flower colors were combined.

387
388

Fig. 3. HPLC chromatograms of isoflavones identified in

D. adscendens, C. ternatea, C. olitorius and
H. sabdariffa.