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Food Sci. Techno!., Int., I (2), 74-76, 1995

Lnuonoid Glucosides in Seeds

of Shiikuwasha ( Citrus depressa HAYATA)

Hideaki OHTA,1 Yolchi NOGATA,1 Koh-Ichi YOZA,1 Ken-Ichi KUSUMOTOI and Shin HASEGAWA2

* Chugoku National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries. Fukuyama, Hiroshima 721,

Ja pa n

2 US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, A Ibany, Cahfornia, 94710 U. S.A.

Received Aprll 26, 1995

Limonoid glucosides extracted from seeds of Shiikuwasha (Citrus depressa HAYATA) grown in Okinawa were

separated by column chromatography and high-performance liquid chromatography (HPLC), and then analyzed by

thin layer chromatography, HPLC and nuclear magnetic resonance spectrometry. The 17-P-D-glucopyranoside

derivatives of nomilin, Iimonin, deacetylnomilin and nomilinic acid were identified. The seeds contained 1.27 g of

total limonoid glucosides per 100 g on a dry weight basis, showing two-fold higher levels than that found in other

common citrus seeds.

Keywords: Iimonoids, Iimonoid glucosides, HPLC, Shiikuwasha, Citrus depressa, citrus

Limonoids are a group of chemically related hlghly

oxygenated triterpenoids present in the Rutaceae and

Meliaceae family plants. Llmonoids are one of two bitter

principles in Citrus. Citrus limonoids have been shown to

have biological activities. They induce increased actlvity of a

detoxifying enzyme, glutathione S-transferase, and inhibit the

formation of tumors in laboratory animals such as mlce and

hamsters (Lam & Hasegawa, 1989; Lam et a/., 1989; Miller et

a/., 1989, 1992). They also possesses antifeedant activity

against Insects and termites (Klocke & Kubo, 1982; Alford &

Bentley, 1986; Alford, et a/., 1987; Ishigaki & Kawaguchi,

1989). Because of the above, the demand for limonoids has

increased significantly in recent years. We are continuously

searching for new sources of limonoids.

Limonoids are present in mature citrus frult tissues and

seeds as both aglycones and glucoside derivatives (Hasegawa

et aZ, 1989). They are synthesized during maturation as one

of the major secondary metabolites in Citrus (Fong et al,

1991). Commercial orange juices, for example, contain an

average of 320 ppm of total limonoid glucosides (Fong et a/.,

1989). Limonin 1 7-~-D-glucopyranoside has also been shown

to possess anticancer activity in hamsters (Miller et a/., 1992).

Limonoid glucosides are practically tasteless and soluble in

water. Therefore, they could be ideal natural food additlves

for cancer prevention. Citrus seeds have been shown to

contain high concentrations of limonoid aglycones and

glucosides (Hasegawa et al, 1980; Ozaki et al, 1991; Ohta et

aZ, 1992). Shiikuwasha, the so-called Hiraml lemon, is a very

popular Citrus in the Okinawa region due to its unique sour

fiavor. In this study, we have examined the limonoid

glucosides in seeds of Shiikuwasha grown in Okinawa.

Materials and Methods

Materials Shiikuwasha (Citrus depressa HAYATA)

was obtained from the Agricultural Product Processing

Factory, Agricultural Cooperative Association of Okinawa,

Japan. Thin layer chromatography (TLC) plates (silica gel)

were purchased from Analtech (Newark, DE, USA). The

analytical high-performance liquid chromatography (HPLC)

column, C18 reversed phase, partisil ODS-3, 2.2x25 cm,

particle size 10 pm, and the preparative HPLC column, 4.6x

250 mm, particle size 5 pm, were purchased from Analtech,

Deerfield, IL. DEAE-Sephacel and XAD-2 resin (20-60

mesh) were obtained from Sigma. Nuclear magnetic reso-

nance (NMR) spectral assignments were made on the basis of

IH-1HCOSY and NOESY, DEPT and 13C-1HCOSY spectra,

using a JEOL EX400 at 400 MHZ for IH and 100 MHZ for

13C. The standard limonoid glucosides used in this study were

Isolated from various citrus j uices and seeds and characterized

by NMR spectrometry.

Extraction of limonoid glucosides Seeds were dried

and ground in a Retsch mill. An allquot of 100 g of seed meal

was placed in a Soxhlet extractor, washed thoroughly with

hexane to remove oily materials, extracted with acetone and

subsequently extracted with methanol. The acetone extract

(fraction 1) contained most of the aglycones, and the

methanol extract (fraction 2) contained the limonoid

glucosides and the remainder of aglycones. This fraction was

evaporated to dryness, and the residue was extracted with a

mixture of dichloromethane and water (1 : 1). The water

fraction (fraction 3) contained glucosides and the dichloro-

methane fraction (fractlon 4) contained aglycones.

Isol a tion and ch aracterization

The limonoid

glucoside fraction (fraction 3), after adjusting its pH to 6.5,

was loaded on a DEAE-Sephacel column (2.5X25 cm) and

eluted with a linear gradient system with increasing NaCl

content in water. This treatment separates the limonoid

glucosides from neutral compounds such as flavonoids. The

glucoside fraction was then desalted through a Dowex 50

column (H+ form, 1.5x20 cm), and refractionated on an

XAD-2 column (2.5x75cm) which was developed with

methanol (Ohta et a/, 1992). After the solvent was evapo-

Lirnonoid Gtucosides in Shlikuwasha

rated, the extract was dissolved in water and fractionated on

a C18 reversed phase preparative HPLC column. Fractions

containing specific limonoid glucosides were obtained and

subsequently characterized by NMR and HPLC analyses

(Hasegawa et al, 1989).

Quantitative analysis Both the acetone (fraction l) and

dichloromethane (fraction 4) fractions were combined and

evaporated to analyze the limonoid aglycones. The dried

material was resuspended in methanol and a portion was

injected into a C18 reversed phase column, the column being

eluted isocratically with acetonitrile-methanol-water ( 10 : 41 :

49). The limonoid aglycones were separated and identified by

monitoring the UV absorption at 210nm as described

previously (Ozaki et a/., 1991).

The limonoid glucoside content was also determined by

TLC and HPLC methods as described previously (Ozaki et

al., 1991). For TLC analysis, the water extract (fraction 3)

was evaporated, dissolved in a measured volume of methanol

and spotted on a plate, which was developed with ethyl

acetate- methyl ethyl ketone-formic acid-water (5 : 3 : 3 : I ).

After drying, the plate was sprayed with Ehrlich's reagent and

color was developed in an HCI gas chamber. The spots were

compared with those of standards, and the total limonoid

glucosides were determined. For HPLC analysis, duplicate

IOO pl injections were made into a C18 reversed phase HPLC

column, the column being eluted at one ml/min with a linear

gradient system, starting with 15% acetonitrile in 3 mM

phosphoric acid and ending with 26% acetonitrile over 33

min. The elution was monitored by UV absorption at 210 nm

and a standard curve was run for each limonoid glucoside.

Each compound was quantified by its peak area.

Results and Discussion

Table I shows the limonoid aglycone and limonoid

glucoside contents in the seed of Shiikuwasha. TLC and

HPLC analyses showed the presence of five limonoid

glucosides. They are identified by NMR analysis as the 17-~-

D-glucopyranosides of lirnonin, deacetylnomilin, nomilin,

nomilinic acid and obacunone. The seeds contained rela- tively high concentrations of limonoid glucosides. They

contained 1.274 g of total limonoid glucosides per 100 g of

seeds on a dry weight basis, which is approximately two fold higher than that of other common citrus seeds (Ozaki et a/.,

1991). As expected, nomilin glucoside was the predominant

limonoid glucoside, followed by the glucosides of

obacunone, nomilinic acid, Iimonin glucoside and deacetyl-

nomilin in order of decreasing concentration. This order is

very similar to that of many citrus species (Ozaki et a/ ., 1991 ).

Because limonoids are biosynthesized in seeds, fruit tissues

and leaves independently (Fong et al, 1992), this is quite

different from that in the fruit tissues where limonin glucoside

is predominant,

The conversion of open D-ring limonoid aglycones to their

corresponding glucosides is catalyzed by UDP-D-glucose

transferase, which has been isolated from the albedo tissues of

navel oranges (Suhayda & Hasegawa, 1994). The glucosida-

tion of aglycones in the seeds begins during the late stages of

fruit growth and continues during maturation until the fruit

is harvested (Hasegawa et a/, 1991). The Shiikuwasha seeds


Table 1. Limonoid aglycones and glucosides in seeds of Shiikuwasha

( Citrus depressa HAYATA)a).


Aglycone of





Glucoside of




Nomilinlc acid



a) See text for analytical conditions.

T* (min)

Content (%)

l 5, l


1 87







O, 1 16


0,0 1 8





1 85



1 96

I .274

contain limonoid glucosides at levels about two-fold higher

than that of other common citrus seeds; therefore it is most

likely that there are higher levels of UDP-D-glucose transfer-

ase activity. The fruits used in this study were harvested during the early season (November). Thus, fruits harvested

during the late season should have higher concentrations of

limonoid glucosides. This work shows that Shiikuwasha

seeds are excellent sources of limonoid glucosides.

Acknowledgments This work was supported by a Grant-in-Aid from

the Science and Technology Agency of Japan and by an integrated

research program for effective use of biological activities to create new

demand (Bio Renaissance Program) from the Ministry of Agriculture,

Forestry and Fisheries (BRP-95-VII-B-2). S.H. was a recipient of

Research Awards for specialists from Japanese Government.


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