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

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,

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/.,

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

mesh) were obtained from Sigma. Nuclear magnetic resonance (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

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

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

(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

Limonoid glucosides are practically tasteless and soluble in water. Therefore, they could be ideal natural food additlves

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

fraction (fraction 3) contained glucosides and the dichloro-

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

methane fraction (fractlon 4) contained aglycones. Isol a tion and ch aracterization The
glucoside fraction (fraction 3), after adjusting its pH to 6.5,

limonoid

popular Citrus in the Okinawa region due to its unique sour

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

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,

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

7,5

rated, the extract was dissolved in water and fractionated on

Table 1. Limonoid aglycones and glucosides in seeds of Shiikuwasha

( Citrus depressa HAYATA)a).

a C18 reversed phase preparative HPLC column. Fractions containing specific limonoid glucosides were obtained and

Limonoids
Aglycone of

T* (min)
l 5, l

Content (%)
O. 1 87

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

Limonin Nomilin

26.3

Obacunone
Total

43.2

0.096 0.045 0.328

O, 1 16
0,0 1 8

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 :

Glucoside of

Limonin
Deacetynomilin

16.2

23.4
29.6
30.5

Nomilin
Nomilinlc acid

0,759
O. 1 85 O. 1 96

49). The limonoid aglycones were separated and identified by

monitoring the UV absorption at 210nm as described

previously (Ozaki et a/., 1991).

Obacunone
Total
a) See text for analytical conditions.

33.8

I .274

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 ).

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-

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.

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.

Results and Discussion

Table I shows the limonoid aglycone and limonoid

glucoside contents in the seed of Shiikuwasha. TLC and

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