Bull. Natl. Mus. Nat. Sci., Ser. B, 36(3), pp. 127–132, August 22, 2010

Flavonoids from two Parasitic and Achlorophyllous Plants,
Aeginetia indica and Orobanche minor (Orabanchaceae)
Tsukasa Iwashina
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, 305–0005 Japan
E-mail: iwashina@kahaku.go.jp(Received 12 June 2010; accepted 23 June 2010)

Abstract Two parasitic and achlorophyllous species, Aeginetia indica and Orobanche minor belonging to the family Orobanchaceae, were surveyed for flavonoids. The flowers and aerial parts ofA. indica were exracted with MeOH and the flavonoids were isolated by various chromatographicaltechniques. Two anthocyanins were obtained from the flowers together with other flavonoids, andcharacterized as cyanidin 3-O-rutinoside and 3-O-glycoside. Other flavonoids were identified asapigenin 7-O-glucuronide, luteolin 7-O-glucuronide, apigenin, luteolin, quercetin 3-O-rutinosideand naringenin 7-O-glucoside. They were also obtained from the aerial parts except for anthcyanins and luteolin. On the other hand, two flavonoids were isolated from the aerial parts of
Orobanche minor and identified as luteolin 7-O-glucoside and 7-O-glucuronide.
Key words : Aeginetia indica, anthocyanin, apigenin, flavonoids, luteolin, Orobanchaceae,
Orobanche minor.

Introduction
Aegineta indica L. is parasitic on roots ofmonocots, especially the Poaceae such as Saccharum officinarum L. and Miscanthus sinensis
Anderss., and is distributed in Indonesia to Japan(Mabberley, 1997). Though the whole plants are
achlorophyllous and therefore brownish or yellow brown, the flowers are pale red purple.
On the other hand, Orobanche minor Sutton isparasitic on roots of the Leguminosae, Trifoliumspecies, and sometimes Asteraceae and Umbelliferae species. Though the species is originally
native to Europe and Africa, it was now widely
naturalized in the world.
A flavone aglycone, apigenin has been reported from A. indica as antihepatotoxic substance(Oshima et al., 1984). A common iridoid, aucubin has also isolated from the whole plants (Endoet al., 1979). On the other hand, a flavone aglycone, tricin has been found in the seeds ofOrobanche sp. as allelopathy compound (Rice,1984). The occurrence of phenylethanoids, e.g.
acteoside, bandioside, orobanchoside and crenatoside, has been reported in the Orobanchaceae(Serafini et al., 1995; Mølgaard and Ravn, 1988;
Cometa et al., 1993; Andary et al., 1982).
In this paper, the flavonoid properties of theflowers and aerial parts of two parasitic plants,
Aeginetia indica and Orobanche minor, are described.
Materials and Methods
Plant materials
Aegineta indica L. (Fig. 1) which is natively
growing in Tsukuba Botanical Garden, National
Museum of Nature and Science, Tsukuba, Japan,
was collected for flavonoid analysis. It was parasitic on roots of Miscanthus condensatus Hack.
(Poaceae).
Orobanche minor Sutton (Fig. 1) was collected
in Tsukuba City, Ibaraki, Japan. The species wasparasitic on roots of Trifolium repens L. (Legu-128

Tsukasa Iwashina

Fig. 1.

Aeginetia indica (left) and Orobanche minor (right).

minosae). Voucher specimens were deposited in
the herbarium of National Museum of Natureand Science, Tokyo (TNS).
Extraction and isolation of anthocyanin andflavonoids in A. indica flowers
Fresh flowers (107 g) were extracted with
MeOH/HOAc/H2O (10 : 1 : 10). The concentratedextracts were applied to preparative paper chromatography (prep. PC) using solvent system:
BAW (n-BuOH/HOAc/H2O4 : 1 : 5, upper phase).
Anthocyanin and other flavonoid bands were cut
out and eluted with 5% HOAc in MeOH (anthocyanins) or MeOH (other flavonoids). Anthocyanins were purified by Sephadex LH-20 column chromatography using solvent system:
HOAc/MeOH/H2O (5 : 70 : 25). On the other
hand, other flavonoids were applied to prep. PC
using solvent systems: 15% HOAc and thenBEW (n-BuOH/EtOH/H2O4 : 1 : 2.2). The
obtained flavonoids were finally purified bySephadex LH-20 column chromatography using
solvent system: 70% MeOH. Two flavonoids (F1and F2) were obtained as pale yellow powders(ca. 10 and 5 mg, respectively).

Extraction and isolation of flavonoids in A. indica and O. minor aerial parts
Fresh aerial parts of A. indica (280 g) and O.
minor (367 g) were extracted with MeOH,respectively. The concentrated extracts werewashed with petroleum ether for removal of
lipophilic compounds, and then applied to prep.
PC using solvent systems: BAW, 15% HOAc andthen BEW. The obtained flavonoids were finallypurified by Sephadex LH-20 column chromatography using solvent system: 70% MeOH.
High performance liquid chromatography(HPLC)
Qualitative HPLC was performed with Shimadzu HPLC systems using Shim-pack CLCODS [I.D. 6.0150 mm (Shimadzu)], at a flowrate of 1.0 ml min1; detection wavelength was
190–350 nm and eluents were MeCN/H2O/H3PO4(20 : 80 : 0.2 for glycosides, and 35 : 65 : 0.2 foraglycones).
Liquid chromatograph-mass spectra (LC-MS)
LC-MS was performed with Shimadzu LCMS systems using Senshu Pak PEGASIL ODScolumn [I.D. 2.0150 mm, Senshu Scientific Co.
Ltd.], at a flow-rate of 0.1 ml min1; ESI 4.5 kV

Flavonoids from Aeginetia and Orobancheand ESI 3.5 kV, 250°C. The eluent was MeCN/
H2O/HCOOH (20 : 75 : 5).
Identification of anthocyanins
The anthocyanins from A. indica flowers wereidentified by characterization of the products obtained by acid hydrolysis (in 12% HCl, 100°C,3 min), visible spectroscopy, and direct PC comparison with authentic sample. PC and UV spectral data of two isolated anthocyanins were asfollows.
Cyanidin 3-O-rutinoside (keracyanin, A1). PC:
Rf 0.22 (BAW), 0.35 (BuH, n-BuOH/conc.HCl/H2O7 : 2 : 5), 0.17 (1% HCl), 0.79 (Forestal,HOAc/conc.HCl/H2O5 : 3 : 30); UV—purplish
red. Visible spectra: l max (nm) 0.1% MeOH–HCl
530 nm.
Cyanidin 3-O-glycoside (A2). PC: Rf 0.28(BAW), 0.30 (BuH), 0.07 (1% HCl), 0.71 (Forestal); UV—purplish red. Visible spectra: l max(nm) 0.1% MeOH–HCl 528 nm.
Identification of other flavonoids
Other flavonoids from A. indica and O. minor
were identified by UV spectroscopy according toMabry et al. (1970), LC-MS, acid hydrolysis (in
12% HCl, 100°C, 30 min), direct HPLC and TLC
comparisons with authentic specimens. TheirTLC, UV and LC-MS data were as follows.
Apigenin 7-O-glucuronide (F1). TLC: Rf 0.64(BAW), 0.54 (BEW), 0.19 (15% HOAc); UV—
dark purple, UV/NH3—dark greenish yellow.
UV: l max (nm) MeOH 267, 333; NaOMe 274,378 (inc.); AlCl3 275, 299, 348, 376; AlCl3/HCl 275, 298, 341, 374; NaOAc 266, 389;NaOAc/H3BO3 267, 340. LC-MS: molecularion peak, m/z 447 [MH] (apigenin1 mol
glucuronic acid), fragment ion peak, m/z 269[M176H] (apigenin).
Luteolin 7-O-glucuronide (F2). TLC: Rf 0.40(BAW), 0.40 (BEW), 0.11 (15% HOAc); UV—
dark purple, UV/NH3—yellow. UV: l max (nm)
MeOH 256, 266, 348; NaOMe 266, 272, 392(inc.); AlCl3 274, 426; AlCl3/HCl 265, 273,295sh, 360, 384sh; NaOAc 257sh, 265, 402;NaOAc/H3BO3 257sh, 262, 374. LC-MS: mol-129

ecular ion peak, m/z 463 [MH] (luteolin1
mol glucuronic acid), fragment ion peak, m/z 287[M176H] (luteolin).
Apigenin (F3). TLC: Rf 0.93 (BAW), 0.96(BEW), 0.80 (Forestal); UV—dark purple, UV/
NH3—dark greenish yellow. UV: l max (nm)
MeOH 267, 339; NaOMe 275, 326, 393 (inc.);
AlCl3 274, 302, 355, 376sh; AlCl3/HCl 276,299, 348, 375sh; NaOAc 275, 311, 388;NaOAc/H3BO3 267, 348. LC-MS: molecularion peak, m/z 271 [MH] (apigenin).
Luteolin (F4). TLC: Rf 0.84 (BAW), 0.87(BEW), 0.55 (Forestal); UV—dark purple, UV/
NH3—yellow. UV: l max (nm) MeOH 257sh, 265,360; NaOMe 267sh, 274, 331, 411 (inc.);
AlCl3 275, 431; AlCl3/HCl 266, 274, 300,362, 395sh; NaOAc 267sh, 274, 326, 397;NaOAc/H3BO3 265, 380. LC-MS: molecularion peak, m/z 285 [MH] (luteolin).
Luteolin 7-O-glucoside (F5). TLC: Rf 0.42(BAW), 0.43 (BEW), 0.07 (15% HOAc); UV—dark purple, UV/NH3—dark yellow. UV: l max(nm) MeOH 255, 266sh, 347; NaOMe 268,392 (inc.); AlCl3 273, 426; AlCl3/HCl 266,274sh, 295, 359, 384sh; NaOAc 260, 405;NaOAc/H3BO3 259, 373.
Acteoside. TLC: Rf 0.60 (BAW), 0.52 (BEW),
0.74 (15% HOAc); UV—blue, UV/NH3—bluegreen. UV: l max (nm) MeOH 246sh, 291, 332;
NaOMe 258, 384 (inc.); AlCl3 262, 300,363; AlCl3 245sh, 290sh, 332; NaOAc 279,289sh, 380; NaOAc/H3BO3 256sh, 295, 355.
LC-MS: molecular ion peak, m/z 623 [MH]
(acteoside).
Results
Anthocyanins and flavonoids in Aeginetia indica
Two anthocyanins, four flavones, and each one
flavonol and flavanone were found in the flowersof A. indica. Of their compounds, major anthocyanin A1 was identified as cyanidin 3-O-rutinoside (Fig. 2) by acid hydrolysis, visible spectral properties and direct PC comparison withauthentic keracyanin from the flowers of Cannageneralis L.H. Bailey (Cannaceae) (Hayashi et130

Tsukasa Iwashina

Fig. 6.

Fig. 2.

Cyanidin 3-O-rutinoside (keracyanin, A1).

Fig. 3.

Apigenin 7-O-glucuronide (F1).

Fig. 4.

Luteolin 7-O-glucuronide (F2).

Fig. 5.

Apigenin (F3).

al., 1953). Another minor anthocyanin was characterized as cyanidin glycoside.
UV spectral properties of two flavone glycosides F1 and F2 showed that they are apigenin
and luteolin 7-O-glycosides, respectively. Acidhydrolysis of them liberated apigenin and glu-Luteolin (F4).

curonic acid, and luteolin and glucuronic acid.
Finally, F1 and F2 were identified as apigenin 7O-glucuronide (Fig. 3) and luteolin 7-O-glucuronide (Fig. 4) by direct TLC and HPLC comparisons with authentic samples from the leavesof Uncarina grandidieri (Baill.) Stapf. (Pedaliaceae) (Yamazaki et al., 2007).
UV spectra of flavonoids F3 and F4 showedthat they are flavones having free 5-, 7- and 4hydroxyl, and free 5-, 7-, 3- and 4-hydroxyl
groups, respectively. Moreover, they were soluble
in diethyl ether, showing that the compounds areflavone aglycones. Their Rf values of TLC andretention times of HPLC completely agreed withthose of authentic apigenin (Fig. 5) and luteolin(Fig. 6) from the pubescence of Glycine max (L.)
Merr. (Leguminosae) (Iwashina et al., 2006),
respectively.
Flavonoid F6 was obtained from the flowers ofA. indica as a minor compound. Its UV spectral
properties clearly showed that F6 is 3-substituted
quercetin. The compound was directly comparedwith authentic rutin (Fig. 7) from the leaves ofBegonia formosana (Hayata) Masam. (Begoniaceae) (Iwashina et al., 2008), and their retention
times agreed with that of each other.
Flavonoid F7 was also isolated from the flowers of A. indica. UV spectral properties of thecompound was those of flavanone, dihydroflavonol or dihydrochalcone (Mabry et al.,1970). Finally, flavonoid F7 was identified as flavanone, naringenin 7-O-glucoside (Fig. 8) by direct HPLC comparison with commercial authentic sample (Carl Roth). Though the presence ofanother flavanone was shown by HPLC survey,
its retention time did not agree with those of authentic naringenin 7-O-neohesperidoside and eri-

Flavonoids from Aeginetia and Orobanche131(Yamazaki et al., 2007), respectively. Apart fromtheir flavonoids, much amount of colorless powder, which was presumed as caffeoyl derivative
by UV spectral properties, was obtained (ca. 100mg). This was identified as phenylethanoid,acteoside, by LC-MS and direct HPLC comparison with authentic sample from the leaves ofPlantago asiatica (Plantaginaceae) (Murai et al.,2009).
Discussion
Fig. 7.

Quercetin 3-O-rutinoside (rutin, F6).

Fig. 8.

Naringenin 7-O-glucoside (F7).

Fig. 9.

Luteolin 7-O-glucoside (F5).

odyctiol 7-O-glucoside.
Flavonoids in Orobanche minor
Two flavonoids were isolated from the aerial
parts of O. minor. They were shown to be luteolin 7-O-glycosides by their UV spectral survey.
They liberated luteolin and glucose, and luteolin
and glucuronic acid by acid hydrolysis, respectively. Finally, they were identified as luteolin 7O-glucoside (F5) (Fig. 9) and luteolin 7-O-glucuronide (F2) by direct TLC and HPLC comparisons with authentic samples from the leaves ofSchmalhausenia nidulans Petrak (Asteraceae)(Iwashina and Kadota, 1999) and Uncarina spp.

In this experiment, two anthocyanins, fourflavones, and each one flavonol and flavanone
were found in the flowers and aerial parts of
Aeginetia indica and completely or partiallycharacterized. On the other hand, two flavone
glycosides were isolated from the aerial parts of
Orobanche minor. Only one flavonoid, apigenin
has been reported from Aeginetia species including A. indica until now (Oshima et al., 1984).
Though the aerial parts of this species is achlorophyllous and therefore brownish or yellow
brown, the flower is pale red purple. In this survey, it was shown that the flower color is due toanthocyanin, cyanidin 3-O-rutinoside, together
with another cyanidin glycoside. Four flavones,
apigenin and luteolin, and their 7-O-glucuronides, a flavonol, quercetin 3-O-rutinoside,
and a flavanone, naringenin 7-O-glucoside, wereaccompanied with anthocyanins in flowers. They
were detected in the aerial parts except for two
anthocyanins and luteolin. Their flavonoids
except for apigenin were reported in Aeginetiaspecies for the first time.
A flavone, tricin has been reported from seedsof Orobanche species as allelopathy compound(Rice, 1984). In this survey, two glycosides, luteolin 7-O-glucoside and 7-O-glucuronide, were
found in the aerial parts of Orobanche species forthe first time.
The flavonoids have been reported from a few
achlorophyllous plants, e.g. quercetin 3-O-glucoside and 3-O-glucuronide from Monotropa uniflora L. (Pyrolaceae) (Bohm and Averett, 1989),
3-hydroxyphloretin and its 4-O-glucoside from132

Tsukasa Iwashina

Balanophora tobiracola Makino (Balanophoraceae) (Ito et al., 1980), vitexin, isovitexin andvicenin-2 from Petrosavia sakuraii (Makino) J.J.
Sm. ex van Steenis (Petrosaviaceae, Liliaceaes.l.) (Iwashina et al., unpublished data). However,their flavonoid composition was comparatively
simple. One of major function of flavonoids isUV shields (Bohm, 1998), so that achlorophyllous plants, which do not perform the photosynthesis, may be not synthesize many kinds andlarge amount of flavonoids. The study of theflavonoid function in achlorophyllous plants is inprogress.
References
Andary, C., Wylde, R., Laffite, C., Privat, G. and Winternitz, F. 1982. Structures of verbascoside and orobanchoside, caffeic acid sugar esters from Orobanche
rapum-genistae. Phytochemistry 21: 1123–1127.
Bohm, B.A. and Averett, J.E. 1989. Flavonoids in some
Monotropoideae. Biochemical Systematics and Ecology 17: 399–401.
Bohm, B.A. 1998. Introduction to Flavonoids. pp. 339–364, harwood Academic Publishers, Amsterdam.
Cometa, F., Tomassini, L., Nicoletti, M. and Pieretti, S.
1993. Phenylpropanoid glycosides. Distribution andpharmacological activity. Fitoterapia 64: 195–217.
Endo, T., Taguchi, H., Sasaki, H. and Yoshida, I. 1979.
Studies on the constituents of Aeginetia indica L. var.
garcilis Nakai structures of three glycosides isolated
from the whole plant. Chemical and Pharmaceutical
Bulletin 27: 2807–2814.
Hayashi, K., Noguchi, T. and Abe, Y. 1953. Studien überAnthocyane, XXIV. Keracyanin; ein Farbstoffprinzip inden feuerroten Blüten von Canna generalis. Pharmaceutical Bulletin 2: 41–45.
Ito, K., Itoigawa, M., Haruna, M., Murata, H. and Fu-rukawa, H. 1980. Dihydrochalcones from Balanophoratobiracola. Phytochemistry 19: 476–477.
Iwashina, T. and Kadota, Y. 1999. Flavonoids from
Schmalhausenia nidulans (Compositae): a taxon
endemic to the Tien Sian Mountains. BiochemicalSystematics and Ecology 27: 97–98.
Iwashina, T. Benitez, E.R. and Takahashi, R. 2006. Analysis of flavonoids in pubescence of soybean near-isogenic lines for pubescence color loci. Journal of Heredity 97: 438–443.
Iwashina, T., Saito, Y., Peng, C.-I, Yokota, M. andKokubugata, G. 2008. Foliar flavonoids from two Begonia species in Japan. Bulletin of National Museum ofNature and Science, Series B 34: 175–181.
Mabberley, D.J. 1997. The Plant-Book 2nd. ed. pp. 13 and511–512. Cambridge University Press, Cambridge.
Mabry, T.J., Markham, K.R. and Thomas, M.B. 1970. The
Systematic Identification of Flavonoids. Springer,Berlin.
Mølgaard, P. and Ravn, H. 1988. Evolutionary aspects ofcaffeoyl ester distribution in dicotyledons. Phytochemistry 27: 2411–2421.
Murai, Y., Takemura, S., Takeda, K., Kitajima, J. andIwashina, T. 2009. Altitudinal variation of UV absorbing compounds in Plantago asiatica. Biochemical Systematics and Ecology 37: 378–384.
Oshima, Y., Kawakami, Y., Kiso, Y., Hikino, H., Yang,L.L. and Yen, K.Y. 1984. Antihepatotoxicity principles
of Aeginetia indica herbs. Shoyakugaku Zasshi 38:
198–200.
Rice, E.L. 1984. Allelopathy. Academic Press, New York.
Serafini, M., Di Fabio, A., Foddai, S., Ballero, M. andPoli, F. 1996. The occurrence of phenylpropanoid glycosides in Italian Orobanche spp. Biochemical Systematics and Ecology 23: 855–858.
Yamazaki, K., Iwashina, T., Kitajima, J., Gamou, Y.,
Yoshida, A. and Tannowa, T. 2007. External and internal flavonoids from Madagascarian Uncarina species(Pedaliaceae). Biochemical Systematics and Ecology
35: 743–749.