Antiproliferative activity of Rhinacanthus nasutus (L.) KURZ extracts and the active moiety, rhinacanthin C

Article abstract of DOI:10.1248/bpb.27.1070

Rhinacanthus nasutus (L.) KURZ (Acanthaceae) is a shrub widely distributed in South China and India. In this study, the antiproliferative activity of the ethanol extract of root and aqueous extract of leaves of R. nasutus, and the supposed active moiety rhinacanthin C was assessed in vitro using the human cervical carcinoma cell line HeLa, its MDR1-overexpressing subline Hvr100-6, human prostate carcinoma PC-3 cells and human bladder carcinoma T24 cells. Rhinacanthin C was chemically synthesized and its content in the R. nasutus extracts was determined by HPLC with a photodiode array detector. The antiproliferative activity of the R. nasutus extracts was also assessed in vivo using sarcoma 180-bearing mice. It was suggested that 1) the in vitro antiproliferative activity of rhinacanthin C was comparable with or slightly weaker than that of 5-FU, 2) rhinacanthin C showed antiproliferative activity for MDR1-overexpressing Hvr100-6 cells, similarly to parent HeLa cells, 3) the in vitro antiproliferative activity of the ethanol extract of root R. nasutus was due to rhinacanthin C, whereas that of the aqueous extract of leaves of R. nasutus was due to constituents other than rhinacanthin C, and 4) both of the R. nasutus extracts showed in vivo antiproliferative activity after oral administration once daily for 14 d.

Full text of DOI:10.1248/bpb.27.1070

1070
Biol. Pharm. Bull. 27(7) 1070—1074 (2004)
Vol. 27, No. 7
Antiproliferative Activity of Rhinacanthus nasutus (L.) KURZ Extracts and
the Active Moiety, Rhinacanthin C
Akinobu GOTOH,a Toshiyuki SAKAEDA,*,b Takashi KIMURA,b Toshiro SHIRAKAWA,c Yoshitaka WADA,c
Atsushi WADA,b Tetsutaro KIMACHI,d Yoshiji TAKEMOTO,e Akira IIDA,e Seigo IWAKAWA,f Midori HIRAI,f
b
Hisako TOMITA,f Noboru OKAMURA,b Tsutomu NAKAMURA,b and Katsuhiko OKUMURA
a Department of Clinical Genetics and International Center for Medical Research, School of Medicine, Kobe University;
c
b Department of Hospital Pharmacy, School of Medicine, Kobe University; Department of Organs Therapeutics, Faculty
of Medicine, Kobe University Graduate School of Medicine; 7–5–2 Kusunoki-cho, Chuo-ku, Kobe 650–0017, Japan:
d Faculty of Pharmaceutical Sciences, Mukogawa Women’s University; 11–68 Koshien 9-Bancho, Nishinomiya 663–8179,
Japan: e Graduate School of Pharmaceutical Sciences, Kyoto University; Yoshida, Sakyo-ku, Kyoto 606–8501, Japan: and
f Kobe Pharmaceutical University; 4–19–1 Motoyamakita-machi, Higashinada-ku, Kobe 658–8558, Japan.
Received February 17, 2004; accepted April 28, 2004; published online May 10, 2004
Rhinacanthus nasutus (L.) KURZ (Acanthaceae) is a shrub widely distributed in South China and India. In
this study, the antiproliferative activity of the ethanol extract of root and aqueous extract of leaves of R. nasutus,
and the supposed active moiety rhinacanthin C was assessed in vitro using the human cervical carcinoma cell line
HeLa, its MDR1-overexpressing subline Hvr100-6, human prostate carcinoma PC-3 cells and human bladder
carcinoma T24 cells. Rhinacanthin C was chemically synthesized and its content in the R. nasutus extracts was
determined by HPLC with a photodiode array detector. The antiproliferative activity of the R. nasutus extracts
was also assessed in vivo using sarcoma 180-bearing mice. It was suggested that 1) the in vitro antiproliferative
activity of rhinacanthin C was comparable with or slightly weaker than that of 5-FU, 2) rhinacanthin C showed
antiproliferative activity for MDR1-overexpressing Hvr100-6 cells, similarly to parent HeLa cells, 3) the in vitro
antiproliferative activity of the ethanol extract of root R. nasutus was due to rhinacanthin C, whereas that of the
aqueous extract of leaves of R. nasutus was due to constituents other than rhinacanthin C, and 4) both of the R.
nasutus extracts showed in vivo antiproliferative activity after oral administration once daily for 14 d.
Key words Rhinacanthus nasutus (L.); rhinacanthin C; MDR1
Rhinacanthus nasutus (L.) KURZ (Acanthaceae), a shrub with a photodiode array detector, and its in vitro antiprolifer-
widely distributed in South China and India, has been re- ative activity was also evaluated and compared with that of
cently cultivated for use in the treatment of hepatitis, dia- the R. nasutus extracts to elucidate whether rhinacanthin C
betes, hypertension and skin diseases in Taiwan.1,2) Indige- was the main constituent for antiproliferative activity.
nous medical practitioners use the roots and leaves pounded
with vinegar or alcohol.1,2) Among various compounds iso- MATERIALS AND METHODS
lated from R. nasutus, including flavonoids, anthraquinones,
triterpenes, and sterols, naphthoquinones have shown some
Materials Both of the R. nasutus extracts, the ethanol
biological activity.1—3) To date, 14 naphthoquinone deriva- extract of root and aqueous extract of leaves, were kindly
tives have been isolated and named rhinacanthin A—D and supplied by ARSOA HONSHA Corp. (Yamanashi). Rhi-
G—P.1—3) Although there is little information on their biolog- nacanthin C was synthesized as described below. Vinblastine
ical activity, rhinacanthin C and D have been demonstrated to sulfate (VLB), paclitaxel (TXL), doxorubicin hydrochloride
show antiviral activity, being comparable with gancyclovir (DXR), daunorubicin hydrochloride (DNR), 5-fluorouracil
and acyclovir.1) During the assay for antiviral activity, they (5-FU), and mitomycin C (MMC) were purchased from
were also found to show cytotoxicity.1) In this study, the an- Wako Pure Chemical Industries, Ltd. (Osaka). Cisplatin
tiproliferative activity of the ethanol extract of root and aque- (CDDP) was from Sigma-Aldrich Chemical Co. (St. Louis,
ous extract of leaves of R. nasutus was assessed in vitro and MO, U.S.A.). Other chemicals were of the highest purity
in vivo. The human cervical carcinoma cell line HeLa-Ohio available.
(HeLa) and its multidrug resistant subline, Hvr100-6,4—7) and
Synthesis of Rhinacanthin C The first total synthesis of
the human prostate carcinoma cell line PC-3 and human rhinacanthin C has been achieved and reported by Awai et
bladder carcinoma cell line T24 were used for in vitro assess- al.10) In the present study, we planned the route of synthesis
ment. The information of antiproliferative activity of new by considering the preparation of a 2-hydroxy-1,4-naphtho-
agents on prostate and bladder carcinoma cells could be use- quinone moiety as a key structure to carry out structure–ac-
ful because benefit of chemotherapy has been limited.8,9) In tivity relationship investigations in the future (Figs. 1, 2). We
vivo antiproliferative activity of the R. nasutus extracts was adopted a convergent route to prepare a suitable precursor
evaluated after oral administration once daily for 14 d using
murine sarcoma 180-bearing ICR mice. Herein, rhinacanthin
C was chemically synthesized by a new route for future
structure–activity relationship investigations with the 2-hy-
droxy-1,4-naphthoquinone moiety as a key structure (Figs. 1,
Fig. 1. Chemical Structure of Rhinacanthin C
2). The amount of rhinacanthin C was determined by HPLC
* To whom correspondence should be addressed. e-mail: sakaedat@med.kobe-u.ac.jp © 2004 Pharmaceutical Society of Japan

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Fig. 2. Synthesis of Rhinacanthin C
(I), which could be converted to rhinacanthin C by demethy- Co., Kyoto, Japan) equipped with a photodiode array detector
lation and air oxidation at the final step, by condensing with (SPD-M10Avp, Shimadzu Co.) and a workstation (CLASS
a stable 1,2,4-trimethoxynaphthalene derivative (II) having a LC10/M10A, Shimadzu Co.). The column was CAPCELL
hydroxyl group, and the olefin-containing conjugated car- PAK C18 UG80 (4.6 mmꢁ250 mm, Shiseido Co., Ltd.,
boxylic acid side chain (III). Thus we selected the 3-substi- Tokyo). The mobile phase consisted of acetonitrile : double
tuted-1,2,4-trimethoxynaphthalene derivative (II) as a suit- distilled water : trifluoroacetic acid at a volume ratio of 80 :
able candidate for the stable 2-hydroxy-1,4-naphthoquinone 20 : 0.1. The flow rate was 1.0 ml/min, and the column tem-
precursor. Starting with 1,2,4-trimethoxynaphthalene (1), a perature was maintained at 40 °C. Ten or 15 ml of aqueous
methoxycarbonyl group was introduced by the metalation- solution of the R. nasutus extracts was injected onto the chro-
substitution reaction in high yield. Reduction by lithium alu- matograph, and the peak areas at 254 nm and 10.7 min were
minum hydride (LAH) and bromination afforded 3-bro- used for quantification after comparing the absorption spec-
momethyl-1,2,4-trimethoxynaphthalene (4), which was treated trum with that of rhinacanthin C. The rhinacanthin C content
with ethyl isobutyrate in the presence of lithium diisopropyl- of the ethanol extract of root and aqueous extract of leaves of
amide to give a-dimethylcarboxylic acid ester (5). Subse- R. nasutus was determined as the average of 3 experiments,
quent reduction by LAH gave a desired precursor (II) in 81% and the CV% was 0.022% and 0.067%, respectively.
yield from 5. On the other hand, the synthesis of monoter-
Cell Culture of HeLa, Hvr100-6, PC-3 and T24 for in
pene carboxylic acid (III) was achieved by using 4-methyl- Vitro Assessment HeLa cells were obtained from Dainip-
trans-4-hexenal (8) according to a previous report.10) 4- pon Pharmaceutical Co., Ltd. (Osaka, Japan). They were
Methyl-trans-4-hexenal (8) was prepared by Swern oxidation maintained in culture medium consisting of Dulbecco’s mod-
of 4-methyl-trans-4-hexenol (CH3CHꢀC(CH3)CH2CH2CH2- ified Eagle’s medium (D-MEM with glucose (4.5 g/l), L-glut-
OH) (7) which was synthesized originally, by the carbon–car- amine (4 mM) and sodium pyruvate (1 mM); Cat. No. 12800-
bon bond forming reaction of 2-lithio-cis-2-butene and O- 017, Invitrogen Corp., Carlsbad, CA, U.S.A.) supplemented
silylated 3-bromopropanol. The derived silyl ether (6) was with 10% fetal bovine serum (FBS; Lot. No. AGM7413, Hy-
exposed to tetrabutylammonium fluoride (TBAF) to afford a Clone, UT, U.S.A.) and 100 mg/l kanamycin sulfate (Invitro-
known alcohol (7) in 81%. Both intermediates II and III were gen Corp., Carlsbad, CA, U.S.A.). Hvr100-6 cells have been
condensed with the help of water-soluble carbodiimide to af- established by the stepwise exposure of HeLa cells to
ford the desired precursor (I) in a good yield (60%). The final VLB.4—7) FACS and real-time quantitative RT-PCR have
demethylation by BBr3 and subsequent air oxidation afforded demonstrated that the multidrug resistant transporter
rhinacanthin C in 65% yield only in the presence of 2- MDR1/P-glycoprotein was induced without alterations of ex-
methyl-2-butene in the reaction mixture to avoid undesirable pression of multidrug resistance-associated protein 1 and 2
bromination at the olefin function. The structures of all com- (MRP1 and MRP2). Hvr100-6 cells were maintained with
pounds and synthetic rhinacanthin C were confirmed mainly 100 nM VLB. PC-3 cells and T24 cells were obtained from
on the basis of NMR spectral data and mass spectroscopic the American Type Culture Collection (Rockville, MD,
analysis (see Appendix).
U.S.A.). These cells were maintained in culture medium con-
Determination of Rhinacanthin C Content in R. nasu- sisting of RPMI1640 (Cat. No. 31800-022, Invitrogen Corp.)
tus Extracts by HPLC with a Photodiode Array Detector supplemented with 10% FBS, 100 units/ml penicillin and
The rhinacanthin C content of the R. nasutus extracts was de- 100 mg/ml streptomycin. HeLa and Hvr100-6 cell lines were
termined by HPLC with a photodiode array detector. The seeded into culture flasks (Corning Costar Corp., Cambridge,
HPLC apparatus consisted of a pump (LC-10A, Shimadzu MA, U.S.A.) at a density of 4 and 12ꢁ104 cells/cm2, respec-

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Vol. 27, No. 7
tively, and PC-3 and T24 cells were seeded in culture dishes the mice were sacrificed, and the antiproliferative activity
(IWAKI No. 3020-100, ASAHI TECHNO GLASS Corp., was assessed as the inhibition rate (%); IR (%)ꢀ[1ꢂ(tumor
Chiba, Japan) at a density of 2 and 1ꢁ106 cells/100 mm dish, weight of treatment group)/(tumor weight of untreated
respectively, grown in a humidified atmosphere of 5% CO2– group)]ꢁ100.
95% air at 37 °C, and subcultured every 3 or 4 d with 0.05%
trypsin–0.02% EDTA (Invitrogen Corp.).
Statistical Analysis The data represent the meanꢄstan-
dard deviation (S.D.) or standard error (S.E.). Multiple com-
In Vitro Antiproliferative Activity of R. nasutus Ex- parisons were performed using one-way analysis of variance
tracts and Rhinacanthin C The in vitro antiproliferative (ANOVA) followed by Scheffé F-test. p values of less than
activity of the R. nasutus extracts and rhinacanthin C was as- 0.05 (two-tailed) were considered significant.
sessed by the WST-1 (tetrazolium salts) colorimetric assay
using a Cell Counting Kit (Dojindo Laboratories, Kuma- RESULTS
moto, Japan). Cells (1000 cells/well) were seeded on 96-well
plates (“Nunclon” flasks, Nalge Nunc International,
Rhinacanthin C Content of R. nasutus Extracts Fig-
Rochester, NY, U.S.A.) in 100 ml of culture medium on Day ure 3 shows typical chromatograms after injection of the
0, and the culture medium was exchanged for that containing aqueous solution of the R. nasutus extracts. By comparison
test material at various concentrations on Day 1. After incu- of absorption spectrum and quantification based on the peak
bation for 3 d at 37 °C (on Day 4), the culture medium was areas, rhinacanthin C content was found to be 0.474 and
exchanged for 110 ml of that containing WST-1 reagent solu- 0.0017 (w/w)% for the ethanol extract of root and aqueous
tion (10 ml of WST-1 solution and 100 ml of the culture extract of leaves of R. nasutus, respectively.
medium), and 3 h later, the absorbance was determined at
In Vitro Antiproliferative Activity of R. nasutus Ex-
450 nm with a reference wavelength of 630 nm using a mi- tracts and Rhinacanthin C Table 1 lists the IC50 values
croplate reader (SJeia Auto Reader II; Sanko Junyaku Co. for the R. nasutus extracts and rhinacanthin C in HeLa and
Ltd., Tokyo, Japan). The 50% growth inhibitory concentra- its MDR1-overexpressing subline, Hvr100-6. The ethanol ex-
tion (IC50) was calculated according to the sigmoid inhibitory tract of root showed similar cytotoxicity in HeLa and
effect model as follows using the nonlinear least-squares fit- Hvr100-6 cells, as did the aqueous extract of leaves. Rhi-
ting method (“WinNonlin”, ver. 2.1, Pharsight Corp., Moun- nacanthin C showed IC50 values of 26.2ꢄ4.08 mM and 11.2ꢄ
tain View, CA, U.S.A.): EꢀEmaxꢁ[1ꢂCg/(CgꢃIC50g)]. E and 4.21 mM, respectively, and these were comparable with or
Emax represent the surviving fraction (% of control) and its slightly weaker than those of 5-FU. Table 2 lists the IC50 val-
maximum, respectively, and C and g represent the drug con- ues for PC-3 and T24 cells. The R. nasutus extracts and rhi-
centration in the medium and the sigmoidicity factor, respec- nacanthin C showed higher cytotoxicity for these cells than
tively. Each series of experiments was conducted in quadru- HeLa and Hvr100-6 cells. The IC50 values of rhinacanthin C
plicate.
In Vivo Antiproliferative Activity of R. nasutus Ex-
tracts The in vivo antiproliferative activity of the R. nasu-
Table 1. In Vitro Antiproliferative Activity of the R. nasutus Extracts and
Rhinacanthin C in HeLa and Hvr100-6 Cells
tus extracts was also assessed using sarcoma 180-bearing
mice. Sarcoma 180 was kindly supplied by the National Can-
IC50
cer Center, Japan, and maintained by the weekly transplanta-
HeLa
Hvr100-6
tion of ascites, 1ꢁ106 cells/0.1 ml, into the peritoneal cavity
of recipient male 5—6 week old ICR mice (CREA JAPAN,
Inc., Tokyo, Japan). A cell suspension of sarcoma 180 was
transplanted into the right femoral muscle at a concentration
of 1ꢁ106 cells/0.1 ml on day 0, and the R. nasutus extracts
were given orally once daily at a dose of 500 mg/kg/d from
day ꢂ7 to day ꢂ1 and day 1 to day 7 (total of 14 d). To
check the validity of in vivo experimental system, antiprolif-
erative activity of MMC was also assessed in sarcoma 180-
bearing mouse. MMC was given intraperitoneally once daily
at a dose of 1 mg/kg/d from day 1 to day 7 (for 7 d). The dos-
ing volume was 0.1 ml per 10 g weight of mice. On day 10,
VLB (nM)
TXL (nM)
DXR (nM)
DNR (nM)
MMC (mM)
5-FU (mM)
CDDP (mM)
R. nasutus extracts
Root (mg/ml)
Leaves (mg/ml)
Rhinacanthin C (mM)
0.135ꢄ0.036
0.136ꢄ0.238
2.94ꢄ0.535
1.09ꢄ0.309
0.0209ꢄ0.00576
2.85ꢄ0.747
67.0ꢄ30.1
562ꢄ255
153ꢄ56.8
216ꢄ87.3
0.187ꢄ0.0132
11.5ꢄ2.33
0.223ꢄ0.0458
0.574ꢄ0.112
1239ꢄ257
1499ꢄ113
26.2ꢄ4.08
977ꢄ152
1582ꢄ101
11.2ꢄ4.21
The data represent the meanꢄS.D. of 4 experiments.
Fig. 3. Typical HPLC Chromatograms of Ethanol Extract of Root of R. nasutus (A) and Aqueous Extract of Leaves of R. nasutus (B)
Based on the peaks indicated by arrows, the rhinacanthin C content of the ethanol extract of root and aqueous extract of leaves of R. nasutus was determined to be 0.474.

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Table 2. In Vitro Antiproliferative Activity of the R. nasutus Extracts and
Rhinacanthin C in PC-3 and T24 Cells
mM and 11.3 mM in solutions of 1239 mg/ml and 977 mg/ml
(the IC50 values in HeLa and Hvr100-6 cells, respectively
(Table 1)) of the ethanol extract of root, and these rhinacan-
thin C concentrations were comparable with the IC50 values,
that is, 26.2 mM and 11.2 mM, respectively, suggesting that
most of the antiproliferative activity of the ethanol extract of
root in these cells was due to rhinacanthin C. In contrast, the
rhinacanthin C content of the aqueous extract of leaves of R.
nasutus was only 0.0017 (w/w)%, and the antiproliferative
activity of the aqueous extract of leaves in HeLa and
Hvr100-6 cells was due to other components than rhinacan-
thin C.
IC50
PC-3
T24
R. nasutus extracts
Root (mg/ml)
567ꢄ135
359ꢄ50.5
1.92ꢄ0.482
373ꢄ65.3
616ꢄ17.9
0.660ꢄ0.188
Leaves (mg/ml)
Rhinacanthin C (mM)
The data represent the meanꢄS.D. of 4 experiments.
Table 3. In Vivo Antiproliferative Activity of the R. nasutus Extracts in
Sarcoma 180-Bearing ICR Mice
R. nasutus extracts and rhinacanthin C showed similar in
vitro antiproliferative activity both for HeLa and for Hvr100-
6 cells. Hvr100-6 cells are a MDR1-overexpressing HeLa
subline. Multidrug resistance is one of the most serious
causes of the failure of chemotherapy, and biological factors
in multidrug resistance have been investigated, including
down-regulation of the uptake or induction of efflux systems
(e.g., MDR1), induction of inactivation enzymes, alteration
of targeted enzymes, changes in DNA repair processes, and
alteration of apoptotic signaling.11) MDR1 expression is the
best characterized of these mechanisms, and the magnitude
of resistance depends on the expression of MDR1 up-regu-
lated by chemotherapy. Using HeLa and Hvr100-6 cells, it
has already been demonstrated that the antiproliferative ac-
tivity of anthracyclines, vinca-alkaloids, taxanes, actino-
mycin D and etoposide decreased extensively according to
the MDR1 expression. R. nasutus extracts and rhinacanthin
C might be effective for tumors with multidrug resistance ac-
quired by chemotherapy.
R. nasutus extracts and rhinacanthin C exhibited in vitro
antiproliferative activity on PC-3 and T24, which are human
prostate and bladder carcinoma cell lines, respectively. Addi-
tion to radiotherapy or surgery, androgen deprivation therapy
and chemotherapy has been used in patients with prostate
cancer. However, chemotherapy has failed to demonstrate a
survival benefit in clinical trials of hormone refractory dis-
ease, though is has resulted in clinical benefit in some stud-
ies.8) While chemotherapy for bladder cancer has been used
after surgery, the effect was limited.9) The information on an-
tiproliferative activity of novel agents on prostate and bladder
carcinoma cells could be useful for the discovery of new
chemotherapeutic agents.
R. nasutus extracts showed in vivo antiproliferative activity
(Table 3). The once daily oral administration of 500 mg/kg/d
of the ethanol extract of root and aqueous extract of leaves of
R. nasutus for 14 d resulted in suppression of sarcoma 180
growth in mice by 52.5% and 44.2%, respectively. As for the
ethanol extract of root of R. nasutus, this is equivalent to
2.37 mg/kg/d of rhinacanthin C. Rhinacanthin C has a molec-
ular weight of 411, which is comparable with that of oral
drugs, suggesting that the in vivo antiproliferative activity of
the ethanol extract of root of R. nasutus was due to rhinacan-
thin C transferred into the blood circulation.
In summary, the antiproliferative activity of the ethanol ex-
tract of root and aqueous extract of leaves of R. nasutus, and
its supposed active moiety rhinacanthin C was assessed in
vitro using HeLa, its MDR1-overexpressing subline Hvr100-
6, PC-3 and T24, and in vivo activity was also assessed using
sarcoma 180-bearing mice. It was suggested that 1) the in
Route Dose (mg/kg/d)
Tumor weight (g)
IR (%)
Control
Root
Leaves
MMC
3.66ꢄ0.21
1.74ꢄ0.20*
2.04ꢄ0.16*
1.73ꢄ0.10*
p.o.
p.o.
i.p.
500
500
1
52.5
44.2
52.7
The data represent the meanꢄS.E. of mice. *: pꢅ0.05, compared with control.
were 1.92ꢄ0.482 mM and 0.660ꢄ0.188 mM, respectively.
In Vivo Antiproliferative Activity of R. nasutus Ex-
tracts The in vivo antiproliferative activity of the R. nasu-
tus extracts was assessed using sarcoma 180-bearing mice.
Table 3 lists the tumor weight with or without treatment
using the R. nasutus extracts (p.o., 500 mg/kg/d for 14 d) or
MMC (i.p., 1 mg/kg/d for 7 d). The activity of MMC was
found, showing that this sarcoma 180-bearing mouse system
is appropriate to assess the activity of rhinacanthin C. The
ethanol extract of root and aqueous extract of leaves of R. na-
sutus showed significant antiproliferative activity with inhibi-
tion of 52.5% and 44.2%, respectively.
DISCUSSION
Fourteen naphthoquinone derivatives isolated from R. na-
sutus are classified into 4 dimethyldihydropyrano-1,4-naph-
thoquinones (rhinacanthin A, B, O and P) and 10 2-hydroxyl-
1,4-naphthoquinones (rhinacanthin C, D, G—N).1—3) Rhi-
nacanthin C and D were isolated by anti murine cy-
tomegalovirus (mCMV) activity-guided fractionation, and
their antiviral activity has been studied extensively using the
viral cytopathic effect (CPE) assay, the plaque-neutralization
(plaque) assay, and the hemadsorption-inhibition (HAI) assay.
In the CPE assay or plaque assay using mCMV, it has been
demonstrated that rhinacanthin C shows cytotoxicity, but rhi-
nacanthin D has weaker cytotoxicity, suggesting that rhi-
nacanthin C might be a promising candidate for development
as a novel anticancer drug. The reason for the difference of
activity is still unknown, but lipophilicity could contribute to
the cytotoxicity.3,10) This study was conducted to assess the
antiproliferative activity of rhinacanthin C as an anticancer
drug and to know the potential of other constituents. It has
been suggested that rhinacanthin C shows antiproliferative
activity, being comparable with or slightly weaker than 5-FU.
Here, the rhinacanthin C content of the ethanol extract of
root of R. nasutus was determined to be 0.474 (w/w)%. Thus,
rhinacanthin C should be included at concentrations of 14.3

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Vol. 27, No. 7
304.1674. LR-MS (EI) m/z 304 (Mꢃ 100), 273 (3), 257 (25),
231 (43), 200 (26), 173 (10).
vitro antiproliferative activity of rhinacanthin C was compa-
rable with or slightly weaker than that of 5-FU, 2) rhinacan-
thin C showed antiproliferative activity for MDR1-overex-
pressing Hvr100-6 cells, similarily to parent HeLa cells, 3)
the in vitro antiproliferative activity of the ethanol extract of
root R. nasutus was due to rhinacanthin C, whereas that of
the aqueous extract of leaves of R. nasutus was due to other
compounds than rhinacanthin C, and 4) both of the R. nasu-
tus extracts showed in vivo antiproliferative activity after oral
administration once daily for 14 d.
4-Methyl-trans-4-hexenol tert-Butyldiphenylsilyl Ether (6):
1H-NMR (270 MHz, CDCl3) d 1.05 (s, 9H), 1.56 (m, 6H),
1.63 (m, 2H), 2.05 (m, 2H), 3.64 (t, 2H, Jꢀ6.4 Hz), 5.18 (m,
1H), 7.25—7.68 (m, 10H). 13C-NMR (125.8 MHz, CDCl3) d
13.2, 23.3, 26.9, 27.7, 30.9, 63.8, 119.1, 127.6, 129.5, 134.2,
135.7. IR (cmꢂ1) n 2920, 2880, 1420, 1100, 820, 700. HR-
MS Calcd for C23H32SiO: 352.22236. Found: 352.2222.
4-Methyl-trans-4-hexenol (7): 1H-NMR (400 MHz, CDCl3
)
d 1.57 (d, 3H, Jꢀ6.6 Hz), 1.61 (s, 3H), 1.67 (m, 2H), 2.05
(m, 2H), 3.62 (t, 2H, Jꢀ6.4 Hz), 5.25 (q.quint, 1H, Jꢀ6.6
Hz, Jꢀ1.3 Hz). 13C-NMR (125.8 MHz, CDCl3) d 13.3, 15.5,
30.9, 36.0, 62.9, 118.8, 135.4. IR (cmꢂ1) n 3400, 2920, 1440,
1380, 1040, 1000. HR-MS Calcd for C7H14O: 114.10452.
APPENDIX
Methyl 1,3,4-Trimethoxy-2-naphthoate (2): Colorless solid
.
1
mp 52—56 °C. H-NMR (500 MHz, CDCl3) d 3.98 (s, 3H),
3.99 (s, 6H), 4.00 (s, 3H), 7.47 (t, 1H, Jꢀ8.2 Hz), 7.54 (t,
1H, Jꢀ8.2 Hz), 8.06 (d, 1H, Jꢀ8.2 Hz), 8.11 (d, 1H, Jꢀ8.2
Hz). 13C-NMR (125.8 MHz, CDCl3) d 52.5, 61.1, 61.6, 63.3,
121.1, 121.9, 122.8, 125.3, 125.5, 127.4, 130.2, 143.5, 145.3,
149.8, 166.6. IR (cmꢂ1) n 2940, 1728, 1620, 1598, 1230,
1060. HR-MS Calcd for C15H16O5: 276.0998. Found:
276.1002.
Found: 114.1044.
4-Methyl-trans-4-hexenal (8): H-NMR (400 MHz, CDCl3
1
)
d 1.57 (d, 3H, Jꢀ6.6 Hz), 1.61 (s, 3H), 2.32 (m, 2H), 2.50
(m, 2H), 5.24 (q, 1H, Jꢀ6.6 Hz). 13C-NMR (125.8 MHz,
CDCl3) d 13.3, 15.7, 31.9, 42.2, 119.6, 202.6. IR (cmꢂ1) n
2920, 2880, 1720, 1440, 1380, 1100. HR-MS Calcd for
C7H12O: 112.08886. Found: 112.08848.
3-Hydroxymethyl-1,2,4-trimethoxynaphthalene (3): 1H-
NMR (270 MHz, CDCl3) d 2.62 (m, 1H), 3.98 (s, 6H), 4.07
(s, 3H), 7.43—7.54 (m, 2H), 8.03—8.12 (m, 2H). 13C-NMR
(125.8 MHz, CDCl3) d 56.4, 61.0, 61.3, 121.7, 122.5, 125.3,
125.5, 125.5, 126.5, 129.5, 143.5, 147.9, 150.7. IR (cmꢂ1) n
3500, 2920, 1620, 1598, 1360, 1060. HR-MS Calcd for
C14H16O4: 248.10488. Found: 248.1055. LR-MS (EI) m/z
248 (Mꢃ 100), 233 (62), 205 (45), 173 (11), 161 (12), 15 (7),
129 (7).
Precursor of Rhinacanthin C (I): 1H-NMR (270 MHz,
CDCl3) d 0.98 (s, 6H), 1.56 (d, 3H, Jꢀ6 Hz), 1.60 (s, 3H),
1.83 (s, 3H), 2.04—2.26 (m, 2H), 2.84 (s, 2H), 3.83 (s, 2H),
3.93 (s, 3H), 3.94 (s, 6H), 5.22 (m, 1H), 6.7 (m, 1H), 7.44
(m, 2H), 7.98—8.10 (m, 2H). HR-MS Calcd for C28H38O5:
454.2720. Found: 454.2724.
1
Synthetic Rhinacanthin C: H-NMR (270 MHz, CDCl3) d
1.02 (s, 6H), 1.55 (d, 3H, Jꢀ6.6 Hz), 1.58 (s, 3H), 1.79 (s,
3H), 2.01 (m, 1H), 2.14 (m, 1H), 2.71 (s, 2H), 3.91 (s, 2H),
5.22 (q, 1H, Jꢀ6.6 Hz), 6.69 (m, 1H), 7.72 (m, 2H), 8.09 (m,
2H). HR-MS Calcd for C25H30O5: 410.2094. Found:
410.2087.
1
3-Bromomethyl-1,2,4-trimethoxynaphthalene (4): H-NMR
(270 MHz, CDCl3) d 3.97 (s, 3H), 4.07 (s, H), 4.13 (s, 3H),
4.83 (s, 2H), 7.44—7.54 (m, 2H), 8.0—8.12 (m, 2H). 13C-
NMR (125.8 MHz, CDCl3) d 23.4, 60.9, 61.2, 62.6, 121.8,
122.6, 123.7, 125.3, 127.0, 130.0, 143.8, 147.4, 151.4. IR
(cmꢂ1) n 2920, 1620, 1590, 1460, 1360, 1060, 1000. HR-
MS Calcd C14H15O3Br: 310.02045, 312.01845. Found:
310.0205, 312.0186. LR-MS (EI) m/z 312 (55), 310 (56),
297 (4), 231 (100), 216 (34), 201 (41), 200 (50), 173 (21),
143 (13).
REFERENCES
1) Sendl A., Chen J. L., Jolad S. D., Stoddart C., Rozhon E., Kernan M.,
Nanakorn W., Balick M., J. Nat. Prod., 59, 808—811 (1996).
2) Wu T. S., Hsu H. C., Wu P. L., Leu Y. L., Chan Y. Y., Chern C. Y., Yeh
M. Y., Tien H. J., Chem. Pharm. Bull., 46, 413—418 (1998).
3) Wu T. S., Tien H. J., Yeh M. Y., Lee K. H., Phytochemistry, 27,
3787—3788 (1988).
4) Iida N., Takara K., Ohmoto N., Nakamura T., Kimura T., Wada A.,
Hirai M., Sakaeda T., Okumura K., Biol. Pharm. Bull., 24, 1032—
1036 (2001).
5) Takara K., Sakaeda T., Yagami T., Kobayashi H., Ohmoto N., Hori-
nouchi M., Nishiguchi K., Okumura K., Biol. Pharm. Bull., 25, 771—
778 (2002).
6) Wada A., Sakaeda T., Takara K., Hirai M., Kimura T., Ohmoto N.,
Zhou J., Nakamura T., Kobayashi H., Okamura N., Yagami T., Oku-
mura K., Drug Metab. Pharmacokin., 17, 467—474 (2002).
7) Sakaeda T., Nakamura T., Hirai M., Kimura T., Wada A., Yagami T.,
Kobayashi H., Nagata S., Okamura N., Yoshikawa T., Shirakawa T.,
Gotoh A., Matsuo M., Okumura K., Pharm. Res., 19, 1323—1329
(2002).
8) Carroll P. R., Benaron D. A., Blackledge G., Coaksley F. V., D’amico
A. V., Higano C. S., Iversen P., Kattan M., Nanus D. M., Nelson J. B,
Oh W. K., Roach M. III, Sellers W. R., Smith M. R., McMann M. C.,
Kantoff P. W., J. Urol., 170, S3—S5 (2003).
9) Cote R. J., Datar R. H., Crit. Rev. Oncol. Hematol., 46, S67—S85
(2003).
10) Awai N., Kuwahara S., Kodama O., Santisopasri V., Biosci. Biotechnol.
Biochem., 59, 1999—2000 (1995).
11) Sakaeda T., Nakamura N., Okumura K., Biol. Pharm. Bull., 25,
1391—1400 (2002).
Ethyl 2,2-Dimethyl-3-(1,3,4-trimethoxynaphthalen-2-yl)-
1
propionate (5): H-NMR (270 MHz, CDCl3) d 1.17 (s, 6H),
1.24 (t, 3H, Jꢀ7.26 Hz), 3.09 (s, 2H), 3.84 (s, 3H), 3.93 (s,
3H), 3.94 (s, 3H), 4.14 (q, 2H, Jꢀ7.26 Hz), 7.44 (m, 2H),
7.99 (m, 1H). 13C-NMR (125.8 MHz, CDCl3) d 14.1, 25.1,
34.4, 43.5, 60.4, 60.8, 61.5, 121.5, 122.4, 123.5, 124.8,
125.2, 125.8, 128.8, 143.2, 148.8, 151.5, 178.0. IR (cmꢂ1) n
2920, 1710, 1590, 1460, 1360, 1140, 1060, 1000. HR-MS
Calcd for C20H26O5; 346.17808. Found: 346.1774. LR-MS
(EI) m/z 346 (Mꢃ 100), 331 (12), 301 (3), 273 (10), 231 (86),
216 (24), 200 (37).
2,2-Dimethyl-3-(1,3,4-trimethoxynaphthalen-2-yl)-
1
propan-1-ol (II): Colorless solid. mp 138—141 °C. H-NMR
(270 MHz, CDCl3) d 0.95 (s, 6H), 2.77 (s, 2H), 3.01 (d, 2H,
Jꢀ6.27 Hz), 3.75 (t, 1H, Jꢀ6.27 Hz), 3.89 (s, 3H), 3.96 (s,
3H), 3.98 (s, 3H), 7.47 (m, 2H), 7.98 (m, 1H), 8.10 (m, 1H).
13C-NMR (125 MHz, CDCl3) d 25.2, 32.0, 38.4, 60.8, 62.0,
69.7, 121.7, 122.2, 124.0, 125.0, 125.2, 126.0, 128.6, 143.6,
148.6, 150.9. IR (cmꢂ1) n 3470, 2920, 1590, 1450, 1360,
1060, 1000. HR-MS Calcd for C18H24O4 304.1675. Found: