|Title of Invention||
"A PROCESS FOR ISOLATING AN ANTITUMOR SUBSTANCE HEDERAGENIN 3-O-α-L-RHAMNOPYRANOSYL (1→2) [β-D-GLUCOPYRANOSYL (1→4)]-α-L-ARABINOPYRANOSIDE FROM PULSATILLAE RADIX"
|Abstract||A process for isolating an antitumor substance hederagenin 3-O-α-L-rhamnopyranosyl (1→2) [β-D-glucopyranosyl (1—>4)]-α-L-arabinopyranoside from Pulsatillae Radix comprising following steps:i)extracting Pulsatillae Radix Powder from 50% aqueous solution of ethanol, drying the same under reduced pressure, adding acetone at 5 to 10 fold, shaking and centrifuging the mixture at 3000 rpm and collecting the precipitate;ii) dissolving the precipitate of step (i) in aqueous solution of methanol , fractionating on Sephadex LH20 Column, subjecting the fractions to thin layer chromatography and collecting a fraction having Rf of 0.48 to 0.50; and iii) isolating hederagenin 3-O-α-L-rhamnopyranosyl (1→2) [β-D-glucopyranosyl (1→4)]-αL-arabinopyranoside from the fraction of step (ii).|
|Full Text||USE OF HEDERAGENIN 3-O-α-L-RHAMNOPYR NOSYL((l->2)-[p-GLUCOPYRANOSYL(
l-»4)]-a-L-ARABINOPYRAN0SIDE OR AN EXTRACT FROM PULSATILLAE RADIX CONTAINING THE SAME AS A THERAPEUTIC AGENT FOR SOLID TUMORS
This invention relates to a use of hederagenin 3-O- α -L-rhamnopyranosyl((l—>2)-
[|3-D-glucopyranosyl(l—»4)]-a-L-arabinopyranoside as represented by the following
or an extract from Pulsatillae radix containing the same as a therapeutic agent for
Pulsatillae radix is a dried root of Pulsatilla species belonging to the
Ranunculaceae family (Ki Hwan Bae, Korean Medicinal Herbs, 1999). According to the
Chinese medicine, Pulsatillae radix is known to have effects of removing heat from the
blood and detoxifying. It has also been used as anti-inflammatory, astringent, hemostatic
and antidiarrhea agents, and for the treatment of hematochezia, malaria, nosebleed, and
bleeding from tooth. Its flower is called as Pulsatillae Flos, and used for the treatment of
malaria, or smallpox. Its leaf is called as Pulsatillae Folium, and used for treatment of
waist pain, edema, or heart pain. In addition, decoction of Pulsatillae radix was reported
to have an antibacterial effect against amoebic dysentery, and a pesticidal effect against
Pulsatillae radix contains about 9% of saponins, and such ingredients as
protoanemonin, anemonin, ranunculin, hederagenin, betulinic acid, and oleanolic acid
derivatives and their glycosides have been isolated therefrom by now as represented by the
following formula (II):
The above ingredients have not yet been extensively studied for their
pharmacological effects, but protoanemonin was reported to have mitotoxicity
(Vonderbank, F., Pharmazie 5, 210, 1950). Li, et al. (Li, R. Z., et al., Yao Hsueh Hsueh
Pao. 28, 326 31, 1993) also reported that ranunculin has cytotoxicity against KB cells, by
inhibition of DNA polymerase.
was isolated from Pulsatilla cerna and P. kqreana by Shimizu, et al.
(Chem. Pharm. Bull., 26, 1666, 1978); from P. chinensis by Yoshihrro, etal. (J. Nat. Pro.,
62, 1279, 1999); and from Serjania salzmanniana Schleeht by Ekabo, et al. (J. Nat. Prod.,
59, 431, 1996). Kang, et al. (Arch. Pharm. Res., 12(1), 42-47, 1989) also isolated it from
P. koreana, and reconfirmed its structure. Yoshihiro, et al. reported that hederagenin and
oleanolic acid derivatives showed cytotoxicity against HL-60 human leukemia cells in the
above article. They reported that hederagenin 3-O- α -L-rhamnopyranosyl(l—»2)-[p-Dglucopyranosy](
l ->4)]-a-L-arabinopyranoside isolated from Chinese Pulsatillae radix
(Pulsatilla clunensis) had week cytotoxicity, i.e. 3.8 ug/ml of ED50, against HL-60 cells.
However, most of saponins and many kinds of natural products commonly show such level
of cytotoxicity, and thus, the above compound cannot be said to have antitumor activity
based thereon. Therefore, it has never been known that hederagenin 3-O-ct-LrhamnopyranosyK
1 —»2)-[p-D-glucopyranosyl( 1 —»4)]- α -L-arabinopyranoside has
antitumor activity, particularly, against solid tumors.
DISCLOSURE OF THE INVENTION
The present inventors isolated deoxypodophyllotoxin from medicinal herbs
including Anthriscus sylvestris Hoffman, Pulsatillae radix, etc., and found that this
substance inhibited the growth of solid tumor cells by inhibiting angiogenesis, and
obtained a Korean patent (Korean Patent Number 315,200) for the same. The present
inventors carried out extensive studies to develop an antitumor agent from medicinal herbs.
As a result, they obtained a fraction which is poorly soluble in an organic solvent, but is
readily soluble in water from Pulsatillae radix, and isolated an antitumor compound from
the fraction, and so completed the present invention.
Accordingly, the purpose of the present invention is to provide a therapeutic agent
for solid tumors comprising an antitumor compound isolated from Pulsatillae radix or a
fraction from Pulsatillae radix containing the-same as an active ingredient.
One aspect of the present invention provides a therapeutic agent for solid tumors
comprising a Pulsatillae radix extract containing hederagenin 3-O-a-Lrhamnopyranosyl(
l-2)-[p-D-glucopyranosyl(l--4)]-a-L-arabin as an active
"Solid tumors", as used herein, refer to any mass tumor except blood cancers, a
representative example of which is lung tumor.
In the present invention, the Pulsatillae radix extract containing hederagenin 3-0-
a-L-rhamnopyranosyl( 1 —>2)-[p-D-glucopyranosyl( 1 —»4)]-a-L-arabinopyranoside can be
obtained by extracting Pulsatillae radix with an aqueous solution of ethanol, and forming
precipitates by adding acetone thereto to obtain a water-soluble fraction (WT). Or, it can
be obtained by extracting Pulsatillae radix with the aqueous solution of ethanol, forming
precipitates by adding acetone thereto to obtain the water-soluble fraction, and passing the
fraction through Sephadex LH20 column to obtain a fraction (SPX3) having Rf of 0.48-0.5,
and developing red color, and then, blue color upon spraying sulfuric acid followed by
Another aspect of the present invention provides a therapeutic agent for solid
tumors comprising hederagenin 3-0- α -L-rhamnopyranosyl(l—>2)-[(3-Dglucopyranosyl(
l—»4)]-a-L-arabinopyranoside as an active ingredient.
Hereinafter, the present invention wi41 be explained in detail.
According to the present invention, Pulsatillae radix extract is extracted with 50%
ethanol to obtain a fraction WT poorly soluble in acetone, and the fraction is further
purified on Sephadex LH20 to obtain fraction SPX3, and from the SPX3 fraction, pure
SB365 is finally obtained. This compound is hederagenin 3-O-a-Lrhamnopyranosyl(
1 —>2)-[p-D-glucopyranosyl( 1 —>4)]-a-L-arabinopyranoside, and exhibits
higher antitumor activity against solid tumors formed with mouse lung tumor cells, LLC
(Lewis Lung Carcinoma) cells, or human lung tumor cells, NCI-H23 cells, than a clinical
In particular, the present process for preparing an antitumor fraction from
Pulsatillae radix and isolating an antitumor substance therefrom is as follows.(1) Preparation of antitumor fraction WT from Pulsatillae radix
Pulsatillae radix powder was extracted with 50% aqueous solution of ethanol, and
dried under reduced pressure. To the obtained dried material was added acetone at 5 to
10-fold amount. The mixture was shaken, centrifuged at 3,000 rpm, and the
supernatant was decanted therefrom to obtain an insoluble part. The above process was
repeated twice. The remaining insoluble part was readily soluble in water, and so
designated as "fraction WT". This fraction showed relatively high antitumor activity
against BDF1 mice transplanted with LLC cells and nude mice transplanted with NCIH23
(2) Preparation of fraction SPX3 from fraction WT
A given amount of fraction WT is dissolved in a given amount of aqueous
solution of methanol at various concentrations, and then fractionated on Sephadex LH20
column stabilized with the same solvent. In this case, the best isolation is achieved with
employing 80% aqueous solution of methanol, and the suitable size of the filled column
was 60*4 cm for 500 mg of fraction WT. As a result, fraction SPX1 (test tube numbers
26-66), SPX2 (test tube numbers 66-91), SPX3 (test tube numbers 91-111), and SPX4 (test
tube numbers 111-138) were obtained. When spraying sulfuric acid onto the fractions
developed on a silica gel plate and heating the plate, the fraction SPX3 developed red color
at first, and blue color with the lapse of time, and contains a sported compound having Rf
of 0.48 to 0.50 as a main ingredient. It was shown to have high antitumor activity on
BDF1 mice transplanted with LLC cells and nude mice transplanted with NCI-H23 cells.
(3) Isolation of SB365 from fraction SPX3
To isolate an antitumor substance from the fraction SPX3 showing antitumor
activity, HPLC was carried out to obtain pure compound, SB365. To identify the
structure of SB365, Lieberman-Burchard reaction, IR, 'H-NMR, I3C-NMR, and
ethanol/sulfuric acid hydrolysis were carried out. As a result, SB365 was confirmed as
which is a saponin ingredient that had already been isolated from
The Pulsatillae radix extracts according to the present invention or pure SB365
compound isolated therefrom have a weak cytotoxicity against solid tumor cells, but
unexpectedly showed excellent antitumor activity in animal experiments. Thus, they can
improve problems caused by previous antitumor agents on clinical use, e.g. reducing
immune responses due to decreasing blood cells. It is also anticipated that they show low
toxicity on rapidly dividing cells like hematopoietic cells, etc.
The Pulsatillae radix fractions and SB365 according to the present invention may
be combined with pharmaceutically acceptable carriers that are conventionally used, and
manufactured into various formulations that are conventional in the pharmaceutical field,
for example, orally administrable formulations like solutions, suspensions, etc.; injectable
formulations like injeetable solution or suspension, ready-to-use injectable dry powder,
etc.; and topically administrable formulations like ointments, creams, and solutions.
Particularly, the active ingredient of the present invention is soluble in water, and may be
dissolved in various solutions such as physiological saline, Ringer's solution, and nutrient
solution, etc. Such pharmaceutical formulations may be intravenously, subcutaneously,
intraperitoneally, or topically administered.
A recommended dosage of the active ingredient of the present invention to human
beings is 3.5-8.0 mg/kg body weight in case of SB365, 20-40 mg/kg body weight in case
of fraction SPX3, or 200-300 mg/kg in case of fraction WT. The. optimal dosage is 6.5
mg/kg body weight in case of SB365, 25 mg/kg body weight in case of fraction SPX3, or
250 mg/kg body weight in case of fraction WT. However, such dosage may be
appropriately adjusted depending on age, body weight, health, severity of disease of
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows silica gel TLC patterns of fraction WT;
Fig. 2 shows silica gel TLC patterns of fraction SPX3 purified on a Sephadex
Fig. 3 is an HPLC chromatogram of fraction SPX3; and,
Fig. 4 is an HPLC chromatogram of SB365.
BEST MODE FOR CARRYING OUT THE INVENTION
This invention will be better understood from the following examples. One
skilled in the art will readily appreciate the specific materials and results described are
merely illustrative of, and are not intended to, nor should be intended to, limit the invention
as described more fully in the claims, which follow thereafter.
Example 1: Preparation of fraction WT
Pulsatillae radix powder of 50 g was extracted three times with 500 ml of 50%
aqueous solution of ethanol, and the extract was dried under reduced pressure to obtain 22
g of dried materials. To this dried materials was added 300 ml of acetone, and the
mixture was shaken and centrifuged at 3,000 rpm. The supernatant was removed
therefrom to give a precipitate. For this precipitate, the acetone treatment was repeated
twice. The acetone layer was discarded, and an insoluble part was dried to obtain 17.8 g
of dried materials (fraction WT). The obtained fraction WT was subjected to silica gel
TLC (developing solvent: butanol : acetic acid : water in the ratio of 4 : 1 : 1, color
reaction: sulftiric acid-spraying followed by heating). The result is shown in Fig. 1. In
Fig. 1, a blue spot having the Rf in thiwange of 0.48 to 0.50 corresponds to the active
ingredient of the present invention as described below. As shown in Experimental
Example 1 below, fraction WT showed relatively high antitumor activity (inhibition rate of
tumor growth: 57%) on BDF1 mice transplanted with LLC cells.
Example 2: Preparation of fractions SPX
Fraction WT of 560 mg was further fractionated on Sephadex LH20 column (200
g, 60> mt/min, and the fraction volume of 0.5 ml/tube. These fractions were spotted on a silica
gel thin layer in order, and developed to obtain factions (developing solvent: butanol :
acetic acid : water in the ratio of 4 : 1 : 1, color reaction: sulfuric acid-spraying followed by
heating). The result is shown in Fig. 2. In Fig. 2, SPX1 (139 mg, 24.8%) was obtained
by collecting test tube numbers 26 to 66, and consisted of 4 major spots, lower one of
which developed yellow color upon reacting with sulfuric acid. SPX2 (344 mg, 61.4%)
was obtained by collecting test tube numbers 66 to 91, and consisted of 2 major spots.
SPX3 (61 mg, 10.9%) was obtained by collecting test tube numbers 91 to 111, and
developed red color at first, and then blue color with the lapse of time, upon spraying
sulfuric acid followed by heating. Fraction SPX3 contained a spot having the Rf value in
the range of 0.48 to 0.50 as its major ingredient. SPX4 (15.7 mg, 2.8%) was obtained by
collecting test tube numbers 111 to 138. Fractions SPX3 and SPX4 had relatively high
purity showing one spot on the thin layer.
As shown in Experimental Example 1 below, SPX3 exhibited 60% of the
inhibition rate of tumor growth on 15 days from its administration. By contrast, SPX1,
SPX2, and SPX4 did not exhibit any action, and so it could be assumed that the substance
developing blue color against sulfuric acid was an ingredient with antitumor activity.
This SPX3 fraction may be used as an antitumor agent in itself.
Example 3: Isolation of SB365
In order to isolate a pure substance from fraction SPX3, HPLC was carried out as
Reverse-phased silica gel (RP-Cig, 250*10 mm, manufactured by Metachem) was
used as the fixed phase, and a mixed solution of methanol and water (80:20) was used as
the mobile phase. The detection wavelength was 210 nm, and the flow rate was 1 ml/min.
The result is shown in Fig. 3. As shown in Fig. 3, SPX3 consisted of 3 major substances.
From the obtained fractionated amounts, peaks at Rt of 8.5 min and 10.4 min contained
small amounts of ingredients, and a peak at Rt of 23.3 min contained the major ingredient.
Thus, it was assumed that the latter would have antitumor activity. As described above,
the substance with R, of 23.3 min that developed blue color with sulfuric acid and would
be the active ingredient was collected to obtain SB365 of 2.8 mg from 31 mg of SPX3.
The collected fraction at R, of 23.3 min was dried, and was subjected to HPLC under the
condition as described above to measure its purity. The result is shown in Fig. 4. From
Fig. 4, it was confirmed that SB365 was a pure substance. The obtained SB365 was
directly used for the structural identification and antitumor activity test below.
As shown in Experimental Examples 1 and 2 below, SB365 exhibited 81% and
82.1% of the inhibition rate of tumor growth on BDF1 mice transplanted with LLC cells
and nude mice transplanted with NCI-H23 cells, respectively, which could be said to be
excellent antitumor activities.
Example 4: Structural identification and confirmation of the active compound SB365
SB365 isolated in the above was the white amorphous form with m.p, 239~241 °C
and [a]n +23.6° (c, 0.2, MeOH), and was positive in Liebermann-Buchard reaction, and so
confirmed as a glycoside. In addition, according to IR (cm"1), peaks were observed at
3400 (br, -OH), 2940 (br, C-H), 1695 (CO), 1455, and 1040 (C-O).- Jt was also assumed
as a glycoside from the absorption peaks in the ranges of 1000-1100 and 3000-3400.
In view of 'H-NMR, it had NMR patterns typical of saponins. Six -CH3 groups
were observed at 0.91, 0.92, 0.98, 1.00, 1.07, and 1.21 ppm, and another -CH3 group was
observed as doublet at 1.64 ppm. It could be seen from this that the compound comprised
one rhamnose group in its sugar groups. Anomeric protons were observed at 6.25 (br.),
5.11 (1H, .1=7.80 Hz), and 4.97 ppm (1H, J=6.66 Hz). Therefore, SB365 was confirmed
as a glycoside having three sugar groups.
According to I3C-NMR, a hydroxymethyl group was observed at 65. 4 ppm (C-
23), and three anomeric carbon signals was observed at 104.2 (C-T), 106.7 (C-l'"), and
101.7 pprn (C-T). Two olefinic carbons were observed at 122.5 ppm (C-12) and 144.8
ppm (C-l3), and one carboxy carbon was observed at 180.2 ppm (C-28). In general,
about 4 Hz of glycosylation upfield shift is shown when sugar is bound at the 28 position
(180.2 ppm—>176.2 ppm). In the present compound, the above phenomenon was not
observed, and so it was confirmed that the compound does not have a sugar group in the 28
Subsequently, the compound was hydrolyzed in ethanol/sulfuric acid to identify
its sugar groups and the structure of aglycone. SB365 was confirmed as hederagenin
after comparing physicochemical data of the hydrolysis product, aglycone, 13C-NMR, and
'H-NMR data. Further, the hydrolyzed sugars were confirmed as rhamnose, arabinose,
and glucose by comparative TLC.
On the basis of the above analysis results and data in published literatures, SB365
was confirmed as hederagenin 3-O-a-L-rhamnopyranosyl((l—>2)-[(3-Dglucopyranosyl(
H-NMR and "C-NMR data of SB365 are as shown in the following Table 1.
Experimental Example 1: Antitumor activity on BDFl mice transplanted with LLC
The mouse species used in this experiment was BDFl, and healthy male mice
with the body weight of 18-25 g were used. These animals were supplied with water and
foods ad libitum at a place of controlled temperature in the range of 23-24 °C, and were
bred with an antibiotic-free mouse feed. LLC cells were subcutaneously cultured in
C57BL/6 mice for 14 days. A LLC cell-containing tissue was taken and thereto was
added sterilized cold physiological saline water (5 ml/g tissue) to prepare a cell suspension.
The cell suspension of 0.2 ml was subcutaneously transplanted to the groin region of BDFl
From 24 hours after transplantation, the above mice were divided into several
groups consisting of 5 mice. Then, samples, fractions WT and SPX fractions, and SB365,
were dissolved in physiological saline, and were injected intraperitoneally at each
concentration of 280 mg/kg (WT), 70 mg/kg (SPX1), 171 mg/kg (SPX2), 30.5 mg/kg
(SPX3), 8.1 mg/kg (SPX4), and 6.4 mg/kg (SB365). To the negative control group was
injected only the physiological saline, and to the positive control group was injected
adriamycin (0.5 mg/kg). The injection was scheduled from 24 hours after tumor
transplantation to administer the samples successively once a day for 7 days, and stopped
for one day, and then, was carried out for 6 more consecutive days.
In order to evaluate toxicity of SB365 on mice, experimental mice were weighed
twice a week. Antitumor activity was calculated after measuring tumor volume of the
control and test groups on 14th and 15th day after sample administration as follows:
Tumor volume (mm3) = length (mm) x width2 (mm2) / 2
Inhibition rate of tumor growth (%) = (C - T) x 100 / C
(C: average tumor volume in the control group, T: average tumor volume in the
The result is shown in the following Table 2.
Inhibition rate of tumor growth (IR, %) of Pulsatillae radix fractions and SB365 on BDFl
mice transplanted with LLC cells
Fractions or compounds WT
a) Days after transplantation of tumor cells
As shown in the above Table 2, fractions WT and SPX3 showed the inhibition rate
of tumor growth of 55% and 60%, respectively, and SB365 showed the inhibition rate of
tumor growth of 79%, higher than adriamycin of 64% on 15th day from transplantation of
Experimental Example 2: Antitumor activity on nude mice transplanted with NCIH23
Female nude mice of the age of 5 weeks weighing 16-25 g obtained from Harlan
Co. (USA) were used as experimental animals in this experiment. The mice were used
after acclimation for 1 week in an aseptic animal room. The animal room maintained the
temperature of 22±2 °C, the humidity of 55±5%, and the light and darkness cycle of 12
hours, which was automatically controlled. Solid feed for experimental animals was
radiosterilized, and drinking water was sterilized in an autoclave. The animals were
supplied with feed and drinking water ad libitum. A human tumor cell line provided by
National Cancer Institute (NCI), USA, and preserved in the Korean Research Institute of
Bioscience and Biotechnology (KRffiB), Korea, was used. Lung tumor cells, NCI-H23
cells, among the human tumor cells, were transplanted to the nude mice. The tumor cells
of 3> body weight. The samples were intraperitoneally injected to the mice every day for 13
days, that is, from 1 day to 14th day except 8th day after tumor cells transplantation. The
size of tumor formed during the injection was measured in each animal, and any change in
its body weight was also measured. On 16th day after tumor cells transplantation, the
nude mice were sacrificed, and the tumor was separated and weighed. To the positive
control group was intraperitoneally injected adiamycin of 0.5 mg/kg body weight on 1st,
5th, 9th, and 14th day. The result is shown in the following Table 3.
Inhibition rate of tumor growth (IR, %) of SB365 on nude mice transplanted with NCIH23
a): Days after tumor cells transplantation
As shown in the above Table 3, SB365 of 6.4 mg/kg showed a high inhibition rate
of tumor growth, 82.1%, on 16th day after tumor cells transplantation.
Experimental Example 3: Cytotoxicity test
Tumor cells A549, SK-MEL-2, and MCF-7 were obtained from the KRIBB, and
used in this experiment. A culture medium was prepared by adding one pack of Lglutamine-
containing RPMI1640 medium, 100 ml of fetal bovine serum (FBS) inactivated
by heating at a water bath of 50 °C for 30 minutes, 2 g of NaHCO3, 100,000 units of
penicillin, and 1-00 mg of streptomycin, to a sterilized distilled water for injection,
adjusting the pH of the mixture with 0.1 N HC1 to a total volume of 1 1, and disinfecting
the mixture with filtration, and stored at 4 °C before use. The cells were maintained by
propagation once every three days, and a solution containing 0.5% trypsin and 2% EDTA
in physiological buffered saline (PBS) was used to detach the cells from wells.
Cytotoxicity on tumor cells was measured according to Sulforhodamine-B (SRB)
method developed by the NCI in 1989 to measure in vitro antitumor activity of drugs.
Specifically, the cells were detached from wells with 0.5% trypsin-EDTA solution
and then, 3--5*104 cells/ml of cell suspension was prepared. Then, the cell suspension
(180 µl/well) was added to 96-well plate, and the plate was incubated in an incubator of 37
"C, 5% CO2 for 24 hours.
The sample was dissolved in dimethylsulfoxide (DMSO) and diluted with the
culture medium or tertiary distilled water to obtain required concentrations for experiment,
and serially diluted to a final concentration of DMSO of 0.2% or less. To each well of
96-well plate were added 20 µl of the serially diluted sample solutions, and then, the plate
was incubated in an incubator of 37 °C, 5% CC>2 for 48 hours. At the point of time to add
the sample solution, Tz (Time zero) plate was collected. Medium was removed from Tz
plate and from each plate after completing the incubation, and to the plates was added 10%
trichloroacetic acid (TCA) (50 µl/well). The resulting plates were allowed to stand for 1
hour at 4 "C to immobilize the cells on the bottom of the plates. After completing the cell
immobilization, the plates were washed 5~6 times with water to completely remove the
remaining TCA solution, and the resulting plates were dried at room temperature to contain
To the completely dried plates was added 50 µl of a staining solution with 0.4%
SRB in 1% acetic acid to stain the cells for 30 minutes. Then, the plates were washed
5-6 times with 1% acetic acid solution to completely remove SRB unbound to the cells.
The plates were dried at room temperature. Thereto was added "100 µl of 10 mM Tris
solution to dissolve the dye. Then, OD (optical density) value was measured by
microplate reader at a wavelength of 520 nm.
ED50 value of the sample on tumor cells [50% effective dose (ng/ml): a
concentration at which tumor cell growth is inhibited by 50%] was calculated as follows.
T7 value was defined as OD value at the time of starting the incubation after adding the
sample, C (control) value as OD value of the well not treated with the sample, and T (test)
value as OD value of the well treated with the sample. From the values Tz, C, and T,
cytotoxicity of the agent was measured by the following formula:
- in the case of Tz > T, (T - Tz) / (C - Tz)x 100
- in the case of Tz From the values as calculated above, ED50 value of the sample was obtained by
using data regression function of Lotus program.
As a result, ED$o value of SB365 on human lung tumor cells, A549 cells, human
melanoma cells, SK-MEL2, and human breast tumor cells, MCF7 was >20 µg/ml, >10
µg/ml, and >10 µg/ml, respectively. Therefore, SB365 had little cytotoxicity on solid
Formulation Example 1: Preparation of an injectable solution containing fraction
WT fraction of 250 mg obtained in Example 1 was dissolved in 10 ml of
physiological saline to prepare an injectable solution.
Formulation Example 2: Preparation of an injectable dry powder containing fraction
SPX 3 fraction of 25 mg obtained in Example 2 was dissolved in 10 ml of Ringer's
solution, sterilized and then, freeze-dried to prepare ready-to-use injectable dry powder.
This powder would be re-constituted with distilled water for injection before use.
Formulation Example 3: Preparation of an injectable solution containing SB365
SB365 of 6.5 mg obtained in Example 3 was dissolved in 10 ml of Ringer's
solution and sterilized to prepare an injectable solution.
Pulsatillae radix factions WT and SPX3, and SB365, hederagenin 3-O- α –L-rhamnopyranosyl[(
1 —>2)-[β-D-glucopyranosyl(l —>4)]-L-arabinopyranoside, isolated
from the fractions according to the present invention, not only have a high inhibition rate
of tumor growth on solid tumor cells, but also can be conveniently used by dissolving in
various solutions including physiological saline, Ringer's solution, or nutrient solution
because it is readily soluble in water, and has low cytotoxicity enough to ameliorate side
effects of previously developed anti-tumor agents. Therefore, it is anticipated to be very
useful as a therapeutic agent for solid tumors.
A process for isolating an antitumor substance hederagenin 3-O-α-L-rhamnopyranosyl (1→2) [β-D-glucopyranosyl (1→4)]-α-L-arabinopyranoside from Pulsatillae Radix comprising following steps:
i)extracting Pulsatillae Radix Powder from 50% aqueous solution of ethanol, drying the same under reduced pressure, adding acetone at 5 to 10 fold, shaking and centrifuging the mixture at 3000 rpm and collecting the precipitate;
ii) dissolving the precipitate of step (i) in aqueous solution of methanol, fractionating on Sephadex LH20 Column, subjecting the fractions to thin layer chromatography and collecting a fraction having Rf of 0.48 to 0.50; and
iii) isolating hederagenin 3-O-α-L-rhamnopyranosyl (1→2) [β-D-
glucopyranosyl (1→4)]-aL-arabinopyranoside from the fraction of step
The process as claimed in claim 1, the said isolation of hederagenin 3-O-α-L-
rhamnopyranosyl (1→2) [(β-D-glucopyranosyl (1→4)]-α-L-arabinopyranoside
compound from fraction is carried out by HPLC.
|Indian Patent Application Number||893/DEL/2003|
|PG Journal Number||13/2009|
|Date of Filing||14-Jul-2003|
|Name of Patentee||KIM, Song-Bae|
|Applicant Address||533-2, BONGGOK-RI, BANPO-MYUN, GONGJU-SHI, 314-920 CHOONGCHUNGNAM-DO, REPUBLIC OF KOREA|
|PCT International Classification Number||A61K 35/78|
|PCT International Application Number||N/A|
|PCT International Filing date|