COMPOSITION FOR PREVENTING OR TREATING CANCER COMPRISING EXTRACTS OF ANEMONE RADDEANA, LONICERA SPECIES, AND ARALIA ELATA CONTAINING HIGH CONCENTRATION OF ANTITUMOR SAPONINS, AND METHOD FOR PREPARING SAME

Abstract

The present invention relates to a composition for preventing or treating cancer, comprising extracts of Anemone raddeana and Lonicera species containing a high concentration of antitumor saponins, and a method for preparing the same, wherein saponins contained in the extracts of Anemone raddeana and Lonicera species, are converted to anticancer saponins having high antitumor activity, thereby producing extracts containing a high concentration of the antitumor saponins, and the extracts thus prepared have been confirmed to show desirable antitumor activity. Accordingly, the extracts containing a high concentration of antitumor saponins of the present invention are promising for use in the development of drugs for cancer treatment.

Claims

1. A pharmaceutical composition comprising a plant extract as an active ingredient for prevention or treatment of cancer, the plant extract being prepared by a process comprising: i) a first step of adding distilled water to at least one plant selected from the group consisting of Anemone raddeana and Pulsatilla koreana and grinding to prepare a plant medium; ii) a second step of adding an organic solvent to at least one plant selected from the group consisting of Lonicera species, Anemone raddeana, Pulsatilla koreana and Aralia elata to prepare an extract thereof; iii) a third step of combining the medium of the plants in the first step with the extract in the second step and fermenting the combination at 37° C. while stirring; iv) a fourth step of adding an organic solvent to the fermented product obtained by fermentation in the third step to form fractions; v) a fifth step of subjecting the fractions of the fourth step to column chromatography to obtain eluates; and vi) a sixth step of adding an organic solvent to the eluates of the fifth step to obtain fractions and drying the fractions to obtain the plant extract.

2. The pharmaceutical composition of claim 1, wherein the plant medium in the first step is prepared by primarily grinding the at least one plant in distilled water in a mixer; and secondarily performing grinding the by sonication.

3. The pharmaceutical composition of claim 1, wherein the plant in the first step is Anemone raddeana, and the plant in the second step is Lonicera species, or a combination of Lonicera species and Aralia elata.

4. The pharmaceutical composition of claim 1, wherein the Anemone raddeana and Lonicera species an anticancer saponin content of the plant extract is 15 to 110 fold improved in anticancer saponin content.

5. The pharmaceutical composition of claim 4, wherein the anticancer saponin is at least one selected from the group consisting of hederagenin 3-O-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.3)-α-L-rhamnopyranosyl-(1.fwdarw.2)-α-L-arabinopyranoside, oleanolic acid 3-O-α-L-rhamnopyranosyl-(1.fwdarw.2)-[β-D-glucopyranosyl-(1.fwdarw.4)]-α-L-arabinopyranoside, and hederagenin 3-O-β-D-glucopyranosyl-(1.fwdarw.3)-α-L-rhamnopyranosyl-(1.fwdarw.2)-α-L-arabinopyranoside.

6. The pharmaceutical composition of claim 1, further comprising an anticancer agent.

7. The pharmaceutical composition of claim 1, wherein the cancer is a solid cancer.

8. The pharmaceutical composition of claim 1, wherein the plant in the first step is Pulsatilla koreana, and the plant in the second step is Lonicera species and Anemone raddeana.

9. A process for preparing a plant extract comprising: i) a first step of adding distilled water to at least one plant selected from the group consisting of Anemone raddeana and Pulsatilla koreana and grinding to prepare a plant medium; ii) a second step of adding an organic solvent to at least one plant selected from the group consisting of Lonicera species, Anemone raddeana, Pulsatilla koreana and Aralia elata to prepare an extract thereof; iii) a third step of combining the medium of the plants in the first step with the extract in the second step and fermenting the combination at 37° C. while stirring; iv) a fourth step of adding an organic solvent to the fermented product obtained by fermentation in the third step to form fractions; v) a fifth step of subjecting the fractions of the fourth step to column chromatography to obtain eluates; and vi) a sixth step of adding an organic solvent to the eluates of the fifth step to obtain fractions and drying the fractions to obtain the plant extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 shows anticancer activities of the compositions comprising an Anemone raddeana and Lonicera species extract as an active ingredient according to the present disclosure.

MODE FOR CARRYING OUT THE INVENTION

[0064] Hereinafter, preferred examples of the present disclosure will be described in detail. The present disclosure may, however, be embodied in many different forms and should not be construed as being limited to the examples set forth herein. Rather, these examples are provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art.

Experimental Example 1. Preparation of Plant Extract and Medium of Plant Per Se

[0065] Selection was made of saponin-containing target plants including Pulsatilla koreana, Pulsatilla chinensis, Pulsatilla cernua, Anemone raddeana, Aralia elata, Akebia quinata, Patrinia scabiosifolia, Lonicera species (Lonicera sp.), which each have hederagenin or oleanolic acid as the aglycone moiety thereof.

[0066] Available portions of the plants were roots from Pulsatilla koreana, Pulsatilla chinensis, Pulsatilla cernua, Anemone raddeana, and Patrinia scabiosifolia, leaves from Aralia elata, seeds or fruits from Akebia quinata, and Lonicera japonica flowers from Lonicera species.

Experimental Example 1-1. Preparation of Plant Extract

[0067] 1) Pulsatilla chinensis Extract (Pc Extract)

[0068] Roots of Pulsatilla chinensis were pulverized into powder in a pulverizer. To 100 g of root powder of Pulsatilla chinensis was added 500 ml of 70% (v/v) ethanol, followed by reflux for 4 hours while stirring. After reflux, a filtrate was separated by filtration through a filter and stored. The plant residues were added with 500 ml of 70% (v/v) methanol and refluxed for 4 hours to separate an additional filtrate which was pooled with the previous filtrate. The 70% (v/v) methanol extract thus obtained was dried in a vacuum and the resulting dried extract was mixed with 70 ml of 95-100% (v/v) ethanol. Only the ethanol layer was separated by filtration and dried to secure a Pulsatilla chinensis extract (6.4 g).

[0069] 2) Pulsatilla cernua Extract (Pcer Extract)

[0070] A Pulsatilla cernua extract (5.9 g) was obtained from Pulsatilla cernua roots in the same manner as the preparation method for 1) Pulsatilla chinensis extract in Experimental Example 1-1.

[0071] 3) Anemone raddeana Extract (Ar Extract)

[0072] An Anemone raddeana extract (4.1 g) was obtained from Anemone raddeana roots in the same manner as the preparation method for 1) Pulsatilla chinensis extract in Experimental Example 1-1.

[0073] 4) Patrinia scabiosifolia Extract (Ps Extract)

[0074] A Patrinia scabiosifolia (4.0 g) was obtained from Patrinia scabiosifolia roots in the same manner as the preparation method for 1) Pulsatilla chinensis extract in Experimental Example 1-1.

[0075] 5) Aralia elata Extract (Ae Extract)

[0076] Young Aralia elata leaves taken in late April were dried in a shade place and finely cut. After being mixed with 300 ml of 95-100% (v/v) of methanol, 100 g of the cut leaves was ground in a mixer and then refluxed for 5 hours using an extractor while stirring. Thereafter, only the methanol layer was separated by filtration using a filter. The plant residues were added with 300 ml of 95% (v/v) methanol and refluxed for 5 hours to separate an additional methanol layer which was then pooled with the previous methanol layer. The methanol extract thus obtained was concentrated to a volume of about 200 ml to which 200 ml of hexane was added and shaken for 10 minutes. The mixture was left and the methanol layer thus formed was separated to give a primary methanol fraction. The primary methanol fraction was added again with 200 ml of hexane, shaken for 10 min, and left. The methanol layer thus formed was separated to secure a secondary methanol fraction. The secondary methanol fraction was concentrated and dried to obtain a solid. The solid was mixed with 70 ml of 100% (v/v) ethanol and left for 30 min, followed by filtration to separate the ethanol layer only. The separated ethanol layer was dried to afford an Aralia elata extract (3.7 g).

[0077] 6) Akebia quinata Extract (Aq Extract)

[0078] In 400 ml of hexane, 100 g of Akebia quinata seeds was ground using a mixer and then left. The hexane layer was separated and filtered to remove hexane. The filtrate free of hexane was mixed with 400 ml of 95% (v/v) methanol and refluxed for 4 hours with an extractor while stirring. After reflux, only the methanol layer was separated by filtration using a filter. The plant residues were added with 500 ml of 95% (v/v) methanol and refluxed for 4 hours to separate an additional methanol layer which was then pooled with the previous methanol layer. The methanol extract thus obtained was concentrated to a volume of about 300 ml to which 300 ml of hexane was added and shaken for 10 minutes. The mixture was left and the methanol layer thus formed was separated by removing the hexane layer. The separated methanol layer was dried in a vacuum. The residue thus obtained was mixed with 70 ml of 100% (v/v) ethanol, left for a predetermined time, and filtered to separate the ethanol layer only. This ethanol layer was dried to afford an Akebia quinata extract (4.4 g).

[0079] 7) Lonicera sp. Extract (Lj Extract)

[0080] Flowers of Lonicera japonica were used. After being mixed with 300 ml of 95% (v/v) of methanol, 100 g of the flowers was ground in a mixer and then refluxed for 3 hours using an extractor. Thereafter, only the methanol layer was separated by filtration using a filter. The plant residues were added with 300 ml of 100% (v/v) methanol and refluxed for 3 hours to separate an additional methanol layer which was then pooled with the previous methanol layer. The methanol extract thus obtained was concentrated to a volume of about 200 ml to which 200 ml of hexane was added and shaken for 10 minutes. The mixture was left and the methanol layer thus formed was separated by removing the hexane layer to give a primary methanol fraction. The primary methanol fraction was added again with 200 ml of hexane, shaken for 10 min, and left. The methanol layer thus formed was separated to secure a secondary methanol fraction. The secondary methanol fraction was concentrated and dried to obtain a solid. The solid was mixed with 70 ml of 100% (v/v) ethanol and left for 30 min, followed by filtration to separate the ethanol layer only. The separated ethanol layer was dried to afford a Lonicera sp. extract (3.9 g).

Experimental Example 1-2. Preparation of Medium of Plant Per Se

[0081] Saponins in plant exist mainly as glycosides. Particularly, in saponins having hederagenin, oleanolic acid, betulinic acid, and 23-hydroxybetulinic acid as aglycone moieties, the acidic group at position 28 is in the form of glycosyl ester. Thus, the ester group must be hydrolyzed in order to obtain anticancer saponins. Chemical hydrolysis may not only influence on the structure of saponins, but also requires complicate separation processes, thus decreasing in economic advantage. In the present disclosure, advantage was taken of the hydrolysis of the ester group by a hydrolase present in plants during the isolation of saponins glycosides from the plants, and a medium of plant per se was prepared with reference to the technical disclosure set forth in Korean Patent Number 0628334 issued to the present inventors.

[0082] Any of the plants used in Experimental Example 1 may be available for preparing a medium of plant per se. For seeds rich in vegetable oil such as Akebia quinata seeds, however, it is complicated to remove the vegetable oil. For Aralia elata leaves, hydrolases may be deactivated during removal of chlorophyll and wax. Hence, the present disclosure utilized Pulsatilla koreana roots, Anemone raddeana roots, and Akebia quinata fruits.

[0083] 1) Medium of Pulsatilla koreana Per Se (Pk Medium)

[0084] Fresh Pulsatilla koreana roots were washed with water and peeled before being finely cut. In 200 ml of distilled water cooled to 4° C., 100 g of the finely cut Pulsatilla koreana roots was ground using a mixer. The roots ground with a mixer were sonicated for 10 minutes at high frequency with a sonicator to prepare a medium of Pulsatilla koreana per se.

[0085] 2) Medium of Anemone raddeana Per Se (Ar Medium)

[0086] A medium of Anemone raddeana per se was prepared from Anemone raddeana roots in the same manner as the preparation method for 1) medium of Pulsatilla koreana per se in Experimental Example 1-2.

[0087] 3) Medium of Akebia quinata Per Se (Aq Medium)

[0088] Use was made of Akebia quinata fruits that remained undivided. In 200 ml of distilled water cooled to 4° C., 100 g of Akebia quinata fruits was ground using a mixer. The fruits ground with a mixer was sonicated for 5-10 minutes at high frequency using a sonicator to prepare a medium of Akebia quinata per se.

Example 1. Preparation of Extract Containing High Concentration of Anticancer Saponin

[0089] An extract containing a high concentration of anticancer saponins according to the present disclosure was prepared using the plant extracts and media of plant per se prepared in Experimental Example 1. In this regard, the media of plant per se were used immediately after being prepared.

Example 1-1. Preparation of an Anemone raddeana and Lonicera Species Extract

[0090] A mixture of 2) medium of Anemone raddeana per se (Ar medium) in Experimental Example 1-2 and 3.9 g of 7) Lonicera species extract (Lj extract) in Experimental Example 1-1 was fermented at 37° C. for 2 hours while stirring. The fermented product was cooled to room temperature, added with 400 ml of 95% (v/v) methanol, and stirred at room temperature for 12 hours. Thereafter, a filtrate was separated using a filter and stored. The solid residue after filtration was added with 400 ml of 95% (v/v) methanol and reacted in the same manner as described above to obtain a filtrate. The filtrates obtained twice were combined and concentrated. The concentrate thus obtained was dried using a desiccator. The resulting solid residue was mixed with 70 ml of 100% (v/v) ethanol and left for a predetermined time, followed by filtration through a filter to separate only the ethanol layer. The separated ethanol layer was transferred into a pyrogen-free vessel and dried to give 5.4 g of a solid.

[0091] In 10 ml of distilled water was dissolved 2 g of the solid which was then left for 20 min, followed by filtration through a filter. The filtrate thus obtained was stabilized with water and added and adsorbed to 300 g of diaion HP-20 resin packed in a column (column size 5×80 cm). After the column was washed by passing 1,500 ml of distilled water over 2 hours therethrough, elution was conducted by sequentially adding 300 ml of 20% (v/v) methanol and 1,000 ml of 90% (v/v) methanol. The eluates were collected by 20 ml. Drops of the collected eluates were placed on silica gel plates and dried. Afterwards, 10% (v/v) sulfuric acid was sprayed over the plates which were then heated to 150° C. to monitor the appearance of white red colors. Only the eluates that appeared white red were pooled, concentrated, and dried. The solid residue was mixed with 20 ml of 100% (v/v) ethanol and left for a predetermined time, followed by filtration through a filter to separate only the ethanol layer. The separated ethanol layer was transferred into a pyrogen-free vessel and dried to afford an Anemone raddeana and Lonicera species extract (0.98 g) containing a high concentration of anticancer saponins according to the present disclosure.

Example 1-2. Preparation of Anemone raddeana, Lonicera Species and Aralia elata Extract

[0092] A mixture of 2) medium of Anemone raddeana per se (Ar medium) in Experimental Example 1-2 and 3.9 g of 7) Lonicera species extract (Lj extract) and 3.7 g of 5) Aralia elata extract (Ae extract) in Experimental Example 1-1 was fermented at 37° C. for 2.5 hours while stirring. The fermented product was cooled to room temperature, added with 500 ml of 95% (v/v) methanol, and stirred at room temperature for 12 hours. Thereafter, a filtrate was separated using a filter and stored. The solid residue after filtration was added with 500 ml of 95% (v/v) methanol and reacted in the same manner as described above to obtain a filtrate. The filtrates obtained twice were combined and concentrated. The concentrate thus obtained was dried using a desiccator. The resulting solid residue was mixed with 100 ml of 100% (v/v) ethanol and left for a predetermined time, followed by filtration through a filter to separate only the ethanol layer. The separated ethanol layer was dried to form a solid to which 70 ml of 100% (v/v) ethanol was then added. Only the ethanol layer was separated by repeating the above process. The separated ethanol layer was transferred into a pyrogen-free vessel and dried to give 7.9 g of a solid.

[0093] From 2 g of the solid, an Anemone raddeana, Lonicera species, and Aralia elata extract (1.09 g) containing a high concentration of saponins according to the present disclosure was prepared in the same manner as with the diaion HP-20 column in Example 1-1.

Example 1-3. Preparation of Anemone raddeana, Lonicera Species, and Pulsatilla koreana Extract

[0094] A mixture of 1) Pulsatilla koreana per se (Pk medium) in Experimental Example 1-2 and 4.1 g of 3) Anemone raddeana extract (Ar extract) and 3.9 g of 7) Lonicera species extract (Lj extract) in Experimental Example 1-1 was fermented at 37° C. for 2.5 hours while stirring. The fermented product was cooled to room temperature, added with 500 ml of 95% (v/v) methanol, and stirred at room temperature for 12 hours. Thereafter, a filtrate was separated using a filter and stored. The solid residue after filtration was added with 500 ml of 95% (v/v) methanol and reacted in the same manner as described above to obtain a filtrate. The filtrates obtained twice were combined and concentrated. The concentrate thus obtained was dried using a desiccator. The resulting solid residue was mixed with 100 ml of 100% (v/v) ethanol and left for a predetermined time, followed by filtration through a filter to separate only the ethanol layer. The separated ethanol layer was transferred into a pyrogen-free vessel and dried to give 8.1 g of a solid.

[0095] From 2 g of the solid, an Anemone raddeana, Lonicera species, and Pulsatilla koreana extract (1.09 g) containing a high concentration of saponins according to the present disclosure was prepared in the same manner as with the diaion HP-20 column in Example 1-1.

Comparative Example 1. Preparation of Comparative Extracts Containing Anticancer Saponins

Comparative Example 1-1. Preparation of Pulsatilla koreana Extract 1

[0096] After 1) the medium of Pulsatilla koreana per se (Pk medium) in Experimental Example 1-2 was prepared, Pulsatilla koreana extract 1 to be compared was prepared in the same manner as in Example 1-1 with the exception that the fermentation process was excluded.

Comparative Example 1-2. Preparation of Pulsatilla koreana Extract 2

[0097] After 1) the medium of Pulsatilla koreana per se (Pk medium) in Experimental Example 1-2 was fermented at 37° C. for 2 hours while stirring, the subsequent process was conducted in the same manner as in Example 1-1 to prepare Pulsatilla koreana extract 2 to be compared.

Comparative Example 1-3. Preparation of Anemone raddeana Extract 1

[0098] After 2) the medium of Anemone raddeana per se (Ar medium) in Experimental Example 1-2 was prepared, Anemone raddeana extract 1 to be compared was prepared in the same manner as in Example 1-1 with the exception that the fermentation process was excluded.

Comparative Example 1-4. Preparation of Anemone raddeana Extract 2

[0099] After 2) the medium of Anemone raddeana per se (Ar medium) in Experimental Example 1-2 was fermented at 37° C. for 2 hours while stirring, the subsequent process was conducted in the same manner as in Example 1-1 to prepare Anemone raddeana extract 2 to be compared.

Comparative Example 1-5. Preparation of Lonicera Species Extract

[0100] A Lonicera species extract to be compared was prepared in the same manner as for 7) Lonicera species extract (Lj extract) in Experimental Example 1-1 with the exception that the fermentation process of Example 1-1 was excluded.

Comparative Example 1-6. Preparation of Pulsatilla koreana and Anemone raddeana Extract

[0101] The 1) medium of Pulsatilla koreana per se (Pk medium) in Experimental Example 1-2 was mixed 4.1 g of 3) Anemone raddeana extract (Ar extract) in Experimental Example 1-1, followed by fermentation at 37° C. for 2 hours while stirring. The subsequent process was conducted in the same manner as in Example 1-1 to prepare a Pulsatilla koreana and Anemone raddeana extract to be compared.

Comparative Example 1-7. Preparation of Pulsatilla koreana and Lonicera Species Extract

[0102] After 3.9 g of 7) Lonicera species extract (Lj extract) in Experimental Example 1-1 was mixed with 1) the medium of Pulsatilla koreana per se (Pk medium) in Experimental Example 1-2, the subsequent process was conducted in the same manner as in Example 1-1 to prepare a Pulsatilla koreana and Lonicera species extract to be compared.

Comparative Example 1-8. Preparation of Anemone raddeana and Lonicera Species Extract

[0103] An Anemone raddeana and Lonicera species extract to be compared was prepared in the same manner as in Example 1-1 with the exception that the fermentation process was excluded.

Experimental Example 2. Content of Anticancer Saponin in Extract

[0104] Contents of anticancer saponins in the extracts prepared in Example 1 and Comparative Example 1 were measured by high performance liquid chromatography (HPLC). As anticancer saponins to be measured for contents, selection was made of 1) hederagenin 3-O-α-L-rhamnopyranosyl-(1.fwdarw.2)-[β-D-glucopyranosyl-(1.fwdarw.4)]-α-L-arabinopyranoside (hereinafter referred to as “H-gra”), 2) hederagenin 3-O-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.3)-α-L-rhamnopyranosyl-(1.fwdarw.2)-α-L-arabinopyranoside (hereinafter referred to as “H-g(1.fwdarw.4)g(1.fwdarw.3)ra”), 3) oleanolic acid 3-O-α-L-rhamnopyranosyl-(1.fwdarw.2)-[β-D-glucopyranosyl(1.fwdarw.4)]-α-L-arabinopyranoside (hereinafter referred to as “O-gra”) and 4) hederagenin 3-O-β-D-glucopyranosyl-(1.fwdarw.3)-α-L-rhamnopyranosyl-(1.fwdarw.2)-α-L-arabinopyranoside (hereinafter referred to as “H-g(1.fwdarw.3)ra”).

[0105] In 3.5 ml of methanol for HPLC, 10 mg of each of the extracts prepared in Example 1 and Comparative Example 1 was dissolved, followed by filtration through a 0.45 μm filter. Then, 50 μl of the filtrate was loaded into Spherisorb® SSODS column on which HPLC was performed using a concentration gradient of acetonitrile:distilled water (3.4:6.6.fwdarw.4:6[v/v]) at a flow rate of 1.5 ml/min. As a result, peaks of retention time were detected at 26.5 min for H-g(1.fwdarw.4)g(1.fwdarw.3)ra saponin and at 43.5 min for H-gra saponin. In addition, HPLC was performed in the same condition except for using isocratic elution with acetonitrile:distilled water (4.3:5.7[v/v]). Peaks of retention time were detected at 18.5 min for H-g(1.fwdarw.3)ra saponin and at 27.1 min for 0-gra saponin. From peaks for each saponin, contents of anticancer saponins in each extract were analyzed. The results are summarized in Table 1.

TABLE-US-00001 TABLE 1 Anticancer saponin content (mg/g) H-g (1.fwdarw.4) g H-g (1.fwdarw.3) (1.fwdarw.3) Extract H-gra ra O-gra ra Example 1-1. Anemone raddeana and 325 205.6 155.7 — * Lonicera species extract Example 1-2. Anemone raddeana, 261.4 146.3 133.3 109.1 Lonicera species and Aralia elata extract Example 1-3. Anemone raddeana, 255.5 156.1 209.8 92.3 Lonicera species and Pulsatilla koreana extract Comparative Example 1-1. 2.2 0.4 1.3 1.1 Pulsatilla koreana extract 1 Comparative Example 1-2. 330 63.69 33 82.2 Pulsatilla koreana extract 2 Comparative Example 1-3. Anemone 4.5 — 3.2 — raddeana extract 1 Comparative Example 1-4. Anemone 330 — 233.9 — raddeana extract 2 Comparative Example 1-5. Lonicera — 10.5 — — species extract Comparative Example 1-6. 324.3 52.1 130 75.6 Pulsatilla koreana and Anemone raddeana extract Comparative Example 1-7. 207.5 177.8 27 69.4 Pulsatilla koreana and Lonicera species extract Comparative Example 1-8. Anemone 4.3 9.5 3.5 — raddeana and Lonicera species extract *: not detected

[0106] It was confirmed that when a combination of the plant extract in Experimental Example 1-1 and the medium of the plant per se in Experimental Example 1-2 was hydrolyzed, contents of anticancer saponins listed in Table 1 were increased although not indicated in Table 1.

Example 2. Preparation of Composition Comprising Extract Highly Rich in Anticancer Saponin and Anticancer Agent in Combination

[0107] Compositions for evaluating anticancer effects upon co-administration of the extracts containing high concentrations of anticancer saponins according to the present disclosure and conventional anticancer agents were prepared.

[0108] As an anticancer agent, deoxypodophyllotoxin (DPT) was used. A 3 μg/ml DPT solution was prepared by mixing 1 ml of a solution of 30 mg of DPT in 100 ml of 95% (v/v) ethanol with 99 ml of 95% (v/v) ethanol. This solution was used as a DPT stock.

[0109] In addition, 10 mg of each of the extracts highly rich in anticancer saponins of Examples 1-1 to 1-3 was dissolved in 9 ml of physiological saline and then mixed with 1 ml of the 3 μg/ml DPT to prepare compositions of Examples 2-1 to 2-3 as listed in Table 2, below.

TABLE-US-00002 TABLE 2 Example Composition Example Example 1-1. Anemone raddeana and Lonicera 2-1 species extract + DPT Example Example 1-2. Anemone raddeana, Lonicera 2-2 species and Aralia elata extract + DPT Example Example 1-3. Anemone raddeana, Lonicera 2-3 species and Pulsatilla koreana extract + DPT

Experimental Example 3. Anticancer Activity

[0110] In order to assay anticancer activities of extracts highly rich in anticancer saponins in Example 1 and Comparative Example 1 and compositions comprising extracts highly rich in anticancer saponins and an anticancer agent in Example 2, solid cancer-induced mouse models were used.

[0111] A solid cancer accounts for a mass of cancer cells. There are a variety of solid cancers and thus, it is difficult in terms of time and cost to evaluate anticancer activities against all kinds of solid cancers. In this regard, solid cancer-induced mouse models which had been established by subcutaneously transplanting cancer cells were employed because many researchers had proven the effectiveness of the models.

[0112] BDF1 male mice with 4 weeks of age, each weighing 20-23 g, were raised at 22±2° C. in the humidity condition 65±5%. The mice were randomly divided into groups of five. In order to induce the onset of a solid cancer, Lewis lung carcinoma cells (LLC) were subcutaneously transplanted in an amount of 1×10.sup.6 cells into the left front armpit of each BDF1 mouse and incubated for 24 hours. After 24 hours, the extracts of Example 1 and Comparative Example 1 or the compositions comprising combinations of extracts and anticancer agent in Example 2 were intraperitoneally injected at a daily dose of 10 mg/kg for two weeks.

[0113] A mouse group which was treated with none of the extracts after transplantation of the cancer cells was used as a negative control. When the cancer volume in the negative control reached about 2 cm.sup.3, cancer sizes were measured.

[0114] Cancer growth inhibition rates were calculated according to the following formula 1. On days 7 and 14 after drug administration, cancer growth inhibition rates were measured and the results are shown in Table 3 below and FIG. 1.

Cancer growth inhibition rate (%)=((average cancer volume in negative control−average cancer volume in administered group)/average cancer volume in negative control)×100  [Formula 1]

TABLE-US-00003 TABLE 3 Cancer No. of Single Dose growth mouse Anticancer Inhibition with saponin DPT Rate (%) cancer Extract (mg/kg) (ng/kg) Day 9 Day 14 extinct Example 1-1. Anemone 10 — 55.6 74.9 0 raddeana and Lonicera species extract Example 1-2. Anemone 10 — 58.7 85.5 1 raddeana, Lonicera species and Aralia elata extract Example 1-3. Anemone 10 — 60.8 82.9 1 raddeana, Lonicera species and Pulsatilla koreana extract Example 2-1. 10 3000 65 82 1 Composition containing extract of Example 1-1 and DPT Example 2-2. 10 3000 60.2 95.2 3 Composition containing extract of Example 1-2 and DPT Example 2-3. 10 3000 76 94.3 2 Composition containing extract of Example 1-3 and DPT Comparative Example 10 — — 25 0 1-1. Pulsatilla koreana extract Comparative Example 10 — 52.3 59.1 0 1-2. Pulsatilla koreana extract 2 Comparative Example 10 — — 13.3 0 1-3. Anemone raddeana extract 1 Comparative Example 10 — — 57.3 0 1-4. Anemone raddeana extract 2 Comparative Example 10 — — — 0 1-5. Lonicera species extract Comparative Example 10 — 53.1 57.6 0 1-6. Pulsatilla koreana and Anemone raddeana extract Comparative Example 10 — 54.5 58.7 0 1-7. Pulsatilla koreana and Lonicera species extract Comparative Example 10 — — 15 0 1-8. Anemone raddeana and Lonicera species extract

[0115] As is understood from the data of Table 3 and FIG. 1, when extracts of Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-8 were administered at equal doses, cancer growth inhibition rates were detected at remarkably higher levels on day 14 after administration in the mice administered the extracts of Examples 1-1 to 1-3 than Comparative Examples 1-1 to 1-8. In the mice to which extracts of Examples 1-2 and 1-3 had been administered, the transplanted cancer cells were found to completely die.

[0116] In addition, when administered, the compositions of Examples 2-1 to 2-3 in which extracts of Examples 1-1 to 1-3 are combined with the anticancer agent DPT exhibited higher cancer growth inhibition rates than the extracts of Examples 1-1 to 1-3 alone, and increased the number of mice in which the transplanted cancer cells had completely become extinct.

[0117] As is understood from the results of Experimental Examples 2 and 3, the composition comprising an Anemone raddeana and Lonicera species extract alone or in combination with an Aralia elata or Pulsatilla koreana extract according to the present disclosure has a potent anticancer activity, with increased contents of anticancer saponins of H-g(1.fwdarw.4)g(1.fwdarw.3)ra, O-gra, and H-g(1.fwdarw.3)ra therein. Furthermore, co-administration of the extract of the present disclosure and a conventional anticancer agent was observed to increase anticancer activity.

[0118] Therefore, it is expected that the Anemone raddeana and Lonicera species extract containing high concentrations of anticancer saponins according to the present disclosure can be used to develop an anticancer agent having potent anticancer activity.

Formulation Example 1. Pharmaceutical Formulation

Formulation Example 1-1. Preparation of Tablet

[0119] With 20 g of the extract of Example 1-2 according to the present disclosure, 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicate were mixed. The mixture was added with a 10% gelatin solution, ground, and filtered through 14-mesh sieves. The filtrate was dried and mixed with 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate before being prepared into tablets.

Formulation Example 1-2. Preparation of Injection

[0120] A solution of 100 mg of the extract of Example 1-2 according to the present disclosure in 100 ml of physiological saline was filtered through a filter capable of removing germs to afford an injection liquid.