JP2007070250A - Anticancer agent containing extract of gentiana spp, health assistant food, cosmetic for medicinal use, and method for producing extract of gentiana spp - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To effectively utilize a root and a leaf of Gentiana triflora which are conventionally discarded. <P>SOLUTION: The anticancer agent as an oral medicine or an external preparation contains a crude extract of a plant of Gentiana spp, or a purified product obtained therefrom as an active ingredient. The Gentiana triflora is preferably used as the Gentiana spp. The root or the leaf, especially the root is preferably used as the part of the plant. The crude extract or the purified product can be used as the health assistant food (supplement) and the cosmetic for medicinal use. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an anticancer agent containing a Gentiana spp plant extract as an active ingredient, a health supplement (supplement) and a medicinal cosmetic, and a gentian extract (having anticancer activity) from a Gentiana spp plant. The present invention relates to a method for manufacturing a product.

  Gentian is a perennial plant that inhabits relatively cold highlands, and its root is a Chinese medicine called “Rondan” (Japanese name: Ryutan, Ryutan). It is said to have a good stomach, increased appetite, increased liver function, and other effects, and has been used as a hot water, powder, pill, and powder coating agent (described in Shinnohonhonso, etc.). Although scientific evidence is not shown for all of these effects, as shown in Table 1 below, several interesting drug effects and pharmacological actions have been reported for gentian roots.

Note 1) PAF: Platelet activating factor. Related to anaphylactic shock. (Huh, H. et al. (1998) Arch. Pharm. Res., 21, 436-439)
Note 2) Aldose reductase enhances corneal damage, a diabetic complication. In some diabetic patients, aldose reductase activity is observed in corneal epithelium (especially basal cells), corneal endothelial cells, and nerve Schwann cells. Clinical improvements have been observed with inhibitors of this enzyme (not gentian). (Zhang, JQ and Zhou, YP (1989) Zhongguo Zhong Yao Za Zhi 14, 557-559, 576) (Chinese)
Note 3) Inhibits the formation of malondialdehyde (indicator of lipid peroxidation) in blood and liver related to liver damage caused by carbon tetrachloride (CCl ₄ ). At the same time, the increase in blood suppresses an increase in enzyme activities such as AST (aspartate transaminase) and ALT (glutamate-pyruvate transaminase), which are indicators of liver damage. (Aktay, G. et al. (2000) J. Ethnopharmacol., 73, 121-129; Kondo, Y. and Hojo, H. (1994) Planta Med., 60, 414-416)
Note 4) Inhibits the growth of Candida albicans. (Tan, RX et al. (1996) Phytochemistry 4, 111-116)

Among the gentian listed in Table 1, G. scabla (Gentiana scabla var. Buergeri) is a kind called sasalindou (or turpentine), which has been used mainly in Chinese herbal medicine. This gentian grows naturally in China, East Russia, South Korea and Japan.
G. lutea is a European flowering gentian with yellow flowers, and this root extract and powder has a mild irritating effect on the stomach and intestinal tract and is used in patients with dyspepsia, anorexia and digestive fluid deficiency. In addition, liqueur dipped in the roots of G. lutea is produced and sold in France and other foreign countries and imported into Japan.
G. olivieri is a gentian used as a folklore medicine in Turkey.
Thus, the roots of Gentianaceae plants are not limited to China, but have a history of being used as a traditional medicine in Europe, and are believed to have some medicinal and pharmacological effects.

  Japan is home to a variety of Gentiana triflora called Gentiana triflora var. Japonica. This gentian grows naturally in Japan, South Korea, and Eastern Russia, but is different from S. gentian (G. scabla) in herbaceous form, flower form and overwintering bud (vegetative breeding bud that sprout in the next year). Breeding has progressed in Japan as a variety for cut flowers, and a considerable amount has been produced. However, there are few examples of medicinal and pharmacological studies on the extract of Ezorindo, and it is not currently used as a material for traditional Chinese medicine. To the extent that novel antibacterial proteins and their genes against plant pathogens are known (see Patent Document 1).

JP 2003-88386 A

  As mentioned above, Japanese native zorindo (Gentiana triflora) has been bred as a cultivar for cut flowers in Japan, and a considerable amount is produced, but its roots are discarded as unnecessary (agricultural waste). ing. The present invention is intended to effectively utilize these ezolin roots that have been discarded.

  As a result of various attempts to make effective use of these Ezorindo roots, the present inventors have found that a substance that inhibits the growth of cultured cancer cells or the growth of solid cancer transplanted into mice in this water-soluble extract of Ezolyndo root The present invention was completed.

  That is, the present invention is an anticancer agent characterized in that a crude extract of a Gentiana spp plant or a purified product obtained therefrom is an active ingredient, and the formulation is an oral preparation or an external preparation.

Here, Gentiana triflora is preferably used as the Gentiana spp plant.
In addition, as a part of a Gentian spp plant, roots or leaves are preferably used, and roots are particularly preferably used.
Further, the medium used for extraction from Gentiana spp plants is preferably an aqueous medium mainly composed of water, and may be a mixed solution with water or an organic solvent soluble in water in addition to water.

  The present invention also provides a health supplement (supplement) containing a crude extract of Gentiana spp plants or a purified product obtained therefrom.

  The present invention also provides a medicinal cosmetic product comprising a crude extract of a Gentiana spp plant or a purified product obtained therefrom.

Furthermore, the present invention provides the following steps (a) to (e):
(A) shredding a Gentiana spp plant;
(B) treating the shredded product with boiling water;
(C) removing (filtering) insoluble matters;
(D) (autoclave) heat treatment under pressure; and (e) drying if necessary;
There is also provided a method for producing a crude gentian extract (having anticancer activity).
The steps (a) to (e) are not necessarily performed in this order, and may be performed in the order of (a)-(b)-(d)-(c)-(e). Further, another purification step may be added after or without performing step (e).

The anticancer agent of the present invention is useful as a cancer therapeutic drug or cancer preventive drug targeting specific cancer cells / tissues.
In addition, the health supplement of the present invention is expected to be useful as a preventive medicine supplement.
The medicinal cosmetics of the present invention are expected to be useful for preventing skin cancer and the like.
By the production method of the present invention, a gentian extract having an anticancer activity can be obtained from a Gentiana spp plant.

Further, the present invention will be described in detail. The crude extract of Gentiana spp plant in the present invention or a purified product obtained therefrom inhibits the growth of cultured cancer cells or the growth of solid cancer transplanted into mice, and therefore can be used as an anticancer agent. It can also be used as a health supplement (supplement), or it can be mixed with cosmetics to make medicinal cosmetics.
The formulation of the anticancer agent of the present invention is an oral preparation or an external preparation as described above. These oral preparations and external preparations may be solid preparations such as tablets, granules, powders and pills, liquid preparations such as liquids and syrups, or semisolid preparations such as ointments, creams and suppositories. it can. In addition, the granule and powder may be in a form in which a unit amount is administered as a capsule, and in the case of a liquid preparation, the dried product may be re-dissolved before administration.
Of these dosage forms, oral preparations and external preparations include, in addition to active ingredients extracted from Gentiana spp plants, binders, excipients, lubricants, disintegrants generally used in pharmaceutical preparations. , Wetting agents, emulsifying agents, suspending agents, stabilizers, buffering agents, flavoring agents, preservatives, fragrances, coloring agents and the like can be added.
In addition, since the dose varies depending on the age of administration subject, administration route, number of administrations, etc., an optimal amount may be appropriately determined according to circumstances.

  The health supplement (supplement) or medicinal cosmetic of the present invention is prepared by a known formulation technique or cosmetic production technique, a preparation (tablet, soft capsule, etc.) containing a crude extract of Gentiana spp plant or a purified product obtained therefrom. ) Or cosmetics (creams, lotions, etc.).

Embodiments of the present invention will be specifically described below with reference to the accompanying drawings.
In addition, PBS and trypan blue in this specification have the following meaning or significance.
PBS: Abbreviation for Phosphate-buffered Saline (saline phosphate buffer).
Trypan blue: A dye used to stain cells, which stains dead cells.

Example 1 Preparation of Crude Extract from Ezorin Dot Root Dry zoelin root is cut into small pieces, and a predetermined amount of boiling water (for example, boiling water of 10 times the dry weight of the root) for a predetermined time (for example, 30 minutes). ) Heat-treated and extracted. The extract was transferred to another container, and the remaining roots were similarly treated with boiling water and extracted again. The extracts obtained by the two extractions were combined, roughly filtered using gauze or the like, and then finely filtered using a sterile filter (for example, a sterile filter having a pore size of 22 μm). The obtained clarified filtrate was freeze-dried, dissolved in an appropriate amount of water or PBS, and further subjected to pressurization and heating (for example, at 120 ° C. for 20 minutes) in an autoclave to obtain a crude extract for demonstration. In addition, the lyophilized product can be used as it is by dissolving it in an appropriate amount of water or PBS without being pressurized and heat-treated in the above autoclave, but by applying pressure and heat-treated in the autoclave, The cell growth inhibitory activity can be increased about twice. FIG. 1 summarizes an example of an extraction procedure from Ezorindo root in a scheme.

Example 2 Evaluation Test I (Inhibitory Activity of Crude Extract on Various Cultured Cancer Cells)
Next, the growth inhibitory activity of the zorindo extract of the present invention was evaluated using various cultured cancer cells. The method was carried out by adding a certain amount of Ezolyndo extract (final concentration: extract dry weight 2 mg / ml medium) to the cultured cell medium and counting the number of viable cells after 24 hours. The results are shown in FIG. 2, and the vertical axis represents the relative value (%) of the number of viable cells in the added medium with respect to the number of viable cells when cultured without the extract.
From this result, it can be seen that the cell growth inhibitory effect of the gentian extract differs depending on the cell type. In addition, the extract of zorindo of the present invention exhibits particularly strong growth inhibition against human Daudi (derived from Burkitt lymphoma), KTC-2 (derived from undifferentiated thyroid cancer) and Y3-Ag (rat myeloma), and SK-Mel28 (Human malignant melanoma) and dRLh84 (rat liver cancer) also showed growth inhibition. However, some cultured cancer cells were not affected at all, such as TTA-1 (a cell line derived from undifferentiated thyroid cancer different from KTC-2). In FIG. 2, the cells derived from thyroid cancer indicated by * are cell lines established from different patients.

Example 3 Evaluation Test II (Inhibition Effect of Crude Extract on Growth of Solid Cancer)
Next, the growth inhibitory effect of the zorindo extract of the present invention was evaluated using cancer cells transplanted into mice. The method is the case where 330 mg gentian extract (PBS solution) per kg body weight was orally administered (once a day, at a fixed time) to BALB / c mice transplanted with mouse solid tumor (renal cell carcinoma Renca) subcutaneously in the back. The growth of the solid cancer was followed for about one month. The results are shown in FIG. 3, and the figure on the left side of FIG. 3 shows that the gentian extract was administered on the same day as the solid cancer transplantation. The figure on the right side of FIG. 3 shows that the gentian extract was administered seven days before the solid cancer transplantation. It was done. In addition, the solid cancer volume was used as the solid cancer growth index, and the solid cancer volume was calculated using the following formula.
<Calculation formula> Solid tumor volume (mm ³ ) = (minor axis) ² × (major axis) × 0.5
A control experiment was performed by orally administering PBS containing no gentian extract in the same volume. From the results of FIG. 3, it can be seen that the growth of solid cancer was suppressed by 30 to 40% by orally administering the gentian extract.

Example 4 Examination of the part of the plant of Ezorindo The presence of each cell growth inhibitory substance was examined for the roots, stems and leaves of Ezolyndo and compared with the dried root extract obtained in Example 1. In the method, the same amount of the extract obtained from each tissue was added to a cancer cell (human Burkitt lymphoma, Daudi) medium, and a cell group after 24 hours of culture was stained with trypan blue to determine the number of dead cells and living cells. This was done by measuring. The result is shown in FIG. 4, and the vertical axis represents the number of viable cells after the extract treatment. It can be seen that the cell growth inhibitory substances are in the order of root>leaf> stem, and the cell growth inhibitory activity in the leaves is about ½ of the root.

Example 5 Purification of Cell Growth Inhibitory Substance from Crude Extract Using the crude extract of Ezorin root obtained in Example 1 as a raw material, this was divided into various fractions by liquid chromatography, and cell growth inhibition in each fraction was performed. The activity was examined. FIG. 5 shows an elution pattern of liquid chromatography and a fraction having a strong cell growth inhibitory activity (peak A fraction: a fraction eluted around 20 minutes elution time). The conditions for liquid chromatography are as follows.
Column: Develosil ODS HG-5
Mobile phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution Mobile phase B: 0.1% TFA acetonitrile solution Flow rate: 1.0 ml / min
-Column temperature: 40 ° C
UV detection condition: 230 nm
Gradient conditions: Table 2

It is a scheme of an example of the extraction procedure from a zelkova root. It is a graph which shows the growth inhibitory activity of the Ezorindo extract of this invention with respect to various cultured cancer cells. It is a graph which shows the growth inhibitory effect of the ezorindo extract of this invention with respect to the cancer cell transplanted to the mouse | mouth. It is a graph which shows the comparison of the plant-body part of an Ezorindo extract with respect to a cultured cancer cell. It is a separation / elution pattern by liquid chromatography using a crude extract of ezolin root as a sample solution.

Claims (8)

An anticancer agent characterized in that a crude extract of a Gentiana spp plant or a purified product obtained therefrom is an active ingredient, and the formulation is an oral preparation or an external preparation. The anticancer agent according to claim 1, wherein the Gentiana spp plant is Gentiana triflora. The anticancer agent according to claim 1 or 2, wherein a part of a Gentiana spp plant is a root or a leaf. The anticancer agent according to any one of claims 1 to 3, wherein the extraction medium is water. A health supplement (supplement) containing a crude extract of a Gentiana spp plant or a purified product obtained therefrom. A medicinal cosmetic comprising a crude extract of a Gentiana spp plant or a purified product obtained therefrom. A method for producing a crude gentian extract having anticancer activity, comprising the following steps (a) to (e):
(A) shredding a Gentiana spp plant;
(B) treating the shredded product with boiling water;
(C) removing (filtering) insoluble matters;
(D) (autoclave) a step of heat treatment under pressure; and (e) a step of drying as necessary. 8. The production method according to claim 7, wherein Gentiana triflora is used as a Gentiana spp plant, and a root or a leaf is used as a site.

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