JPS5936890B2 - anticancer drug - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has excellent effects on the prevention and treatment of cancer and its precursor symptoms, including ulcerated tumors, and also has effects on the prevention and treatment of liver disorders. Furthermore, the present invention relates to a unique anticancer agent that is substantially non-toxic, as can be understood from the fact that it is a green juice component of natural grass grass leaves.

Knowing that the green juice component of the green leaves of barley before the ripening stage is rich in a wide variety of useful natural ingredients, we developed this green juice component as a stable powder that retains its original state in the green juice. Having succeeded in obtaining the patent, the inventor of the present application previously obtained Japanese Patent No. 645378 (Japanese Patent Publication No. 46-38548) [corresponding U.S. Patent No. 3787591, British Patent No. 1358052].
In [2006], we proposed a method for using wheat green leaf powder.

According to this proposal, the pH of green juice obtained by separating and removing coarse solids from mechanically crushed green leaves of wheat before the ripening stage is
By spray-drying or freeze-drying the neutralized product in steps 9 to 9, a stable powder of the green juice component of young barley leaves can be obtained.

It also states that it is useful in a wide range of fields such as foods including luxury goods, pharmaceuticals including health drugs and cosmetics, and examples of health drugs include green juice powder, windbreak, bedding extract powder, starch, and lactose. , talc, magnesium stearate, and ethyl alcohol, and discloses that this agent can be taken for the prevention and treatment of arteriosclerosis.

However, the proposal does not mention at all the anticancer effect of the powder containing green juice components of green leaves of barley before the ripening stage or any knowledge suggesting such a pharmacological effect.

As a result of further research, the present inventor discovered that t-)! is known to exhibit strong mutagenic activity. We have discovered an interesting new fact that when a green juice component of green leaves of wheat before the ripening stage is applied to a J-butophane combustion product, the mutagenic activity of the mutagenic substance is significantly reduced.

Furthermore, for example, when the organophosphorus pesticide malathion is added to the green juice component of Banmugi related leaves and incubated at 35°C, 1
00 ppm malathion becomes about 10 ppm in 120 minutes
We discovered the surprising fact that sorbic acid is decomposed to a similar extent, and that it also exhibits a similar decomposition-promoting effect on the food additive preservative sorbic acid.

Furthermore, an interesting new fact has been discovered that dibutylhydroxytoluene, a known food additive (antioxidant), exhibits a similar decomposition promoting effect.

Based on this new knowledge, we investigated the pharmacological actions and effects of the green juice components of barley green leaves in the field of anticancer drugs.

As a result, the green juice component has remarkable anticancer activity against painful symptoms or their prodromal symptoms, including ulcerative tumors, whose pharmacological targets are completely different from those of the health drug for arteriosclerosis proposed above. We discovered a new fact that shows that.

For example, in a mouse intraperitoneal administration test using murine leukemia P388 cells, the median survival in days of the drug administration group/the median survival in days of the non-administered control group (T/C) exceeded 125% after administration at 400 μ/ky/day. For example, in vitro against the above P388 cells.
As a result of the test, the 50% inhibitory concentration (I C
50) has a high activity of about 20 to about 3 Or (tdl/ml), and furthermore, it has a remarkable suppressive effect in the experimental gastric ulcer tumor formation suppression test conducted by administering it to Shay rats, for example. It was discovered that it exhibits a number of unique anticancer drug activities.

Furthermore, the acute toxicity LD5o of the green juice component is 12. ．． 000
■/ky (oral, mouse) and is virtually non-toxic;
The results of the subacute toxicity test of continuous administration (oral, mouse) of 000 ■/ky showed that virtually no toxicity or side effects were observed, and conventionally known anticancer drugs have both pharmacological effects and low toxicity or no side effects. However, the green juice component of barley green leaves of the present invention has a practical anticancer effect, is substantially non-toxic, and can be administered in large doses. It has been found that this is an extremely unique anticancer drug that has both the following properties:

Moreover, a decrease in the weight of the liver spleen was observed in the results of a test for experimental liver damage using rats, and even during this time, it is an anticancer agent with expected medicinal efficacy, and it also supports the previous proposal of the present inventors. Not only the powder of the green juice component of the green leaves of barley, the six-time dissolution product, but also the supernatant liquid obtained by removing the insoluble part from the redissolved product, and the hydroalcoholic extract of the powder, etc. It was found that the green juice components of green leaves can be used advantageously.

Therefore, an object of the present invention is to provide a unique, substantially non-toxic anticancer agent that has a practical anticancer effect and can be taken for a long period of time and administered in large quantities.

The above objects as well as many other objects and advantages of the present invention will become more apparent from the following description.

The anticancer agent of the present invention can be used to treat cancer including ulcerated tumors and the preceding 1
It should be understood that it is useful for the prevention and treatment of prodromal symptoms, and that this is what is meant by the term anticancer agent in the present invention.

It is also expected to be useful for liver disorders, and together with its substantially non-toxic advantage, it can be said to be an extremely unique anticancer agent.

The active ingredient in the agent of the present invention is a green juice component of green leaves of barley before the ripening stage. Obtainable.

Green leaves of naturally sourced wheat before the ripening stage, preferably from the beginning of tillering to the heading stage, such as barley, naked wheat, oats, and even green leaves of pigeon barley (a generic term for stems and leaves) is squeezed, preferably by mechanical means, without undue thermal denaturation, coarse solids are removed to obtain a green juice, preferably further centrifuged, the supernatant liquid is collected, and this is It can be used after being sterilized and filtered.

Prior to the above-mentioned juice extraction, it is preferable to sterilize the green leaves with a disinfectant such as sodium hypochlorite and then perform the juice extraction treatment.

In addition, a green juice component obtained by neutralizing the green juice obtained as described above to about pH 5 to 9 and pulverizing it by a method that does not cause substantial thermal denaturation, such as spray drying or freeze drying, can also be preferably used.

In the present invention, the green juice component of the green leaves of barley before the ripening stage obtained in this way is used, but in addition to the powder, the powder may be further dissolved in water, or the green juice component in the redissolved product may be used. A solution from which insoluble matter has been removed may be used, or a solution obtained by extracting the powder with an aqueous alcohol, such as a 50% aqueous methanol solution, or a powder thereof may also be used.

In any case, it is best to use green juice made from fresh green leaves of wheat without subjecting it to undue heat history or chemical denaturation.

If you wish, you can use the squeezed green juice component-containing liquid as it is, or
For example, milk, skim milk, other colloidal protein-containing substances, sweeteners, etc. can be mixed and administered orally, but the above spray-dried or freeze-dried products of constant quality and good stability, and furthermore, the redissolved products thereof It is preferable to use a liquid or an extract.

The anticancer agent of the present invention may contain various additives, if desired, and may be in various dosage forms.

Such additives include additives for freeze drying or spray drying as well as pharmaceutical additives for forming the desired dosage form.

Examples of these additives include ascorbic acid,
Nutrients such as biotin, calcium pantothenate, carotene, choline chloride, magnesium oxide, niacin, pyridoxine chloride, riboflavin, sodium pantothenate, thiamine hydrochloride, tocopherol, vitamin A1 vitamin B12, vitamin D2, etc.; sodium metaphosphate, phosphoric acid Sodium (primary, secondary, tertiary salt)
, sodium pyrophosphate, sodium tripophosphate, etc.; preservatives such as calcium chloride, benzoic acid, methyl parahydroxybenzoate, sodium benzoate, etc.;
Gum arabic, tragacanth, sodium alginate, methylcellulose, carboxymethylcellulose, calcium alginate, aluminum silicate, calcium silicate, mannitol, sorbitol, lactose, fructose, soluble starch, amino acids, glucose, sugar, honey, sucrose, fatty acid ester Other additives and diluents can be mentioned.

The anticancer agent of the present invention is most commonly administered orally,
Therefore, it can be in any dosage form suitable for oral administration, such as powders, granules, pellets, tablets, coatings, capsules, liquid tablets, or any other oral dosage form.

Furthermore, depending on the location of the affected area, a crude drug type or an external preparation type can also be adopted.

The dose is approximately 200 mI? /kg/day or more is normal, and can be appropriately selected depending on the type and severity of the symptoms.

Generally about 2001n9/kg/day to about 30001n9/kg/day
nI? An example of a dosage range is approximately 1 kg/day.

If desired, the agent of the present invention can be administered in large amounts since it is substantially non-toxic and causes no side effects.

(1,1 The active ingredient of the present invention, the green juice component of green leaves of barley before the ripening stage, has a strong effect on amino acid and protein combustion products that are known to have strong mutagenic activity or factors. It was discovered that it exhibits the unique effect of significantly reducing its mutagenic activity.

A test will be described using a t-t')butophane combustion material as a representative example, which is said to have the highest mutagenic activity.

The test was carried out by centrifuging a green juice of barley grass and an aqueous suspension of the spray-dried powder of the green juice, and sterilizing the supernatant through filtration.
A test sample of the species was used.

The details are as follows.

Sample A1: Young barley leaves (starting stage of panicle formation, plant height 15-25CrrL)
Approximately 500 kg of leaves and stems were collected, thoroughly washed with water and drained, and then squeezed using a grinding type juicer.The obtained juice was centrifuged in a perforated wall centrifuge covered with F cloth. Separation treatment was carried out to remove coarse solids mainly consisting of fibrous material, yielding Juice 4004 of young barley leaves.

A portion of the obtained green juice was taken and centrifuged for 20 minutes.
After centrifugation at 1,000 G for 15 minutes, the supernatant was collected.

This supernatant liquid was passed through a Millipore filter to remove germs, and the P solution was used as a green juice sample of young barley leaves (sample Al).

Sample A2 The remaining portion of the above-mentioned green juice was adjusted to pH approximately 7 with soda carbonate, and then spray-dried using a disc-type spray dryer to obtain 20 kg of spray-dried powder.

Take a portion of this powder, suspend it in 50mM HEPES buffer to a concentration of approximately 20% w/v, centrifuge it using a centrifuge at 7.0OOG for 20 minutes, and then collect the supernatant. was collected.

This supernatant liquid was passed through a Millipore filter to remove bacteria, and the P solution was used as a spray-dried powder liquid sample of barley grass green juice (sample! 2).

As a mutant system, Salmonella enterica TA of Ames et al.
A test was conducted using -98 cells using histidine (his+) reversion mutation as an indicator.

Homocyne-Bs-9m in PCB-induced rat liver
ix) was used for metabolic activation of mutagens in vitro.

The procedure was to mix the t-to'J butophane combustion mutagen (Try-P) and the sample, react with shaking at 37°C for 60 minutes, heat treatment at 100°C for 10 minutes, and after cooling. , mix a suspension of S-9m1x and TA-98 cells in a soft agar tube, pour this into a selective planar medium,
After incubation at 37° C. for 2 days, the number of reverted mutation colonies was examined.

As a result of the above test, both of the above two test samples showed that the mutagen (Tr) compared to the control culture.
It was observed that the mutagenic activity of y-P) was significantly reduced.

The results are shown in Table 1 below.

0.3m1oTry-P; 100 μg/plate In addition, more potent mutagenic products, Tri-P-1 and Tr, separated and purified from Try-P
In a test conducted in the same manner as above except for using y-P-2, the agent of the present invention showed an excellent mutagenic activity suppressing effect.

The results are shown in Table 1 (continued) below.

The active ingredient of the present invention, the green juice component of the green leaves of barley before the ripening stage, has also been shown to inhibit the proliferation of murine leukemia P388 cancer cells in vitro at a level of 50. % inhibitory concentration (IC,.

) was discovered to exhibit the activity of inhibiting.

The tests conducted using the following test samples will be described.

Sample (A) Spray-dried powder of green juice of barley grass obtained from Sample A2 of [0] above.

Sample [F]) Spray-dried powder of green juice of young barley leaves obtained in the same manner as Sample A2 of CI) except that young barley grass leaves (plant height 20 to 30 CIfL) were used.

Sample (0-knee bare wheat grass (starting stage of panicle formation, plant height 15-25CrrL)
Spray-dried powder of green juice of naked wheat grass obtained in the same manner as Sample 2 of CI) above, except that .

The test consisted of the following standard culture solution composition: Commercially available synthetic medium RTMI・1640 5.25g (Nippon Suisan Nippon Co., Ltd. product) Calf serum (Cotyledon Serum Institute product) 751111 Kanamycin sulfate 50ml (Meiji Seika Co., Ltd. product) Sodium bicarbonate 0.625 g (
Wako Pure Chemical Industries, Ltd. product) was brought to a total volume of 500 TLl with double-distilled water, 10 μmol of mercaptoethanol was added, and the pH at the start of culture was neutral (approximately 6.8 = by blowing carbon dioxide into the above solution, RTMI.16
Neutralize the indicator phenol red contained in 40 until the red color changes to yellow, indicating neutrality.

), and the culture temperature was 37°C.

The above standard medium and the above sample A in the standard medium.

B and C are respectively 40γ and 2 Or (μji/mi
), and added mouse leukemia P388 cancer cells to a total of 4 types of standard medium and sample-added medium, and 7 types of sample-added medium, 2 types of each concentration above. 1.0X10 per 1rrLl of medium
The cells were inoculated at 5 cells and cultured at 37°C for 72 hours.

For culture, the culture medium was inoculated into a rectangular culture bottle made of hard glass with a height of about 8 crrL and a capacity of 50 m1, and then the bottle was sealed with a rubber stopper and cultured on static soil.

After 72 hours, the increased number of P388 cancer cells was counted using a Thoma hemocytometer under a light microscope.

The growth rate of P388 cancer cells in standard medium was reduced to 10
0%, and the concentration at which the proliferation rate in the culture medium supplemented with Samples A, B, and C is 50%, that is, the 50% inhibitory concentration (
IC50) was as shown in Table 1 below.

The results in Table 1 below are the average values of each triplicate test.

Table 1 50% inhibitory concentration (IC50) against P388 cancer cells
Sample A-supplemented medium: 24γ (μg/ml) Sample B-supplemented medium: 25γ Sample C-supplemented medium: 27γ As shown in the results of the above in vitro test, the active ingredient of the present invention has a concentration of approximately 30γ, which is determined to be effective in the above test. Indicates an IC5° value below the order of magnitude.

The active ingredient of the present invention, the green juice component of the green leaves of barley before the ripening stage, was found to be effective in animal tests using P388 cancer cells. Group median survival day value) is, for example, 400 m97 kg.
/day was found to be 127% effective.

The test is CDF, mouse [i.e. (DBA/z
Ba1b/c) F17 mice], P388 cancer cells 1.
Inoculate 0x106.

From the day after vaccination, 9 times a day, 400~/kg
A physiological saline suspension of sample B was administered intraperitoneally to the sample at a dose of /day, and the median survival day value (MST: me
T/C was calculated from the MST of a control group of 6 animals per group to which no drug was administered.

As a result, the MST of the sample B administration group was 14 days, and the MST of the control group was 14 days.
ST was 11 days, T/C was 127%, and it was judged to be effective as an anticancer effect.

It has been discovered that the active ingredient of the present invention, a green juice component of green leaves of barley before the ripening stage, also has an anti-ulcer effect.

For example, a remarkable anti-ulcer effect was observed against experimental gastric ulcers in rats induced by ligation, and a decrease in the weight of the liver and spleen was also observed in experimental liver damage in rats induced by administration of carbon tetrachloride. Ta.

An example of pharmacological effects on experimental gastric ulcers will be described below.

The test was conducted according to the method of Shay using 5 to 8 male Wistar rats of 6 weeks old (body weight 170±10 g) per group.

That is, after fasting the test rats for 48 hours, the junction between the stomach pylorus and duodenum was ligated under ether anesthesia, and each rat was housed in a metal wire mesh cage.

Thereafter, the animal was left without food for 16 hours, and then the abdomen was opened under ether anesthesia, the esophagus just above the cardia was ligated, and the stomach was separated.

After removing the stomach contents, an incision was made along the dog's side and the state of the ulcer was observed.

Maeyama's Sample A was used as a physiological saline suspension as a test drug, and the dosage of Sample A was 200,400°800rnIi.
Three dose groups of I/kg were tested.

Separately, the control group received only physiological saline, each weighing 100 g.
Adjust the dose to 0.5 ml per dose, and perform pylorus ligation at 30 mL.
After 1 minute, the drug was orally administered once through a gastric probe.

During the fast, drinking water (tap water) was available ad libitum.

The test results were as shown in the table below.

In the above test, gastric juice volume, pH 1 free hydrochloric acid, and pepsin were also measured, and the differences in these in the administration group were minimal compared to the control group, and no significant changes were observed.However, regarding ulcer formation, As shown in Table 2 above, the inhibition of ulcer formation was strongest in the 800 my1kg group, and changes in the gastric mucosa were mild. Significantly suppresses the occurrence of 5o
In oTn9/ky, there was a tendency for the occurrence of both large and small ulcers to be significantly reduced.

As shown above to give a few examples, the anticancer agent of the present invention containing as an active ingredient the green juice component of the green leaves of barley before the ripening stage exhibits excellent medicinal efficacy against cancer and its prodromal symptoms. It can be said to be an extremely unique anticancer agent, since it is substantially non-toxic and has low toxicity and side effects, which were difficult to achieve with the drug efficacy of conventionally proposed drugs of this type.

Thus, according to the invention, the gastrointestinal system, blood, lungs, liver,
A unique anticancer agent is provided that exhibits excellent medicinal efficacy against cancer or its precursor symptoms in the mammary gland, uterus, etc., and is extremely nontoxic and has no side effects.

Claims (1)

[Claims] 1. An anticancer agent characterized by containing as an active ingredient a green juice component of green leaves of barley before the ripening stage.