JP2012092081A - Process for producing melia azedarach extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a process for producing a Melia azedarach extract which can thoroughly extract the water-soluble/non-water-soluble active ingredients included therein, the Melia azedarach extract enabling expectation as the conventional drug as a matter of course and expectation of an effect of preventing/treating influenza as well.SOLUTION: The process for producing a Melia azedarach extract comprises adding an acid 12 and an alkali 14 to finely cut raw material Melia azedarach 10 to destruct and neutralize fiber cells, then adding a mineral 16 to the destructed fiber cells to subject the mixture to heat sterilization, thereafter adding aspergilli, yeast, citric acid bacteria, lactic acid bacteria, and acetic acid bacteria and a fiber decomposition enzyme 26 to the heat-sterilized raw material Melia azedarach, and maintaining the resulting mixture at 35-40°C to perform fermentation, adding an organic acid 30 having a carboxyl group selected from citric acid, lactic acid, acetic acid, tartaric acid, and malic acid to the obtained fermented Melia azedarach 28, maintaining the resulting mixture at 35-40°C to subject the mixture to fermentation and aging, and furthermore extracting the fermented and aged liquid by bactericidal filtration.

Description

  The present invention relates to a method for producing Sendan extract. More specifically, the active ingredient of Sendan can be efficiently extracted, and not only as a folk medicine / herbal medicine that has been conventionally used, but also has an effect of preventing and treating influenza. The present invention relates to a promising method for producing a sendan extract.

  Sendan is an evergreen or deciduous tree of the family Meridaceae. The leaves of the sendan are odd-numbered wings, the leaflets are symmetrical, needle-shaped, the tip is thin and sharp, and the edges are serrated. The flowers are white and have a scent of honey. The fruits are berries, green to orange-yellow, with one seed in the center. The seeds are wrapped in soft flesh, the color is light gray, the surface is smooth and silky, and the taste is bitter.

And the neem that grows in India grows to 12-15m in height, its leaves are used as an astringent, parasite prevention, antidote, tumor and insect bite medicine, and leaf juice It is also used as an anthelmintic and dermatological drug, and the fruit is effective against urinary illness, elephants, intestinal parasites, etc., and the seed is widely used as an antiemetic, laxative, anthelmintic, etc. Has been.
Also, in Japan, there are the same companions Sendai (Tatsudan) and Tousendan (Tang Tatsudan), and these root barks and bark are called “bitter skin” or “Rinko” in Chinese medicine, The leaves are also used as insecticides.

  Examples of the active ingredient of such sendan include various essential oil components. For example, in the case of azadirachtin, which is abundant in seeds, the dried seeds are crushed into a coarse powder, It is used by extracting the powder with various solvents such as methanol, ethanol, methylene chloride, chloroform and hexane.

  However, since the seeds of Sendang have a high water content and the active ingredient and water have the same solubility, the solvent for extracting the active ingredient will also extract the water contained in it. The high concentration of water, which is a factor of decomposition of the active ingredient in the solution, has a problem to be solved in terms of storage stability of the preparation.

  Therefore, the ground seed of sendan is extracted with a solvent such as hexane to prepare an extract solution of an active ingredient containing water, and then the synthetic zeolite is formed by forming an infinite number of 3 to 4 pores by removing crystal water. A proposal has been made to reduce the water content by adding / contacting a separating agent or adsorbent to this extraction solution (Patent Document 1), but the amount of separating agent or adsorbent added is the active ingredient. A new problem has been pointed out that it is troublesome because it needs to be adjusted according to the initial amount of water in the extraction solution containing.

By the way, influenza viruses that are widely distributed in animals such as humans, horses, pigs, and birds have caused great damage since ancient times.
In particular, A-type viruses with roots in birds are deeply rooted in the human territory and are prevalent worldwide, especially in the winter, and the damage is immeasurable.
In the avian world, the harm is no exception. For example, the epidemic of the H5 avian influenza in the chicken region has resulted in significant economic losses.
Furthermore, the appearance of this virus in the human world has become a great fear, and not only such type A viruses but also type B viruses are prevalent every year in the human domain.

Virus infections in the human field are the basic strategy for vaccine prevention rather than treatment. Recently, in addition to vaccine prevention methods, treatments such as Tamiflu, Amandadine, and Rimandadine are also used. Although it has become possible to do so, it has not been able to be used as a useful therapeutic agent in reality because of the rapid emergence of resistant viruses against these drugs.
In addition, since an effective influenza preventive drug has not yet been developed in the world today, the development of a preventive or anti-influenza substance effective for human and avian influenza, as well as B influenza is awaited.

In such a situation, according to the latest research etc., it has been proposed that the active ingredient of Sendan is effective as a preventive anti-influenza substance (Patent Document 2).
The prophylactic anti-influenza substance derived from Sendan according to this proposal is obtained by extracting the raw Senden with hot water of about 60 ° C. and then purifying it with a filtration membrane. It is not intended for use.

JP-A-3-38505 Japanese Patent No. 4307659

  Therefore, in the present invention, regardless of whether it is water-soluble or water-insoluble, it is possible to extract all the active ingredients of Sendan, and as a folk medicine / herbal medicine that has been used in the past, the effect of preventing and treating influenza is sufficient. An object of the present invention is to provide a method for producing a sendan extract that can be expected.

  By the way, the present inventor has been practiced for a long time in Japan, focusing on the fermentation technology of miso with origami and soy sauce for safety, and pulverizing and mixing raw materials such as starch and / or grains and seeds and eggshell. Later, we developed a method to efficiently extract and dissociate mineral components contained in raw materials by hydrolyzing and fermenting and ripening with koji mold, and the ionized mineral-containing liquid obtained by this method is The water molecular population is subdivided by adding only a small amount to normal water or the target material, and as a result, a single population of water molecules surrounds and disperses mineral ions, and together with the subdivided water, calcium and It is confirmed that not only the absorption of magnesium and other mineral ions can be dramatically improved, but also the effectiveness of ingredients in raw materials can be improved. It has been.

  Therefore, in the present invention, by adopting a procedure for fermenting raw material sendan added with minerals using koji mold and fiber-degrading enzyme and the like, ripening by adding organic acid to the obtained fermentation sendan, and further sterilizing and filtering , Trying to achieve the intended purpose.

  Specifically, it is preferable to add acid and alkali to the chopped raw material sendan to destroy and neutralize the fiber cells, and then add 3% to 5% by weight of mineral of this raw material sendan and then heat By sterilizing and then heat-sterilized raw material sendan, koji mold, yeast, citric acid bacteria, lactic acid bacteria, acetic acid bacteria alone or a mixture of two or more of them and fiber-degrading enzyme are added and kept at 35-40 ° C. Fermentation is carried out, and an organic acid having a carboxyl group selected from citric acid, lactic acid, acetic acid, tartaric acid and malic acid alone or a mixture of two or more thereof is added to 35 to 40 ° C. A procedure for holding and aging the fermentation and further sterilizing and extracting the obtained fermentation aging solution is adopted.

  In this case, it is preferable to use an ionized mineral as a mineral. Specifically, a raw material obtained by mixing starch and / or cereals, seeds and eggshells at a predetermined ratio is pulverized, and then purified water is added thereto. Fermentation ripening liquid obtained by filtering after dissociating minerals such as calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, etc. contained in the raw material by adding and koji mold and fermentation aging Is to use.

  The present invention also includes the sendan extract itself obtained by the above-mentioned method / procedure.

  According to the method of the present invention, the water-soluble and water-insoluble active ingredients contained in Sendan can be extracted without leaving a simple procedure, so that they can be used efficiently and effectively as conventional folk medicines and herbal medicines. Is something that can be done.

  In addition, as can be seen from the test results, the obtained sendan extract can achieve inactivation of influenza virus, so that it can be used as an inactivating agent for influenza virus, including for prevention, as well as for dilute solutions. It can be preferably used. Specifically, sprays for prevention against influenza that invades from the nasal cavity, sprays for masks, gargles for prevention of influenza, sprays for prevention of influenza for home use, large scales such as companies and hospitals, stations and poultry farms It can be used as a disinfectant in facilities.

It is a schematic procedure explanatory drawing which shows suitable embodiment of the manufacturing method of the sendan extract which concerns on this invention.

Next, a preferred embodiment of a method for producing a sendan extract according to the present invention will be described in detail below.
In FIG. 1, in the method for producing a sendan extract according to the present invention, first, a dried sendan 10 is prepared as a raw material, and this dried sendan is finely cut using a known cutting machine or the like, and then an acid 12, for example, Hydrochloric acid (HCL) and alkali 14, such as caustic soda (NaOH), are added and mixed and stirred to destroy and neutralize the fiber cells of the raw senden 10.
In this case, the ratio of the raw material sentane, acid and alkali is preferably set to 1.0: 1.1: 1.0 by weight.
In addition, as a raw material for the sendan, dried leaves, stems, branches, bark, root bark, seeds, seeds, etc. of the plant family can be used as appropriate, and the shape and size thereof are not particularly limited. Even some crushed material can be used without any problem.

Next, sterilization treatment is performed by adding 3% to 5% by weight, preferably 4% by weight of mineral 16 to the raw material sendan 10 in which the fiber cells have been destroyed in this way, and then heating at 95 ° C. for about 20 minutes. I do.
In this case, as the mineral 16, for example, a raw material obtained by mixing starch and / or cereals, seeds and eggshells at a predetermined ratio is pulverized, and then purified water and koji molds are added thereto, followed by fermentation and aging. Use ionized mineral 20 consisting of fermentation aging liquid obtained by filtering after dissociating minerals such as calcium, magnesium, sodium, potassium, iron, copper, zinc, and manganese contained therein. Can do.
Moreover, if the mineral 16 added to the raw material sendan 10 is 3% by weight or less, it will be difficult to achieve sufficient elution of the active ingredient, and if it is added 5% by weight or more, there will be a problem in terms of cost effectiveness. .

  Such treatment of the raw sendan 10 not only sterilizes the sendan but also suitably dissolves water-soluble active ingredients contained in the sendan, potassium, manganese, some calcium, and water-soluble dietary fiber. It is something that can be done.

When the temperature of the sterilized raw material sendan 10 is lowered to about 30 ° C. to 35 ° C., koji molds such as sake lees, soy sauce koji, miso, etc., yeast, citric acid bacteria, lactic acid bacteria, acetic acid The treated Sendan 22 is fermented by adding the fungi alone or a mixture of two or more of these and a fiber-degrading enzyme 26 such as cellulase and holding at 35 ° C to 40 ° C.
In this case, it is preferable that the koji mold, yeast, citric acid bacteria, lactic acid bacteria, acetic acid bacteria, or a mixture 24 of these two or more and the fiber-degrading enzyme 26 are appropriately adjusted according to the fermentation status of the treated sendan 22, The amount is preferably set in the range of 10% by weight to 20% by weight.
Note that when the amount of the koji mold or the mixture 24 such as koji mold and the fiber-degrading enzyme 26 is less than 10% by weight, not only the fermentation cannot be performed sufficiently, but also the fermentation takes a long time. There are too many, and economic efficiency will fall.
In addition, when the koji mold or the koji mold mixture 24 is added, approximately the same amount (10 wt% to 20 wt%) of sugar is added to the koji mixture.
Furthermore, the fiber-degrading enzyme 26 can be replaced with, for example, a koji mold containing cellulase.

Next, a predetermined amount of an organic acid solution 30 having a carboxyl group selected from, for example, citric acid, lactic acid, acetic acid, tartaric acid and malic acid is added to the fermented senda 28 thus obtained, and 35 ° C. by a heater. Various active ingredients contained in Sendan as a raw material, matured by carrying out electrolytic magnetization treatment and solar light irradiation while slowly stirring in an electrostatic magnetic field and far-infrared irradiation atmosphere while being kept at ˜40 ° C. Is eluted and fermented (ionized).
In this case, the amount of the organic acid solution 30 added to the fermentation senda 28 is preferably set to 2 to 10 times the amount of the fermentation raw material (fermentation sendan 28), and the organic acid solution 30 is used as the fermentation raw material (fermentation sendan). If the amount is less than twice the amount of), a long time is required for fermentation and ripening. If the amount exceeds 10 times, the amount is too large and the aging cannot be performed well, resulting in poor economic efficiency and a strong acid odor.
Further, in the present embodiment, the organic acid solution 30 is added to the obtained fermentation raw material 28. Alternatively, a cell solution in which acetic acid bacteria, lactic acid bacteria, etc. are cultured may be added. Although it depends on the number, it is preferable that the content is about 10% to 20% by weight of the fermentation raw material.

Then, the fermentation ripening liquid thus obtained is sterilized again by means such as heating or ultraviolet irradiation, allowed to stand for 3 to 4 weeks, and then subjected to microfiltration to extract Sendan mineral extract 32.
Sendan mineral extract 32 obtained in this way includes minerals such as water-soluble fiber, calcium, magnesium, potassium and sodium, as well as trace minerals such as iron and zinc, as well as azadirachtin, which is inherent to Sendan. It is prepared as a “whole extract” containing abundant active ingredients such as essential oil components.
In addition, although the residue after extracting the Sendang mineral extract 32 was left at room temperature for about 6 months, there was no inconvenience such as spoilage even though it was exposed to various aerial bacteria.

Absorbency test

Next, the absorbability of Sendan mineral extract 32 obtained in this way and conventional Sendan extract was compared.
Specifically, first, 210 cc of pure water (water from which chlorine and minerals have been removed from tap water as much as possible using a reverse osmosis membrane as much as possible) was added to 100 g of the cut raw material sendan and then heated at 95 ° C. by an appropriate heating means. A comparative hot water extraction Sendan extract A is prepared by heating for about a minute and then naturally cooling it.
On the other hand, tasteless odorless test water (pure water) prepared by removing chlorine and minerals from tap water in advance as much as possible and using a reverse osmosis membrane is prepared.

And first, when the electrical conductivity (electrical conductivity) of the test water (pure) was measured using a water quality tester (manufactured by Hanna Instruments Japan Co., Ltd.), it was 0.49 μS / cm (20 ° C.). there were.
Next, 100.0 cc of the test water is prepared in each of the two containers X and Y, and 1.0 cc of the sendan mineral extract 32 obtained by the method of the present invention is added to the test water of the container X and the container Y Similarly, 1.0 cc of hot water extraction Sendan extract A was added to the test water, and each was stirred and allowed to stand for 30 minutes, and then the conductivity at 20 ° C. of the test water in each container X and Y was measured.
As a result, the conductivity of water in the container X in which the sendan mineral extract 32 was dissolved showed 1640 μS / cm, whereas the conductivity of water in the container Y to which the hot water extraction sendan extract A was added was 20.4 μS / cm. Met.

That is, the conductivity of the water (Y water) to which the hot water extraction Sendan extract A was added was only about 40 times higher than the conductivity (0.49 μS / cm) of the original test water (pure water). On the other hand, the conductivity of water added to Sendan Mineral Extract 32 obtained by the method of the present invention (water in container X) increases to more than 3300 times the conductivity of the original test water (pure water). Yes.
This fact indicates that the Sendan Mineral Extract 32 dissolved in the water of the container X is water-soluble and water-insoluble, including minerals such as calcium, magnesium, potassium, sodium, iron, zinc and other trace minerals. The various active ingredients are dissolved and dissociated (ionized) in a much larger amount than the hot water extraction sendan dissolved in the water of the container Y through the fermentation process and the aging process.

  Further, comparing the electrical conductivity of the water in the container Y in which the hot water extraction Sendan extract A is dissolved and the electrical conductivity in the container X to which the Sendan mineral extract 32 is added, the water in the container X is the water in the container Y. The numerical value of 80 times or more is shown. Therefore, it is clear that the absorbability of the Sendan mineral extract 32 is remarkably improved.

Inactivation test

  Next, an inactivation test for influenza virus of Sendan mineral extract 32 prepared by the above-described manufacturing method was performed (Japan Food Analysis Center).

  The outline of this test is that the Sendan Mineral Extract (specimen) stock solution and the sample diluted solution diluted with purified water added to this stock solution are used as test solutions, and the virus suspension of influenza virus is added to and mixed with these test solutions. The virus infectivity titer of the working fluid at room temperature after a predetermined time is measured. It was confirmed by a preliminary test that the virus infection titer can be measured without being affected by the specimen by diluting the diluted solution 10 times).

[1] Preparation of virus suspension (1) Cell growth medium (Nissui Pharmaceutical Co., Ltd. Eagle MEN medium plus 10% fetal calf serum) used cells [Dainippon Pharmaceutical Co., Ltd .; MDCK (NBL-2) cell ATCCCCL-34] was cultured in a monolayer in a flask for tissue culture. (2) After the monolayer culture, the cell growth medium was removed from the flask, and the test virus [influenza virus type A (HINI)] was removed. Vaccinated.
Next, a cell maintenance medium was added and cultured in a carbon dioxide incubator (CO2 concentration: 5%) at 37 ° C. ± 1 ° C. for 1 to 5 days.
Composition of cell maintenance medium Eagle MEN medium (Nissui) 1000ml
14% 10% NaHCO3
L-glutamine (30 g / l) 9.8 ml
100 x MEN vitamin solution 30ml
20% 10% albumin
0.25% trypsin 20ml
(3) After culturing, the morphology of the cells was observed using an inverted phase contrast microscope, and it was confirmed that morphological changes (cytopathic effect) occurred in the cells. Next, the culture solution was centrifuged (3000 rpm / min. 10 minutes), and the resulting supernatant was used as a virus suspension.

[2] Test operation A sample stock solution (Sendan mineral extract) and a sample dilution solution (10-fold dilution of a sample prepared using purified water as the sample stock solution) were used as test solutions.
0.1 ml of the virus suspension was added to 1 ml of the test solution and mixed to obtain a working solution.
It was allowed to act at room temperature and diluted with cell maintenance medium after 1 minute, 5 minutes, and 15 minutes (however, the working solution was diluted at a concentration confirmed by a preliminary test).
Purified water was tested in the same manner as a control, and measurement was performed at the start and after 15 minutes.

[3] Measurement of virus infectivity titer Using cell growth medium, the used cells were cultured in a monolayer in a tissue culture microplate (98 holes), then the cell growth medium was removed, and 0.1 ml of cell maintenance medium was added. . Next, 0.1 ml of the working solution dilution was inoculated into each of four wells and cultured in a carbon dioxide incubator (CO2 concentration: 5%) at 37 ° C. ± 1 ° C. for 4-7 days.
After culturing, the presence or absence of cell morphological changes (cytopathic effect) was observed using an inverted phase contrast microscope, and the 50% tissue culture infectious dose (TCID50) was calculated by the Reed-Muench method to determine the virus per ml of working solution. When converted into the infectivity value, the measurement results of the virus infectivity value of the working fluid were as shown in Table 1.

From the results shown in Table 1, it was confirmed that the anti-influenza virus effect is extremely significant even if Sendan mineral extract 32 is not only a stock solution but also a diluted solution.
While the benefits of the availability of influenza treatments such as vaccines and Tamiflu to date are certainly a major advance, stopping infections in advance is still more important. In this sense, considering the above findings as a basis, it is strongly suggested that the Sendan mineral extract 32 of the present invention containing an active ingredient of Sendan has the ability to inactivate influenza viruses. It is speculated that the invading virus can be inactivated directly, and can be used to develop various types of influenza preventive drugs.
Specifically, it can be used as a propellant for preventing influenza that invades from the nasal cavity, a spray for masks, a gargle for preventing influenza, and the like.
In addition, there is a good possibility that it can be used as a spray for home influenza prevention, as a disinfectant in a large-scale facility such as a company or hospital, or a station or poultry farm. As an inactivating substance, its utility value is extremely high.

  In addition, when using the Sendan mineral extract 32 of the present invention as an influenza preventive / therapeutic agent, in addition to the Sendan mineral extract, various pharmaceutically acceptable substances for preparation, such as excipients, diluents, and disintegrants Needless to say, binders, coating agents, lubricants, lubricants, lubricants, flavors, sweeteners, solubilizers and the like can be included as auxiliary agents. Specific examples include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol, glycerol and the like.

Since the Sendan mineral extract obtained by the method of the present invention can be diluted with water and used for drinking, it can also be used as an anti-atopic substance by applying a stock solution or an appropriately diluted Sendan mineral extract.
Moreover, since it is a natural substance, it can be used as a cosmetic material.

10. Sendan (raw material)
12 ·· Acid 14 · · Alkali 16 · · Mineral 20 · · Ionized mineral 22 · · Treated Sendan 24 · · Mixture 26 of Aspergillus or Neisseria gonorrhoeae · · Fiber-degrading enzyme 28 · · Fermentation Sendan 30 · · Organic acid solution 32 ..Sendan mineral extract

Claims (4)

  The raw material sterilized by adding acid and alkali to the chopped raw material sendan to destroy and neutralize the fiber cells, then adding 3 wt% to 5 wt% mineral of this raw material sendan and then heat sterilizing Fermentation sendan obtained by fermenting koji mold, yeast, citric acid bacteria, lactic acid bacteria, and acetic acid bacteria to sendan alone or by adding a mixture of two or more of these and fiber-degrading enzyme and holding at 35 ° C to 40 ° C In addition, an organic acid having a carboxyl group selected from citric acid, lactic acid, acetic acid, tartaric acid, malic acid alone or a mixture of two or more of these is added and held at 35 ° C. to 40 ° C. for fermentation aging, A method for producing a sendan extract, which comprises subjecting the obtained fermentation ripening liquid to sterilization filtration and extraction. The method for producing a sendan extract according to claim 1, wherein an ionized mineral is used as the mineral. The ionized mineral is calcium contained in the raw material by pulverizing the raw material in which starch and / or cereal, seeds and eggshell are mixed at a predetermined ratio, and then fermenting and ripening with purified water and koji mold. The manufacturing method of the sendan extract of Claim 2 which consists of fermentation aging liquid obtained by dissociating minerals, such as magnesium, sodium, potassium, iron, copper, zinc, manganese, and filtering. A sendan extract obtained by the method according to claim 1.

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