JP2001106635A - Anti-rotavirus agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-rotavirus active substance which is derived from a natural product and hardly exhibits adverse drug reactions and to prevent or treat infectious diseases of human infants and infants of animals for industrial use, pets or the like. SOLUTION: The extract from stevia plant bodies, mainly stems and leaves, inhibits the adhesion or invasion of rotavirus into the cell and exhibits resistance to gastric acid. The active ingredient contained in the extract is present in a fraction obtained by eluting the extract with water by the use of ODS column and then eluting the resultant eluate with 20-70% ethanol. The ingredient is an acidic polysaccharide with a molecular weight of 20,000-80,000.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an anti-rotavirus agent comprising a hot water extract of stevia leaves and stems.

[0002]

2. Description of the Related Art Most humans (about 90%) are infected with rotavirus in infancy and exhibit symptoms such as vomiting, diarrhea and fever, and 5 to 10% of infected persons are hospitalized for dehydration. It costs. Although the human mortality rate is considered to be very low in Japan, many industrial animals and pets such as monkeys, horses, pigs, cattle, chickens, dogs, cats, rabbits, rats, etc. become infected during infancy, When these animals become infected, mortality is high and industrial losses are high.

[0003]

Although a live attenuated vaccine was approved in the United States in 1998, the treatment was symptomatic, and no effective causal therapy has yet been developed. An object of the present invention is to provide an anti-rotavirus agent derived from food products that are derived from natural products and are also taken by general healthy people for health promotion.

[0004]

The present invention has been completed by finding out that a 220-fold diluted solution of Stevia plant, mainly stem and leaf extracts, inhibits the growth of rotavirus by 50%. The anti-rotavirus activity was determined to be 20 to 7 after elution with water using Stevia extract stock solution and ODS column.
It is found in the fraction eluted with 0% ethanol and is an acidic polysaccharide. This suggests that when rotavirus infects cells, the active ingredient in the stevia extract binds to the virus and inhibits infection.

[0005] The present inventors have screened various substances in search of substances having anti-rotavirus activity.
Stevia extract taken as a health drink, especially this solution is fractionated with water as an extract by YNMC-ODS reverse phase column chromatography, then eluted with 30% ethanol and then 60% ethanol. The present invention has been completed by finding that it has rotavirus activity. The substance of the present invention is selectively effective against rotavirus and has no effect on viruses such as influenza and herpes.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Stevia, which is a raw material of the present invention, is a perennial plant of the family Asteraceae native to South America, Stevia rebaudiana Bertoni and related plants. As a result of the experiment, the active ingredient is contained abundantly in the leaves and stems, especially in the stem before having buds and in the stems of mature plants, but the active ingredient is contained in roots, flowers, and young plants It was confirmed. Stevioside and revadioside, which are known as sweet components of stevia, did not show anti-rotavirus activity. Therefore, the present invention only needs to contain the extract of the whole plant tissue of Stevia, and may be mixed with other substances.

The extraction method is not particularly limited as long as the components contained in the plant tissue of Stevia can be eluted.
Extraction with cold water or extraction with a lower alcohol or the like can also be used. Although the active ingredient cannot be specified, it is considered to be a hydrophilic substance, and thus is extracted with lower alcohols such as methyl alcohol, ethyl alcohol, n-propyl alcohol, and i-propyl alcohol. The active ingredient is assumed to be an acidic polysaccharide having a molecular weight of 20,000 to 80,000.

For extraction, only the leaves are picked, or the aerial part of the plant is cut off, dried or finely ground as it is. Since the stem is hard, it may be cut before pulverization. In order to facilitate the elution of the active ingredient, it is preferable to carry out fine pulverization. The extraction is boiled with hot water for 30 minutes or more. In some cases, when the residue obtained by separating the extraction water obtained in the first extraction step is put into new hot water for extraction and re-extracted, the active ingredient is almost completely extracted. Also, the active ingredient is sufficiently eluted in the supernatant obtained by immersing the fine powder in water at room temperature for 2 to 3 days, and in the supernatant immersed in water at 30 to 50 ° C. for 3 hours. Stevia extract is concentrated to a solid concentration of 15-20.
Used as a stock solution of W / V%. Generally, 0.3 to 3 liters of an undiluted extract is obtained from 1 kg of finely divided stevia.

[0009] The anti-rotavirus activity is measured by MT, which measures cell death and its inhibition associated with virus infection as a color reaction.
This was performed using the T method. That is, living cells reduce yellow tetrazolium to produce reddish purple formazan, and the reddish purple intensity is reduced to 2 at 690 nm and 540 nm.
It is measured by absorbance according to wavelength. The MTT method selects a cell line in which complete cell death occurs due to virus infection, and if an appropriate cell concentration and rotavirus concentration are selected, rotavirus is cultured at 37 ° C for 5 days to kill all cells and to remove tetrazolium. It does not reduce upon addition and remains yellow. That is, a well that does not develop color even after the addition of tetrazolium after completion of culture is a well in which all cells have died or most of the cells have died due to rotavirus or the toxicity of the drug.

[0010] Using a plate in which a number of wells are aligned, a substance to be tested for anti-HIV activity is repeatedly subjected to 5-fold serial dilution.
These are sequentially added to wells arranged and cultured, and the effective dilution concentration and cytotoxic concentration can be calculated. The amount of rotavirus used in the assay is as follows: the number of cells surviving virus infection is one to the amount of cells that should originally grow.
The virus concentration is determined in advance so as to be 0 to 20%, and this amount of virus is used every time. Therefore, it is good conditions to assay so that a maximum of 10-20% survive in wells without antivirals.

[0011]

EXAMPLES Preparation of Stevia Test Solution Stevia seedlings were planted in the spring and the above-ground parts were cut off before having buds. After cutting, it was air-dried and separated into leaves and stems. The leaves are pulverized to 10 μm or less by a pulverizer to produce stevia leaf powder, and the stem is cut to a size of 5 cm.
It was pulverized to 0 μm or less to produce Stevia stem powder. The stevia leaf powder and the stevia stem powder thus obtained were mixed in a ratio of 3: 7
And uniformly mixed to obtain a concentrated raw material of the mixed powder of Stevia.

1 kg of stevia mixed powder was boiled for 1 hour using 10 liters of water. After cooling, pomace was separated by a squeezing machine and boiled down for 3 hours to obtain 1.3 liters of a stevia extract stock solution. When this undiluted extract was put in a container and allowed to stand at room temperature, fermentation proceeded and it was necessary to intermittently release carbon dioxide. The fermentation is carried out violently in the beginning, 3-4
After a month, it gradually decreases, but after one year,
The fermentation continued after more than three years. In this example, a stevia extract stock solution fermented for half a year was used. Therefore, the stevia liquid in this example corresponds to 0.77 g / cm ³ of the raw material stevia mixed powder.

The stevia stock solution obtained by the above method is
Fractionation using YNMC-ODS reverse phase column chromatography using water, fractions 1 to 5 in order from the fraction eluted at the beginning, and then a 30% methanol eluate (fraction 6) and a 60% ethanol eluate (fraction) The anti-rotavirus activity of each of the 7) and 100% ethanol eluate (fraction 8) and the stock solution was measured by MTT cytotoxicity assay. Undiluted solution,
Anti-rotavirus activity was confirmed in the 30% ethanol eluate and the 60% ethanol eluate.

Anti-rotavirus activity test Anti-rotavirus activity was measured using the MTT method. The cell line was MA104 strain (African green monkey cells), and cells were subjected to suspension culture at 37 ° C. for 120 hours using a medium adjusted to contain 10% fetal calf serum (FCS). Stevia extract was repeatedly added to the stock solution by a 5-fold serial dilution method, and the culture was inoculated with 100 PFU of rotavirus Wa strain from the beginning of culture using a microplate. Wells were tested to determine the effective drug concentration. After completion of the culture, the addition of the tetrazolium solution evenly, the color development of red purple formazan measured at absorbance two wavelengths 690nm and 540 nm, 50% inhibitory concentration against viruses (EC ₅₀₎ and cytotoxic concentrations (CC ₅₀₎ Calculated. EC of stevia extract
₅₀ was a 220-fold dilution of the stock solution, and CC ₅₀ was a 105-fold dilution.

For human and animal applications, it must not be inactivated by stomach acid. Therefore, add hydrochloric acid to adjust pH
After being adjusted to 2 and allowed to stand for 30 minutes, neutralization was performed and anti-rotavirus activity was measured, but no decrease in rotavirus activity was observed.

[0016]

EFFECT OF THE INVENTION Since the anti-rotavirus active substance according to the present invention has almost no side effects and has been conventionally taken by humans and animals, it prevents the morbidity and death of rotavirus in humans, industrial animals and pets. Can be used with confidence.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C086 AA01 AA02 EA24 GA17 MA01 MA04 NA14 ZA71 ZB33 4C088 AB26 AC05 BA09 CA05 CA06 CA14 CA25 MA52 NA14 ZA71 ZB33

Claims (3)

[Claims] 1. An anti-rotavirus agent comprising an extract of a Stevia plant and inhibiting the entry of rotavirus into animal cells. 2. The anti-rotavirus agent according to claim 1, which is present in a stevia plant extract in a fraction eluted with 20-70% ethanol after elution with water using an ODS column. 3. The anti-rotavirus agent according to claim 1, which is an acidic polysaccharide having a molecular weight of 20,000 to 80,000.