COMPOSITIONS CONTAINING BUTTERBUR-EXTRACT
Technical Field
The present invention relates to butterbur-extract for protecting brain function and enhancing memory and a method of processing the butterbur-extract. The present invention also relates to pharmaceutical compositions and healthful foods comprising the butterbur-extract, for improving brain function.
Background Art People in a society of a flood of fast information changes live under a stress to absorb much information. The stress induces a decrease of memory due to accelerated aging of brain 'hippocampus'. Human intelligence function means central processes having a definite influence on activities of general life, and includes concentration ability, short term memory (working memory), long term memory, reasoning, motor regulation ability, planning of work etc. Accordingly, the central processes have influence on Quality of Life (QOL) since the central processes are directly related to our everyday mental activity.
Conventional pharmaceutical compositions such as a derivative of γ-amino butyric acid, acetylcholine precursor, receptoragonist, nicotine, etc., for improving brain function and conventional pharmaceutical compositions such as tacrine, aricept (donepezil), etc., preventing decomposition of neurotransmitter for improving human intelligence function were introduced. However, the above-
mentioned compositions have a temporary effect on improving brain function and serious side effects..
Vitamin C or E, β-carotin, seniline and ginkgo biloba extract (Egb 761) may be expected to have an effect on brain function disorder due to a function of preventing oxidation, however more researches need to be done on the effect.
Further, medications for improving human intelligence function and medications for anti-amyloid etc., were developed. The above-mentioned mediations, however, may only have an effect when the medications pass blood brain barrier of neurologic diseases. Research on blood brain barrier of pharmaceutical compositions has not been conducted sufficiently to prove the effects.
Now, a method for solving the problem of blood brain barrier may be a verification of an effect through vivo experiment. Therefore, inventions using the effect of herb that has been used for thousand years such as herb medicines, healthful foods, etc., for treating disease related to brain function may be expected for a development.
Bbutterbur belongs to the family Compositae. Leaves and flowers of butterbur are used for food, and are effective on cough, expectoration, bronchial asthma, stomachic disorder, fever, end organ, vulnus of being bitten by snakes or worms, burn, intoxication (globefish) and alcoholism. However, research on
activity and effect of butterbur has been poor butterbur.
Inventors of the present invention solved the problems of blood brain barrier by verifying an effect of butterbur of herbs without a side effect through vivo experiment, and confirmed that the butterbur has an effect of protecting brain function and enhancing memory.
Disclosure of the Invention Technical problem It is an object of the present invention to provide butterbur-extract for protecting brain function and enhancing memory and a method of processing the butterbur-extract.
It is another object of the present invention to provide pharmaceutical compositions and healthful foods, both of which comprise butterbur-extract for improving brain function.
Technical solution
To accomplish the objects, the present invention provides butterbur-extract for protecting brain function and enhancing memory by passing through blood brain barrier and a method of processing the butterbur-extract.
The present invention also provides pharmaceutical compositions and healthful foods, both of which comprise the above-mentioned butterbur-extract for improving brain function.
Hereinafter, the present invention is described in detail. The present invention provides butterbur-extract for protecting brain function and enhancing memory by passing through blood brain barrier and a method of processing the butterbur-extract.
A butterbur includes Petasites japonicus and Petasites officinalis. Butterbur-extracts include not only a crude extract of butterbur processed by extracting the butterbur with water and alcohol, but also a fraction extract of butterbur processed by extracting the crude extract of butterbur with ethylacetate, eliminating ethylacetate soluble fraction and separating the fraction extract of butterbur from aqueous layer.
The crude extract of butterbur is processed by extracting the butterbur with water and boiling-water-extracting, or extracting the butterbur with alcohol and enfleuraging or heating in low temperature to produce an extract, evaporating the extract under reduced pressure, and drying the evaporated extract. Preferably, alcohol used when the crude extract of butterbur is processed may be methanol or ethanol in a range from about 5% to about 100% by weight, the more preferably ethanol of about 70% by weight. The fraction extract of butterbur is processed by extracting the butterbur with water or alcohol aqueous solution to produce a first extract, extracting the first extract with ethylacetate, eliminating ethylacetate soluble fraction and separating the fraction extract of butterbur from aqueous layer.
The fraction extract of butterbur has an absorption in dark brown resulted from a column chromatography test in which a Rf is in a range from about 0.5 to about 0.8 when a mobile phase mixed in a ratio of bufanol: methanol: ammonium
hydroxide = 3: 2: 1 : 2 is flown in a column and a ultraviolet ray having a wavelength of about 254nm is applied to the fraction extract of butterbur. A dark brown spot is resulted in which a Rfis about 0.6 when the fraction extract of butterbur is soaked in sulphuric acid solution of about 10% by weight and heated at a temperature of about 100°C. Spot is resulted in which a Rfis in a range from about 0.5 to about 0.8 when the fraction extract of butterbur is colored by adding iodide. A negative is resulted from colorimetric reactions of ninhydrin regent and dragendrop.
Further, the fraction extract of butterbur has solution pH of in a range from about 5.0 to about 8. The fraction extract of butterbur has insolubility in hexane and solubility in distilled water, solubility of about 0.1% by weight in methanol and insolubility in hexane, ethylacetate, ethanol, acetone, ether and benzene.
Pharmaceutical compositions for preventing and treating brain function disease are related to change of long term potentitation (LTP), brain derived neurotropic factor (BDNF) at hippocampus and inferior temporal cortex, and activity of TrkB receptor. Deprivation of BDNF damages learning and memory since BDNF is required in memory and learning. Further, CREB (cAMP Responsive Element Binding Protein) is known for protecting brain damage as well as a regulation factor of forming various memories such as space, social learning, etc. The crude extract of butterbur was administrated to rats, the rats was learned by Morris Water Maze and then a result of the crude extract of butterbur on brain function was shown a significant increase of long term potentiation of electro physiologic parametin of memory (refer to FIGS. 1 and 2) and an increase of expression of BDNF and CREB regarded to signaling pathway of memory and learning (refer to FIGS. 3 and 4).
Further, The crude extract of butterbur and the fraction extract of butterbur
were administrated to rats and the result of having influence on brain function showed an effect of enhancing memory (refer to FIG. 5). The fraction extract of butterbur had an effect better than that of the crude extract of butterbur such that an activity content of enhancing memory may be emiched in the fraction extract of butterbur (refer to FIGS. 9 to 11). Further, the crude extract of butterbur was administrated to old rats that were learned by Morris water maze and the result was shown to have enhanced memory (refer to FIGS. 6 to 8).
Therefore, the butterbur-extract may be used on the objects of the young, adults and the old for improving brain function as a medicine-material that had an effect on protecting brain function and enhancing memory by passing through blood brain barrier. In future, the butterbur-extract may be invented as medicines or healthful foods for preventing and treating senile brain disease (amnesia, desanimania, Alzheimer's disease).
Further, the present invention provides the pharmaceutical compositions and the healthful foods, both of which comprise the butterbur-extract, for improving brain function.
The pharmaceutical compositions for improving brain function comprise the butterbur and may be processed by mixing with replenishments, extenders, binders, humectants, diluents such as surfactant etc., excipients etc.. Further, the pharmaceutical compositions may be used as formulations of tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, solutions, etc., for oral administration or as formation of injections processed by a conventional method of dissolving in distilled water for injection.
The healthful foods for improving brain function may be processed as main or sub-materials or by adding the butterbur-extract to chewing gums, noodles such
as Chinese noodles, etc., candy, confectionaris, cereals, bread, tea, drink, etc. Then, stiologically acceptable additive may be used.
The butterbur-extract may be administrated in a dose of about 0.8g to about 3.2g on individual (60kg in weight) by dividing into one to several times, preferably in a dose of about 1.6g to about 3.2g.
According to the results of acute and sub acute toxicity testing, the butterbur- extract was confirmed as a safe material.
Advantageous Effects
The butterbur-extract of the present invention has an effect on protecting brain function and enhancing memory due to passing through blood brain barrier by verifying an effect of butterbur of herbs without a side effect in vivo experiment such that the butterbur-extract may be used for a pharmaceutical material for improving brain function due to solving the problem of blood brain barrier.
Further, the pharmaceutical compositions and the healthful foods comprising the butterbur-extract according to the present invention have an effect on protecting brain function and enhancing memory due to passing through blood brain barrier as known in electro physiologic study related to evaluate degree of induced LTP and influence on Brain Derived Neurotropic Factor (BDNF) expression and camp Responsive Element Binding Protein (CREB) expression such that the pharmaceutical compositions and the healthful foods may improve brain function. The pharmaceutical compositions and the healthful foods may be used on all ages of persons from young to adults and to old for improving brain function, and may be invented as medicines or healthful foods for preventing and treating senile brain disease (amnesia, desanimania, Alzheimer's disease).
Brief Description of the Drawings
FIG. 1 represents degree of induced Long Term Potentiation (LTP) of rat by administrating orally the crude extract of butterbur compared with control. FIG. 2 represents degree of induced Long Term Potentiation (LTP) of rat by administrating feed comprising the crude extract of butterbur compared with control.
FIG. 3 represents influence on Brain Derived Neurotropic Factor (BDNF) expression of inferior temporal cortex by administrating the crude extract of butterbur. FIG. 4 represents influence on cAMP Responsive ElementBinding Protein
(CREB) expression of hippocampus by administrating the crude extract of butterbur.
FIG. 5 represents a result of Morris Water Maze learning by administrating drink comprising the crude extract of butterbur into a normal rat.
FIG. 6 represents a result of Morris Water Maze learning by administrating drink comprising the crude extract of butterbur into a normal old rat.
FIG. 7 represents an effect on working memory by administrating the butterbur-extract into a normal old rat.
FIG. 8 represents picture of swimming trajectory by administrating the crude extract of butterbur into a normal old rat. FIG. 9 represents a result of Morris Water Maze learning by administrating drink comprising the crude extract of butterbur into a normal rat.
FIG. 10 represents a result of Morris Water Maze learning by administrating
drink comprising the fraction extract of butterbur into a normal rat.
FIG. 11 represents a passive avoidance test by adrninistrating drink comprising the fraction extract of butterbur into a Mongolian gerbil.
Best Mode For Carrying Out the Invention
The present invention is described in more detail with the following examples, yet they do not limit the scope of the invention.
Example 1: Preparation of a crude extract of butterbur About 1.8 tons of butter were cleaned and cut into a length of about 10cm.
The cut butterbur was dried, and about 98kg of the dried butterbur were obtained. The dried butterbur was subjected to extraction with about 370L of 70% ethanol by weight for about 8 hours at a temperature of about 55°C to about 60°C and then extraction again with an equal amount of 70% ethanol by weight. The extract was subjected to concentration at a reduced pressure using a rotary evaporator such that about 42kg(yield about 42%) of a crude extract of butterbur that contains about 31%(67~68°Brix) by weight of moisture was obtained.
Example 2: Preparation of an fraction extract of butterbur A suspension of a mixture of about 4kg of the crude extract of buterbur prepared by the Example 1 in about 20L of distilled water was prepared, the crude extract of buterbur was extracted two times with about 18L of ethylacetate and then
ethylacetate soluble fraction (about 36L) was eliminated. The separated aqueous layer was subjected to concentration at a reduced pressure at a temperature of about
50°C such that about 2.8kg (yield about 70%) of a fraction extract of butterbur (pH:
6.7) was obtained. The fraction extract of butterbur was wet in silicagel TLC (Merk) and then flowed with mobile phase in a ratio of butanol: methanol: ammonium hydroxide = 3: 2: 1: 2.
The fraction extract of butterbur has an absorption as dark brown resulted in which a Rfis in a range from about 0.5 to about 0.8 when a ultraviolet ray having a wavelength of about 254nm is applied to the fraction extract of butterbur, dark brown spot resulted in which a Rfis about 0.6 when the fraction extract of butterbur is soaked in sulphuric acid solution of about 10% by weight and is heated at a temperature of about 100°C, spot resulted in which a Rfis in a range from about 0.5 to about 0.8 when the fraction extract of butterbur is colored by adding iodide and negative resulted from colorimetric reactions of ninhydrin regent and dragendrop.
Example 3: Change experiment of LTPfLong Term Potentiation) by administrating the butterbur-extract
(1) A change experiment of LTP by administrating the butterbur-extract
A. Experimental material and animal
The crude extract of butterbur prepared by Example 1 was used as an experimental material and normal adult rats (Sparague Dawley rat) were used as an
experimental animal.
B. Experimental method
To observe LTP related to influence on enhancing memory by administrating the butterbur-extract, drink (about 25%(2.5 g/1) by weight) comprising the crude extract of butterbur was administrated to the experimental animal for about 3 weeks.
The experimental animal had learned by Morris Water Maze since in about 4 weeks and the change experiment of LTP in hippocampus was performed in 5 weeks.
The Morris water maze learning was performed by a method that the experimental animal was swam regularly twice in a day for about 5 days so that the rats started from two points of four cardinal points and arrived to a mark of platform.
After administrating urethane of 20% by weight (about 7ml/kg, Sigma,
USA) into abdominal cavity of the experimental animal learned by Morris water maze and anesthetizing, the anesthetized experimental animal was fixed at stereotaxic instrument (Stoelting, USA) and then had an operation of craniotomy.
To evaluate LTP change, a tungsten-microelectrode for recording was located at CAl cell in hippocampus and an electrode for stimulating was located at granulocyte in dentate gyrus. The electrode for stimulating was located at Schaffer collateral route and the electrode for recording was located at dendrite part in CAl nerve cell. To induce LTP, spasmodic stimulation was given through the electrode for stimulating and then degree of induced LTP was observed and recorded for 2 hours when LTP was induced.
C. Experimental result
Referring to FIG. 1, a group of administrating drink not comprising the butterbur-extract as control (n=8, n indicates a number of experimental animal) induced an increase of potentiation by about 9.42±13.79% and a group of administrating drink containing the crude extract of butterbur (n=8) induced an increase of potentiation by about 25.10±4.4%. Both of the two groups maintained difference of potentiation significantly (*p<0.05, **p<0.01 vs control) for experimental term.
An arrow ( ) on the FIG. 1 indicated a point of spasmodic stimulation, data represented mean ± standard deviation, and significance was approved by student's t-test. '*' indicated p<0.05, '**' indicated pO.Ol and 'M' indicated the crude extract of butter.
(2) A change experiment of LTP after administrating feed containing the butterbur-extract
A. Experimental material and animal
Feed at a ratio of the following Table 1 was used as an experimental material.
[Table 1]
Rats in Example 1 were used as an experimental animal.
B. Experimental method
To evaluate influence of the crude extract of butterbur prepared by Example 1 on LTP, experimental animal was anesthetized and LTP change in hippocampus was recorded in 5 weeks. An experiment was performed by the method of Example 1.
C. Experimental result
Referring to FIG. 2, after administrating the feed for about 4 weeks, a result recording the LTP change (in about 5 weeks) in hippocampus of the experimental animal represented that a group of administrating normal feed as control (n=8) increased by about 10.55±5.57%, a group of administrating feed comprising soybean oil, wheat germ oil, Vitamin mix, α-tocopherol, etc., and not comprising the crude extract of butterbur prepared by Example l(n=6) increased by about 22.53±3.91%, a group of administrating feed comprising the crude extract of butterbur of table 1 (n=6) increased by about 37.89±7.04%, respectively. LTP induced after stimulatance (TBS) was maintained for greater than or equal to about 2 hours on the group of administrating normal feed and the group of administrating feed comprising the butterbur-extract. The result tended to be same as a result comparing a group of administrating normal drink with a group of administrating drink comprising the crude extract of butterbur.
An arrow ( ) on the FIG. 2 indicated a point of spasmodic stimulation after writing base-line for 10 minutes, data represented mean ± standard deviation, and
significance was approved by student's t-test. '*' indicated p<0.05, '**' indicated p<0.01 and 'M' indicated the crude extract of butter.
Accordingly, the butterbur-extract had an effect on enhancing memory according to the experiment for evaluating LTP change and might be used as a new pharmaceutical material for protecting brain function and enhancing memory.
<Example 4> Influence on creating BDNF and CRED expression of animal brain by administrating the butterbur-extract It was confirmed that administrating the butterbur-extract had an influence on molecular mechanism of activity-dependent regulation of CREB and BDNF expression for learning memory.
A. Experimental material and animal The crude extract of butterbur prepared by Example 1 was used as an experimental material and normal adult rats (Sparague Dawley rat, Wister rat, 180~200g weight) were used as an experimental animal.
B. Experimental method Drink comprising the crude extract of butterbur was administrated (in 0.5%
CMC) to experimental animal for about 3 weeks, LTP data of experimental animal was written and analyzed in about 4 weeks. After LTP was recorded, cerebral cortex
and hippocampus were quickly severed from brain and total RNA was separated from the severed sample and an amount of the RNA was measured with spectro photometer. Further, the RNA was obtained from the sample, a cDNA was synthesized using MMVL reverse transcriptase and degree of BDNF and CREB expressions was respectively measured using the cDNA.
C. Experimental result
As may be seen from FIGS. 3 and 4, a group of administrating the crude extract of butterbur (n=7) had an effect on increasing of BDNF (cortex, refer to FIG. 3) and CREB (hippocampus, refer to FIG. 4) expressions compared with control.
Accordingly, the butterbur-extract of the present invention related to expressions of
BDNF and CREB mRNA such that the butterbur-extract may be used as a new pharmaceutical material for protecting brain function and enhancing memory so as to pass through blood brain barrier.
<Example 5> An effect on enhancing learning ability and memory by administrating the butterbur-extract
A. Experimental material and animal The crude extract of butterbur prepared by Example 1 was used as an experimental material and normal adult rats (Sparague Dawley rat, Wister rat, 180~200g weight) were used as an experimental animal.
B. Experimental method
Drink comprising the crude extract of butterbur (35% (2.5g.l) concentration) was administrated to the experimental animal for about 3 weeks and then the experimental animal was learned by Morris Water Maze (4 trials/day) that was identical to experimental method of the Example 3 after about 4 weeks.
After learning of the experimental animal, after about 24 hours were passed from when the escape time (that is a time for the experimental animal to reach the platform) was within about lOseconds, a probe trial test (that includes measuring and comparing time and distance that a rat stayed inside about 1/4 part or about 25cm of target around) was carried out once. Working memory or short term working memory created when the platform was moved after the first learning was compared and measured, the result was represented in Table 5.
As may be seen from FIG. 5, the crude extract of butterbur had an effect on enhancing learning and working memory. Thus, the butterbur-extract was used as a new pharmaceutical material for enhancing memory by passing through blood brain barrier.
'M' indicated the crude extract of butterbur and 'C indicated control, in Table 5.
<Example 6> Effect of enhancing learning and memory ability on the old rat
The example measured an effect on enhancing learning memory of the old rat in order to know that the butterbur-extract has an effect of enhancing memory not
only on the young but also on the adults and the old.
A. Experimental material and animal
The crude extract of butterbur prepared by Example 1 was used as experimental material and about 22 month-old rats (Sparague Dawley rat, n=16) were used as an experimental animal.
B. Experimental method
Drink comprising the crude extract of butterbur (25%(2.5g/l) concentration) was administrated to the experimental animal and then the prove trial test as the first test in Morris Water Maze learning was performed by the method same as Example
5, working memory was measured and spontaneous locomotor activity was compared with control for about 4 hours after about 24 hours from prove trial test.
C. Experimental result
As may be seen from FIG. 6, significant memory difference was shown (n=7~8, * p<0.05 vs control) in Morris Water Maze learning.
Referring to FIG. 7, working memory performed by prove trial test was also increased by about 1.4 times compared with control. Further, FIG. 8 showed that swimming distance per hour of the experimental animal in platform quadrant was increased significantly. Difference of weight and amount of drinking between two groups was not observed for administration term.
'MH' indicated the crude extract of butterbur in Table 6, 7 and 8.
Therefore, the butterbur-extract of the present invention had an effect on enhancing memory on all the individual and might be used as a medicine or a healthful food for preventing and treating diseases regarding to decreasing brain function.
<Example 7> Effect on enhancing learning ability and short term memory by administrating the crude extract of butterbur
A. Experimental material animal
The crude extract of butterbur prepared by Example 1 was used as an experimental material and normal adult rats (Wister rat, male) were used as an experimental animal.
B. Experimental method
Drink comprising the crude extract prepared by Example 1 per dose (about 200mg/kg, about 400mg/kg) of butterbur was administrated to the experimental animal for about 3 weeks and then the experimental animal was learned by Morris Water Maze after about 4 weeks.
The Morris Water Maze learning was measured by comparing difference (Learning index) of time (Escape time) that Morris Water Maze learning was
performed after moving platform to the other side after about 24hours from swimming-learning of the experimental animal repeatedly about 4 times in a day for about 1 week in order to find the platform within 10 seconds.
C. Experimental result
In Example 7, a short-term memory was elevated to increase with Learning Index. Further, as may be seen from Learning Index time of FIG. 9, the efficiency on enhancing memory was increased by about 1.38 times in administration dose of about 200mg/kg and about 1.87 times in administration dose of about 400mg/kg, respectively, compared with control such that the crude extract of butterbur had an effect on enhancing short term memory dependent on dose (n=8, *p<0.01 vs control, **<0.05 vs control).
'M' indicated the crude extract of butterbur and 'C indicated control, in Table 9.
<Example 8> Effect on enhancing learning ability and short term memory by administrating the fraction extract of butterbur A. Experimental material and animal
The fraction extract of butterbur prepared by Example 2 was used as an experimental material and normal adult rats (Sparague Dawley rat, male) were used as an experimental animal.
B. Experimental method
Dr nk comprising the fraction extract prepared by Example 2 per dose (about 120mg/kg, about 240mg/kg, about 480mg/kg) of butterbur was administrated to the experimental animal for about 4 weeks and then the experimental animal was learned by Morris Water Maze after about 5 weeks.
The Morris Water Maze learning was measured comparing difference
(Memory index) of time (Escape time) that Morris Water Maze learning was performed after moving platform to the other side after about 24hours from swimming-learning of the experimental animal repeatedly about 4 times in a day for about 1 week in order to find the platform within about 10 seconds.
C. Experimental result
As may be seen from FIG. 10, memory index time of control was about 16.58±5.56(sec), memory index time of administration dose of about 120mg/kg was about 32.46±15.15(sec), about 240mg/kg was about 45.28±5.90*(sec) and about 480mg/kg was about 33.48±17.06(sec). So, the fraction extract of butterbur had an effect on enhancing short-term memory dependent on dose (n=5, *p<0.05 vs control) to about 240mg/kg.
'MW' indicated the fraction extract of butterbur, in Table 10. As a result compared Example 7 with Example 8, groups administrating the crude extract of butterbur dose of 200mg/kg and 400mg/kg had an effect on increasing by each about 1.38 times and about 1.87 times, respectively, and groups
administrating the fraction extract of butterbur dose of about 120mg/kg and about 240mg/kg had an effect on increasing by each about 1.96 times and about 2.73 times, respectively, such that a dose (about 240mg/kg) of a group administrating the fraction extract of butterbur having the most remarkable effect was administrated by twice less than a dose of a group administrating the crude extract of butterbur, but an effect on enhancing memory in a dose of about 240mg/kg of the fraction extract of butterbur was increased by about 2.73 times compared with control and was better than the effect of a group of administrating the crude extract of butterbur by about 1.5 times.
<Example 9> Effect on enhancing memory by step down latency test after administrating the fraction extract of butterbur
A. Experimental material and animal The fraction extract of butterbur prepared by Example 2 was used as an experimental material and normal adult rats (Mongolian gerbil, male) were used as an experimental animal.
B. Experimental method Drink comprising the fraction extract prepared by example 2 per dose (about
125mg/kg, about 250mg/kg) of butterbur was administrated to the experimental animal for about 5 weeks and the experimental animal was performed by step down
latency test (passive avoidance test) after about 6 weeks.
The step down latency test was measured comparing time (Latency time) that experimental animal was memorized by giving a electric shock of about 0.4mA when the experimental animal came down to grid, after about 24hour, the experimental animal stayed at platform and didn't came down to the grid.
C. Experimental result
In Example 9, memory was elevated to increase with latency time. As may be seen from FIG. 11, latency time of control was about 54.7±17.6(sec) but latency time of a group administrating in a dose of about 125mg/kg of the fraction extract of butterbur was about 101.6±21.5*(sec), about 250mg.kg was about 133.6±39.2*(sec) such that the fraction extract of butterbur had an effect on enhancing memory significantly dependent on dose (n=10, *p<0.05 vs control).
'MW' indicated the fraction extract of butterbur, in Table 11.
<Example 10> Safety (acute toxicity and subacute toxicity) test
To confirm that the butterbur-extract was toxic to a body, animal experiment was performed as follows.
(1) Acute toxicity test
A. Experimental material and animal
The crude extract of butterbur prepared by Example 1 was used as an
experimental material and 14 ICRrats were used as an experimental animal.
B. Experimental method
The experimental animal was administrated orally the crude extract of butterbur to dependent on dose (moisture content 23%; about 2g/kg, about 4g/kg), weight change and survival or not of the experimental animal for about 72 hours before and after administration were observed.
C. Experimental result All of the experimental animals were survived. Weight gain was each about
3.3g and about 2.5g in groups administrating the crude extract of butterbur dose of about 2g/kg and about 4g/kg such that data of the weight gain belonged to error range of normal weight gain. Further, LD50 was not observed in a dose of about 6g/kg b.w. such that the butterbur-extract was stable.
(2) Subacute toxicity test
A. Experimental material and animal
The crude extract of butterbur prepared by Example 1 was used as an experimental material and 13 ICRrats were used as an experimental animal.
B. Experimental method
The experimental animal was administrated orally with a dose of about
2g/kg bw/day and weight change was observed.
C. Experimental result
Change in a range of weight gain of the experimental animal was not observed and all of the experimental animals were survived.
Accordingly to the safety test, the butterbur-extract of the present invention may be almost free of toxicity.