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(51) International Patent Classification: gcheongnam-do 356-882 (KR). LEE, Kwang Gill
A23L 1/076 (2006.01) BOW 15/00 (2006.01) [KR/KR]; 201, 522-1, Guun-dong, Gwonseon-gu, Su
A23L 1/015 (2006.01) B O 39/00 (2006.01) won-si, Gyeonggi-do 44 1-819 (KR). PARK, Kwan Kyu
[KR/KR]; 105-701, Songjeong Town Apt., Beommul 2-
(21) International Application Number:
dong, Suseong-gu, Daegu 706-915 (KR).
PCT/KR20 12/003 139
(74) Agent: NA, Dong Kyu; 302, Oh-Sung Bldg., 1508, Dun-
(22) International Filing Date:
sanl-dong, Seo-gu, Daejeon 302-120 (KR).
24 April 2012 (24.04.2012)
(81) Designated States (unless otherwise indicated, for every
(25) Filing Language: English
kind of national protection available): AE, AG, AL, AM,
(26) Publication Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
(30) Priority Data: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
10-2012-0001015 4 January 2012 (04.01 .2012) KR HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KZ,
(71) Applicant (for all designated States except US): REPUB¬ LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG,
LIC OF KOREA(MANAGEMENT : RURAL DEVEL¬ MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
OPMENT ADMINISTRATION) [KR/KR]; 250, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, SE,
Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do 441- SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT,
857 (KR). TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
(72) Inventors; and (84) Designated States (unless otherwise indicated, for every
(75) Inventors/Applicants (for US only): HAN, Sang Mi kind of regional protection available): ARIPO (BW, GH,
;;;;;;;;;;;;;;
;;;;;;;;;;;;;; [KR/KR]; 354, Mowol-ri, Inji-myeon, Seosan-si, Chun- GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
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(57) Abstract: Disclosed is a method for the purification of bee venom on a
mass scale. It comprises dissolving crude bee venom in a solvent to form a
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form a mixture; and removing the adsorbent through filtration to afford a
pure bee venom solution. By the method of the present invention, crude bee
water
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'4é--- venom collected from bee farms can be purified to a purity of 99% or higher
on a mass scale, without inducing a change in the composition thereof, and
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thus the purified bee venom can be used as a material for medicines, cosmet
ics and foods.
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II I II
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, Published:
RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, .t . t . , ,
DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, ~ w1t1h mternatl onal search report (Art. 21(3))
LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS,
SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM,
GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
wo 2013/103178 PCT/KR2012/003139
1
Description
Title of Invention: METHOD FOR PURIFYING BEE VENOM ON
MASS SCALE
Technical Field
[1] The present invention relates to a method for the purification of bee venom on a mass
scale. More particularly, the present invention relates to a method for purifying bee
venom without inducing a change in the composition thereof, thereby producing highly
pure bee venom on a mass scale.
Background Art
[2] Bee venom refers to the toxic substance that is produced by workers bees and has
been used in the therapy of diseases.
[3] For the application of bee venom to the body, previously, worker bees which had
been caught were forced to sting an affected site or only the stingers, after being
detached from worker bees, were inserted into an affected site. However, it is difficult
to control the injection amount or concentration of bee venom in this manner, which
makes the objectification and generalization of the bee venom therapy impossible and
may generate side effects such as shock.
[4] If given the ability to adjust the amount and concentration of drug depending on the
body condition and constitution of the patient, bee venom therapy can be safely used
without inducing any side effects. Bee venom therapy has spread to some degree over
the U. S. and the Europe. In the early 1990s, the subcutaneous injection of purified bee
venom for the treatment of inflammation and pain was approved by the FDA, and it is
produced under the brand name of Apitoxin. Recently, bee venom collectors have been
located over a vast area in bee farms and thus, a large amount of bee venom is
collected.
[5] However, the bee venom collected via the collectors of bee farms contains significant
amounts of contaminants such as dust, soil, etc., and impurities such as honey, pollen
and propolis. These foreign materials change the composition of bee venom, degrading
the physiological activity of bee venom.
Disclosure of Invention
Technical Problem
[6] It is an object of the present invention to provide a method for purifying bee venom
without inducing a change in the composition thereof, thereby producing highly pure
bee venom on a mass scale.
[7] The objects to be accomplished by the present invention are not limited to the above-
mentioned object, and other objects that are not stated may be clearly understood to
wo 2013/103178 PCT/KR2012/003139
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those having ordinary skill in the art from the following description.
Solution to Problem
[8] In order to accomplish the above-mentioned object, the present invention provides a
method for purifying bee venom on a mass scale, comprising: solubilizing crude bee
venom in a solvent to form a bee venom solution; applying an adsorbent to the bee
venom solution to form a mixture; and removing the adsorbent by filtration to afford a
pure bee venom solution.
[9] In one embodiment, the bee venom solution may be filtered before the adsorbent is
applied thereto.
[10] In this regard, the bee venom solution is filtered through filter paper which may
preferably be a cellulose filter.
[11] In the method, the solvent may be water.
[12] According to another embodiment, the crude bee venom is used in an amount of
from 0.02 to 0. 1 % by weight, based on a total weight of the bee venom solution.
[13] In another embodiment, the adsorbent is PSA (primary secondary amine).
[14] In another embodiment, the adsorbent is added in an amount of from 0.1 to 10 g per
liter of the bee venom solution.
[15] In another embodiment, the filtration for removing the absorbent may be vacuum
filtration and may be conducted using filter paper which may preferably be a cellulose
filter.
[16] The method may further comprise filtering the pure bee venom solution to yield a
filtrate and lyophilizing the filtrate to produce a fine powder.
[17] In this regard, the filtering step is conducted by vacuum filtration.
[18] According to another embodiment, the lyophilizing step is conducted at -20°C or
lower.
Advantageous Effects of Invention
[19] By the method of the present invention, crude bee venom collected from bee farms
can be purified to a purity of 99% or higher on a mass scale, without inducing a change
in the composition thereof, and thus the purified bee venom can be used as a material
for medicines, cosmetics and foods.
Brief Description of Drawings
[20] FIG. 1 is a flow chart illustrating a method for purifying bee venom on a large scale
according to the present invention.
[21] FIG. 2 is a flow chart illustrating a method for purifying bee venom on a large scale
to produce pure bee venom as a fine powder suitable for storage for a long period of
time in accordance with the present invention.
[22] FIG. 3 shows chromatograms comparing the contents of main ingredients of bee
wo 2013/103178 PCT/KR2012/003139
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[38] That is, after the removal of non-water soluble foreign substances, the water-soluble
foreign substances, such as honey or pigment, which have dissolved in the bee venom
solution, are removed by adsorption.
[39] For this, an adsorbent is added and mixed homogeneously to remove the water-
soluble foreign substances which remain dissolved in the solution.
[40] So long as it adsorbs water-soluble foreign substances, such as pigments, any
adsorbent may be used in the present invention. Preferred is PSA (primary secondary
amine), which is one of the most potent adsorbents known to date.
[41] The adsorbent may be preferably added in an amount of from 0. 1 to 10 g per liter of
the bee venom solution. When the amount of the adsorbent is less than 0. 1 g per liter of
the bee venom solution, the adsorption ability is too poor to separate pure bee venom.
An amount of the adsorbent exceeding 10 g per liter causes a change in composition,
making it difficult to separate bee venom at a purity of 99% or higher.
[42] In step S3, the mixture is filtered to separate pure bee venom from the adsorbent and
foreign substances adsorbed to the adsorbent, whereby bee venom can be purified on a
mass scale.
[43] The filtration is preferably vacuum filtration which is more preferably preformed
using filter paper, in order to afford bee venom at a purity of 99% or higher.
[44] In this context, the adsorbent to which the water-soluble foreign substances have
adhered (particle retention (liquid)) ranges in size from 0.45 to 0.8 m, and the filter
paper is preferably a cellulose filter which is suitable for filtering out the adsorbent.
[45] Turning to FIG. 2, there is a flow chart illustrating a method for purifying bee venom
on a mass scale in the form of fine powder in another embodiment of the present
invention, whereby it can be stored for a long period of time.
[46] In addition to the purification procedure (SI, S2 and S3) of FIG. 1, as seen in this
flow chart, the purification method according to this embodiment of the present
invention comprises a step of secondary filtration and freeze drying (S4) by which the
pure bee venom is produced as a fine powder free of bacteria and fungi and thus can be
used as a material for medicines, cosmetics and foods and be stored for a long period
of time.
[47] The secondary filtration is preferably vacuum filtration which utilizes filter paper and
more preferably a cellulose filter with a pore size of 0.2 µτα or smaller. The resulting
filtrate is lyophilized at -20°C or colder to produce pure bee venom as a powder.
[48] As described above, the purification method of the present invention can remove
foreign substance from crude bee venom without inducing a change in the composition
of bee venom to produce bee venom at a purity of 99% or higher on a mass scale,
which can be used as a material for medicines, cosmetics and foods.
[49]
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[50] A better understanding of the present invention may be obtained through the
following examples which are set forth to illustrate, but are not to be construed as
limiting the present invention.
[51]
[52] EXAMPLE 1: Purification of Bee Venom 1
[53]
[54] 1. Removal of non- water- soluble foreign substances
[55] 1). The crude bee venom collected using bee venom collectors was treated under the
following conditions to dissolve its water-soluble components in water.
[56] Conditions and methods
[57] a. Solvent: deionized water
[58] b. Bee venom content: 0.02-0.1 % by weight (based on total weight)
[59] c. Time: Bee venom was lightly stirred for 15 sec in the solvent using a glass rod.
[60] 2) After the water-soluble ingredients were dissolved in such a manner, the solution
was filtered through filter paper under the following conditions. The filtrate was used
in the next step whereas non- water- soluble foreign impurities such as sand, soil, bee
glue, pollen, etc. were left behind on the filter paper and discarded.
[61] * Filter paper
[62] - Type: cellulose filter (glass microfiber filter is excluded from being used)
[63] - Particle retention (liquid): 20-30 µm
[64] - Air flow rate: 5- 10 s/100 mL/in 2
[65]
[66] 2 Removal of water-soluble foreign impurities
[67] 1) After the removal of non- water-soluble foreign impurities, the water-soluble
foreign impurities which have dissolved in water were removed in the presence of a
primary secondary amine (PSA) under the following conditions.
[68] Conditions and method
[69] a. The bee venom solution was added to a beaker containing primary secondary
amine.
[70] b. The solution was stirred to allow ingredients other than sugars and bee venom to
be adsorbed to the adsorbent, followed by the separation of pure bee venom using filter
paper.
[71] c. Loading rate: Primary secondary amine was used in an amount of 0. 11 g per liter
of the bee venom solution.
[72] 2) After the removal of water-soluble foreign impurities, vacuum filtration was
conducted using filter paper under the following conditions to afford bee venom 99 %
pure.
[73] * Filter paper
wo 2013/103178 PCT/KR2012/003139
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[Table 1]
[115]
[116] Taken together, the data obtained above indicate that the purification method of the
present invention can purify crude bee venom collected from bee farms to a purity of
99 % or higher.
[117] Accordingly, bee venom with a purity of 99% or higher can be produced on a mass
wo 2013/103178 PCT/KR2012/003139
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scale by the method of the present invention and is useful as a material for medicines,
cosmetics and foods.
[118]
[119]
[120] While the present invention has been particularly shown and described with reference
to the foregoing preferred and alternative embodiments, it should be understood by
those skilled in the art that various alternatives to the embodiments of the invention
described herein may be employed in practicing the invention without departing from
the spirit and scope of the invention as defined in the following claims. It is intended
that the following claims define the scope of the invention and that the method and
apparatus within the scope of these claims and their equivalents be covered thereby.
This description of the invention should be understood to include all novel and non-
obvious combinations of elements described herein, and claims may be presented in
this or a later application to any novel and non-obvious combination of these elements.
wo 2013/103178 PCT/KR2012/003139
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Claims
[Claim 1] A method for purifying bee venom on a mass scale, comprising:
dissolving crude bee venom in a solvent to form a bee venom solution;
applying an adsorbent to the bee venom solution to form a mixture; and
removing the adsorbent through filtration to afford a pure bee venom
solution.
[Claim 2] The method of claim 1, further comprising filtering the bee venom
solution before the applying step.
[Claim 3] The method of claim 2, wherein the filtering step is conducted using a
filter paper.
[Claim 4] The method of claim 3, wherein the filter paper is a cellulose filter.
[Claim 5] The method of claim 1, wherein the solvent is water.
[Claim 6] The method of claim 1, wherein the crude bee venom is used in an
amount of from 0.02 to 0. 1 % by weight, based on a total weight of the
bee venom solution.
[Claim 7] The method of claim 1, wherein the adsorbent is PSA (primary
secondary amine).
[Claim 8] The method of claim 1, wherein the adsorbent is added in an amount of
from 0. 1 to 10 g per liter of the bee venom solution.
[Claim 9] The method of claim 1, wherein the filtration is vacuum filtration.
[Claim 10] The method of claim 9, wherein the vacuum filtration is conducted
using a filter paper.
[Claim 11] The method of claim 10, wherein the filter paper is a cellulose filter.
[Claim 12] The method of claim 1, further comprising filtering the pure bee venom
solution to give a filtrate and lyophilizing the filtrate to produce a fine
powder.
[Claim 13] The method of claim 12, wherein the filtering step is conducted by
vacuum filtration.
[Claim 14] The method of claim 13, wherein the lyophilizing step is conducted at -
20°C or lower.
wo 2013/103178 PCT/KR2012/003139
1/3
[Fig. 1]
Bee toxin
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1O 20 30 40 - 1O 20 40
Time(min} Time(min)
(A) Befare purification (B) After purification
[Fig. 4]
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According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
A23L 1/076; A61P 17/02; A23L 1/00; G OIN 33/00; G01N 33/18; C07K 14/435; A61K 35/64
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Korean utility models and applications for utility models
Japanese utility models and applications for utility models
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
eKOMPASS(KIPO internal) & Keywords: bee venom, purifying, absorbent, PSA, primary secondary amine
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
* Special categories of cited documents: "T" later document published after the international filing date or priority
"A" document defining the general state of the art which is not considered date and not in conflict with the application but cited t o understand
to be of particular relevance the principle or theory underlying the invention
"E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be
filing date considered novel or cannot be considered t o involve an inventive
"L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone
cited t o establish the publication date of citation or other "Y" document of particular relevance; the claimed invention cannot be
special reason (as specified) considered to involve an inventive step when the document is
"O" document referring t o an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination
means being obvious t o a person skilled in the art
"P" document published prior t o the international filing date but later "&" document member of the same patent family
than the priority date claimed
Date of the actual completion of the international search Date of mailing of the international search report