TW460523B - Manufacturing method for natural cross-linker genipin - Google Patents

Abstract

The present invention relates to a manufacturing method for natural cross-linker genipin, comprising the steps of: (A) the extraction of geniposide is that the crushed gardenia fruit is placed into an extractor and the lipid in the fruit is removed with a solvent. Ethyl acetate is added into the extractor and the crystal powder of geniposide can be obtained through heating, recycling extraction and cooling; (B) the conversion of geniposide into genipin using beta-glucosidase is that the extracted geniposide crystal powder is dissolved in a buffer solution. The beta- glucosidase is added into the solution for stripping the glucose molecules from geniposide and converting geniposide into genipin; (C) the organic extraction and crystallization of purified genipin is that adding another solvent into the mixture of geniposide and genipin formed in step (B), genipin is extracted into water phase and a highly purified genipin crystal powder can be obtained when the water extraction is concentrated.

Description

4 6 0 5 2 3 a7 B7 V. Description of the Invention () Field of Invention The present invention is related to the natural cross-linking agent genipin suitable for biomedical materials. In particular, it relates to a method for manufacturing a natural cross-linking agent genipin. . 5 References

Araujo, JD, et al., J. Cardiovasc. Surg., 28: 434-439, 1987. Fujikawa, S., et al., Biotechnology Letter, 9: 697-702, 1987. Iguidbashian, JP, et al. , Ann. Thorac. Surg., 55: 1022-1024, 1993. 10 Imamura, E., et al., J. Cardiac. Surg., 4: 50-57, 1989.

Lohre, J.M., et al. ≫ Artif. Organs, 16: 630-633, 1992.

Lohre, J.M., et al., ASAIO J., 39: 106-1 13, 1993.

Noishiki, Y., et al., Int. J. Artif. Organs, 16: 192-198, 1993. Nimni, ME, et al., In Collagen Vol. Ill, Florida, ME 15 Nimmi (ed.) CRC Press Boca Raton, 1988, 1-38.

Okoshi, T., et al., Trans. Am. Soc. Artif Intern. Organs, 34: 532-537, 1988.

Okoshi, T., et al., ASAIO J., 36: M411-414, 1990. Printed by the Consumer Cooperatives of the Central Bureau of Procurement, Ministry of Economic Affairs (please read the notes on the back before filling in, page 4)

Rosenberg, D., New York, in Vascular Grafts, P.N. Sawyer 20 and M.J. Kaplitt (eds.), Appleton-Century-Crofts, 1978, 261-270.

Sung, H.W., et al., Asian Cardio. &Amp; Thorac. Annals., 1: 104-109, 1993a.

Sung, HW, et al., ASAIO J *, 39: 532-536, 1993b. -3- This paper size applies to the Chinese National Standard (CNS) A4 (210X297 mm) 460523 A7 B7 Staff Consumption of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative cooperative printing V. Description of invention ()

Sung, H.W., et al., Int. J. Artif. Organs, 16: 199-204, 1993c. Sung, H.W., et al.s J. Biomater. Sci. Polymer Edn., 8: 587-600, 1997.

Sung, HW, et al., J. biomed. Mate. Res., 42: 560-576, 1998. 5 Sung, HW, et al., J. Biomater. Sci. Polymer Edn., 10: 63-78, 1999.

Trowbridge, E.A., et al., J. Thorac. Cardiovasc. Surg., 95: 577-585, 1988.

Uva, M.S., et al., Ann. Thorac. Surg., 54: 89-92, 1992. 10 Wang, C.J., et al., Caner Lett, 60: 95-102, 1991.

Xi, T., et al., J. Biomed. Mat. Res., 26: 1241-1251, 1992. Yamano, T., et al., Fd. Chem. Toxic, 28: 515-519, 1990. Invention Background Animal tissues, collagen, gelatin, chitosan, and 15 synthetic polymers containing amine groups are used in clinical surgery applications as implants in vivo, such as artificial heart valves, artificial blood vessels, and artificial patches. Films, artificial ligaments (Iguidbashian, 1993; Trowbridge, 1988; Uva, 1992), cartilage, interstitial cells, biogels, wound dressings, and drug delivery vehicles. However, before these biomedical materials are implanted into the human body, they must undergo a cross-linking treatment with a cross-linking agent. The main purpose of the cross-linking treatment is to increase the mechanical strength of the material, and to reduce the body's immune system and enzymes' attack on these biomedical materials. . The cross-linking agents that have been used to cross-link these biomedical materials are formaldehyde (Nimni, 1988), glutaraldehyde (Nimni, 1988), dialdehyde starch (Rosenberg, 1978), and ePoxy. CNS) A4ii (210X297mm) I ^ nn I line {Please read the note on the back first and then fill in the _ title page) · Printed by the Central Bureau of Standards, Bureau of Standards, Shellfish Consumer Cooperative 460523 at B7 V. Description of the invention ( ) Compound (Sung, 1996), etc. Among these biomedical material cross-linking agents, glutaraldehyde is most commonly used in clinical practice, but biomedical materials treated with glutaraldehyde (Trowbridge, 1993; Okoshi, 1988; Araujo, 1987) cross-linking in the past clinical experience, but Encountered problems such as tissue sclerosis, calcification5, and fibrosis. "In recent years, epoxy compounds (Sung, 1993a; Sung, 1993b; Noishiki, 1993; Sung, 1993c) have been tried. It was used to crosslink biomedical materials, and experimental results found Biomedical materials crosslinked with epoxy compound are softer and less calcified than biomedical materials crosslinked with glutaraldehyde (Imamura, 1989; Okoshi, 1990; Xi, 1992). However, the degree of cross-linking of biomedical materials treated with epoxy compound is not as good as that of glutaraldehyde (sUng, 1997), so their mechanical strength and ability to resist enzyme decomposition are not as good as those of ghitaraldehyde. Processed biomedical materials. In addition, formaidehyde, 15 glutaraldehyde, and epoxy compound are chemically synthesized cross-linking agents. The results of past experiments have also confirmed that they have a relatively high cytotoxicity (Lohre, 1992; Lohre, 1993). Medical materials often elicit a stronger and sustained immune response. The fruit of gardenia is often used in traditional Chinese medicine to treat various immune diseases and liver diseases. Yamano's research group in an animal experiment (Yaman〇, 1990) has reported that genjp〇side extracted from the vibrator fruit can reduce serum triglyceride, lipid peroxide, and phospholipid in blood. The mechanism by which geniposide is used to treat liver disease has also been published in the literature (Wang, 1991). Since genipin can be used with amino acids or proteins from -5- this standard is applicable to the Chinese National Standard (CNS > A4 size (210X297 mm)-III n I n ^ r line (please read the precautions on the back first) Envy again. This page) 460523 Α7 Β7 Central Standards of the Ministry of Economic Affairs, Employees' Cooperatives, Consumer Cooperatives, India Ben V. Invention Description ()-Natural reactions form dark blue products, and these dark blue products are often used as natural food coloring (Fujikawa, 1987). Fujikawa's research group (Fujikawa, 1987) also pointed out that genipin is a good natural cross-linking agent for proteins, so it should also be used to cross-link biological tissues with materials containing free amine 5 groups. Since genipin and its related derivatives are quite successful in the application of traditional Chinese medicine and food coloring, we speculate that its cytotoxicity should be quite low. In a cytotoxicity experiment (Sung, 1999), we did confirm The toxicity of genipin and its cross-linked biological tissues is much lower than that of 10 glutaraldehyde and its cross-linked biological tissues.

Genipin can be produced by extracting the geniposide from gardenia fruit by removing glucose molecules from the enzyme -glUC0sidase (Fujikawa, 1987). As far as the first practice is concerned, the step of extracting geniposide from gardenia fruit generally uses 15 aqueous solutions of ethanol, methanol or acetone as a solvent. After extraction, the organic solvent is concentrated and recovered, and then subjected to chromatography such as charcoa 丨 or Silica gj, etc. The pigment was separated from the glycoside genipoyde. However, the purity of genip0side obtained by this method is often not high (40-70%), which cannot be directly converted by / 3-glucosidase, and because the solution is not high in purity, the reaction is slow and the conversion rate is low, it must be ethyl acetate. Only 20 crystals can reach a purity of more than 90%. In other words, the conventional techniques for generating genipin have the disadvantages of complicated processes, large amounts of solvents, low conversion rates and high costs, which are not suitable for commercial mass production applications. β In view of this, the main purpose of the present invention is to provide a method for manufacturing a natural cross-linking agent, genipin, which has a simple process to facilitate the commercialization of a large number of raw materials. (210X297) ^^------- ^ ------ '玎 ------. ^ (Please read the note on the back and then this page) A7 B7 460523 V. Invention Explanation (Producer β The present invention-the purpose is to provide a natural cross-linking agent genipin manufacturing method, which does not require the use of a large amount of organic solvents, and a high conversion rate helps reduce manufacturing costs. 5 In order to achieve the above objectives, The natural cross-linking agent genipin provided by the present invention The manufacturing method includes the following steps: (A) extraction of genip〇side, the crushed vibrator fruit is placed in an extractor, washed with a solvent to remove oil and fat from the locust fruit, and then added ethyl acetate After heating cycle extraction and cooling, geniposide crystal powder can be obtained; (B) 10i ^ -glueosidase enzyme is used to convert geniposide into genipin. The extracted geniposide crystal powder is dissolved in a buffer solution and added to yS- glucosidase enzyme, used to remove the genip〇side from glucose molecules and convert it into genipin; (C) organic solvent extraction and purification of genipin crystals, the geniposide and genipin mixed solution in step (B), and then add another 15 solvents After extracting the genipin in the aqueous phase, and then concentrating the extract, a high-purity genipin crystal powder can be obtained. In order to enable the review committee to understand the detailed process and characteristics of the present invention, the following examples are listed, and It is illustrated with the following illustrations, in which: The first diagram is a geniposide crystalline powder 20 and an HPLC analysis chart of a preferred embodiment of the present invention. According to a preferred embodiment of the present invention, the geniposide is converted to genipin by 10,000-glucosidase enzyme. The third chart is a genipin crystal powder and a HPLC analysis chart of a comparative example of the present invention. The paper scale is applicable to Chinese national standards. (CNS) Ad specifications (210 x 297 mm) II I ^ I * I n I— III line (Please read the precautions on the back before reading? (This page) 4 Printed by the Consumers 'Cooperative of the Central Government Bureau of the Ministry of Economic Affairs 460523 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () Detailed description of the invention I KA Unless otherwise explained, the following terms are as follows: Definition. 5 " genipin " refers to natural compounds with structure 1 (shown below), their stereoisomers, and mixtures related to other substances. "Crosslinking agent" is interpreted as a kind of biochemical materials such as animal tissues, collagen, gelatin, chitosan, heme, DNA, enzymes, antibodies and synthetic polymers containing amine groups. Free amines, chemicals cross-linked by chemical bonds 10. "Biomedical materials" are interpreted as materials for the manufacture of medical devices that are used to insert or implant tissues in the body and can be maintained for a certain period of time or permanently implanted. For example, it is widely used to make drug delivery vehicles, blood vessels, heart valves, ligaments 'software, skin grafts' patches, interstitial cells and biosensors, etc., 15 animal tissues, collagen translation, gelatin, Chitosan, heme, DNA, enzymes, antibodies and synthetic polymers containing amine groups. Preparation and properties of II genipin The molecular structure of Genipin is shown below (Structure I). It is a kind of glucoside geniposide (as shown in the following structure II) present in the fruit of Gardenia japonicus, and a glucose molecule is removed by cold-20 glucosidase enzyme get. Because the gluco-glucosidase enzyme is also a free amine-containing protein, when / 5-glucosidase enzyme is used to convert geniposide to genipin, β-glucosidase will generate unnecessary cross-linking reactions with genipin. In order to overcome this problem, we fixed jS-glucosidase to alginate-coated -8-, »Thread (please read the notes on the back of the waiver first and then 1 * this page) This paper size applies Chinese National Standard (CNS) A4 Specifications (210X297 males) 460523 A7 B7 5. Description of the invention () in the small ball. 5

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I ----.----- ^ ------- IT ------. ^ (Please read the notes on the back first and then fill in this page.) The quantification of the geniposide and genipin extracted from the fruit of the vibrator by the consumer cooperative Neem can be analyzed by a high-efficiency liquid chromatography. The analytical method was performed on a reverse-phase C-18 chromatography column at 35 ° C, while the UV detection wavelength was 238 nm. In HPLC analysis, we can use acetonnitrile: H20: perchloric acid = 17: 83: 0.1 as the mobile phase, and the flow rate is 1.0 mL / min. In the HPLC analysis, the retention times of geniposide and genipin were 4.33 and 8.04 minutes, respectively. -9- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 460523 A7 B7 Printing policy of the Consumers ’Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () Although the structure I shows the genipin Natural structure, but referring to the foregoing description, 'any of its stereoisomers or mixtures of stereoisomers can also be used as a cross-linking agent. According to the above, 5 genipin is a good cross-linking agent for biomedical materials in vivo, such as cross-linked bioartificial organs and other implants, biogels, wound dressings, and drug carriers. And blood substitutes. HI · Example Explains one of the preferred embodiment 10 of the manufacturing method of the natural cross-linking agent genipin of the present invention, which mainly includes the following steps: (A) Geniposide stubble first crush the provincial gardenia fruit and place it in Soxhlet The extractor, then washed twice with aerobic or n-hexane to remove the fat component in the fruit of gardenia, then poured in an appropriate amount of ethyl acetate, and then placed the Soxhlet extractor on a heated 15 plate to heat and extract for 6 hours, Then, the extract was naturally cooled at room temperature to obtain a crystalline powder of geniposide. The first figure shows the geniposide crystal powder and its HPLC analysis spectrum after the fruit of gardenia is extracted with ethyl acetate. The experimental results show that the purity of geniposide extracted from the fruit of the vibrator is as high as 20 98%. (B) Using yg -glucosidase to convert geniposide into genipin

The extracted geniposide crystal powder is dried and then dissolved in acetic acid or acid buffer solution at pH 4.0 to pH 6.0, and then at 35 ° C -10- This paper standard is applicable to Chinese Standard (CNS) Α4 Specifications (2 丨 0X297mm) ---------- t .------ ΐτ ------ line (please read the note t ·· on the back before reading this page) ) Printed by the Central Standards Bureau of the Ministry of Economic Affairs and the Industrial Cooperatives Cooperative Association 4 6 0 5 23 a? _ B7 V. Description of the invention () The immobilized / 5-glucosidase enzyme was used to deglucose the molecule and then convert it into genipin. The second picture is the HPLC analysis chart of geniposide converted to genipin by yS -g 丨 ucosidase enzyme. From the area of the map, we know that the conversion rate of 5 genipin is about 60%, so we must extract and separate the water-soluble solution with a suitable organic solvent.泮 Genipin. (C) Organic solvent extraction and purification of genipin crystals. The mixed solution of geniposide and genipin in step (B) is extracted with a suitable organic solvent to extract the genipin in the aqueous phase. High purity crystalline powder. The choice of organic solvents depends on the distribution coefficients of organic solvents and water in the genipin and geniposide. The greater the difference between the two distribution coefficients, the better the effect of this organic solvent in separating the genipin and geniposide in the aqueous phase. In the experiments, we used high-efficiency liquid 15-phase chromatography (HPLC) to test the partition coefficients of organic solvents such as butanol, diethyl ether, chloroform, and toluene with water. Table 1 shows the partition coefficients of genipin and geniposide between different organic solvents and water measured by HPLC. As shown in the table, the partition coefficients of geniPin and geniposide between ether and water are quite different (about 40 20 times), and the boiling point of ether is also lower (35.0. It is more volatile and removed, so we chose Yimei to Extract the geniPn from the aqueous solution, and then concentrate and remove the ether to obtain the crystalline powder of genipin. The third figure is the crystalline powder of genipin and its HPLC analysis chart. From the area of the map, we know that this genipin crystal The purity of the powder is more than 98%. -11- This paper size is used by the Chinese National Car (CNS) Λ4 size {210X297 cm.) Binding line (please note the precautions on the back before Ji · this page) Standards Bureau ’s Work and Consumer Cooperation Du printed 460523 A7 ____B7 V. Description of the invention (Table 1) Partition coefficients of genipin and geniposide between different tested organic solvents and water

Geniposide Genipin BP # (° C) Butanol 1.26 * 16.45 118 Ether 0.04 1.63 35 Gas-form 0.11 1.52 61 Toluene 0.01 0.30 111 * The number in the table is the distribution coefficient: Csolvent / CH20 #BP: The boiling point of each tested organic solvent Using the genipin produced by the above-mentioned manufacturing method, as an identification experiment of the ability to cross-link animal tissues: First we retrieved fresh pig pericardium from the slaughterhouse, and removed the fat on the surface of the pig. 0.625% (w / w) glutaraldehyde or 0.625% (w / w) genipin was crosslinked at 37 ° C for 3 days (pH 7.4). After cross-linking, the biological tissues were tested for their cross-linking index, denaturation temperature, and mechanical strength to understand the ability of genipin to cross-link animal tissues. 15 1. Cross-linking index The cross-linking index represents the percentage of free amine groups on biological tissues that are reacted by cross-linking agents. This index uses ninhydrm (HHN) assay to measure the content of unreacted free amine groups on biological tissues. Column formula to calculate:

Fixation lndex (%) = [{NHN revive a min eJfu: ecJ] χ] 〇〇 (NHN reactive amine) fresh -12- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)- ------- ^ ------ 1T ------ 0 (Please read the precautions on the back before 栌: this page) 460523 Shellfish Consumer Cooperative, Central Standards Bureau, Ministry of Economic Affairs, printed A7 B7 V. Description of the invention () ^ Among them, (NHN reactive amine) fresh is the content of free amine groups before tissue crosslinking, and (NHN reactive amine) fixed is the content of free amine groups remaining after tissue crosslinking. Because ninhydrin reacts with free amine groups to produce a blue-violet product, we use the relationship between the absorption at this wavelength of 570nm 5 (0D value) and the concentration of the product to determine the concentration of ninhydrin in biological tissues. Free amine content of the reaction. Before doing this analysis, the biological tissue must be dried under vacuum for 24 hours, and then an appropriate amount of sample is weighed (approximately 3 mg). The dried tissue is immersed for about 1 hour by adding water, and then heated by adding ninhydrin reagent at 100 ° C for about After 10 minutes, the content of free amine groups in the biological tissues after being cross-linked was quantified using a UV / visible spectrometer at a wavelength of 570 nm. 2. Denaturation temperature test Denaturation temperature is the temperature at which the structure of collagen in a biological tissue suddenly changes during the heating process. Generally speaking, the degeneration temperature of 15 degrees can be used as the degree of cross-linking of biological tissues. If the degeneration temperature of biological tissues is higher, it means that the biological tissues are more cross-linked. For the measurement of biological tissue denaturation temperature, we used a thermal differential scanning card meter. This method has been widely used for the thermal transition measurement of collagen. During the experiment, we used a sealed 20 aluminum pan to seal the sample, and heated the sample at a heating rate of 5t per minute. The tissue heating range was (Td-25 ° C) < T < Td + K) ° C. ), Where Td is the desired denaturation temperature. In terms of instrument calibration, we used the thermal phase change of the melting point (156.6 ° C) of indium (purity of 99.999%) to calibrate the thermal differential scanning caliper, and the heating rate during calibration was still 5 ° C per minute. -13- This paper size applies to Chinese National Standard (CNS) Λ4 specification (210X297 mm) ---------- installation ------ tr ------ line (please read the back first (Notes on this page, .6 page) 460523 A7 B7 V. Description of the invention () 3. Mechanical strength test For the test of the mechanical strength of the crosslinked tissue, we first cut the test tissue into dog-bone test pieces, and then use the material to test Machine to measure the ultimate load of the tissue test piece at a tensile rate of 50mm / min. 5 Table 2 shows the comparison of the cross-linking index, denaturation temperature, and mechanical strength of animal tissue cross-linked by genipin and glutaraldehyde. . As shown in the table, the cross-linking index of genipin and glutaraldehyde cross-linked animal tissues is quite high, and the denaturation temperature and mechanical strength are also significantly higher than those from freshly cross-treated animal tissues. The experimental results confirmed that 10 genipin and glutaraldehyde are both very strong cross-linkers for animal tissues. Table 2: Comparison of cross-linking index, denaturation temperature, and mechanical strength of animal tissues cross-linked with genipin and glutaraldehyde (n = 5, SD)

Fresh1 Glutaraldehyde2 Genipin3 Crosslinking index (%) 0 98_6 ± 0.1 97 · 4 ± 0 · 3 Denaturation temperature (° c) 59 · 5 ± 0 · 6 86 · 7 ± 0.3 80 · 5 ± 0 · 4 (mechanical strength (N ) 9.5 ± 1'1 17.6 ± 1.8 20.5 ± 0.5 ^ resh: fresh tissue without cross-linking treatment 15 2Glutaraldehyde: glutara 丨 dehyde cross-linked treatment tissue 3 Genipin: genipin cross-linked treatment tissue In summary, the present invention The manufacturing method of natural cross-linking agent genipin has simple process and high conversion rate, which helps to reduce costs and commercialized mass production applications. Experiments have proved that the genipin obtained by this method is indeed 20 good biomedical materials. Crosslinking agent, so the practicability and advancement of the present invention are undoubted, and the related documents have not been disclosed before after inspection. Now, in order to protect the rights of the applicant, a patent application has been filed according to law. 14- This paper size is applicable to China National (CNS) A4 specifications (—210X297 Gong ChuΊ -------- ^ --- ^ ------ tT ------ 0 (please first W Read the notes on the back of the refill / this I). Central Standards Bureau of the Ministry of Economic Affairs *: Industrial and consumer cooperation Du Yince 4 6 0 5 23 A7 B7 V. Description of the invention ) Brief description of the diagram: The first diagram is a geniposide crystalline powder and a HPLC analysis chart of a preferred embodiment of the present invention. The second diagram is a geniposide of a preferred embodiment of the present invention converted into genipin by a glucosidase enzyme-5 The third chart is the genipin crystal powder and its HPLC analysis chart of a preferred embodiment of the present invention. ----; ----- ^ ------ t * * (Please read first Note on the back again, ν This page] Printed and printed on paper by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Online Economics The paper size applies the Chinese National Standard (CNS) Α4 specification (210X297 mm)

Claims (1)

Bulletin 460523 A8 B8 C8 D8 Years Λ Application Patent Scope No. 88108258 Amendment Page 90.6 15 Intellectual Property Bureau of the Ministry of Economic Affairs P 'Industrial and Consumer Cooperation Printing 20 I A natural cross-linking agent manufacturing method of Genipin The method includes the following steps: (A) Extraction of gardenia genin sugarberry (genip〇side), the crushed gardenia fruit is placed in an extractor, and a solvent for washing and removing fat is added. In the device, the yakisaki fat in the gardenia fruit is washed and removed, and then ethyl acetate is added. After heating cycle extraction and cooling, a crystalline powder of gardenia geniposide can be obtained; (B) using type B- The enzyme yg-glucosidase converts Geniposide into Genipin, which is a crystalline powder of Geniposide extracted from Geniposide to buffer the solution. Dissolve it and add a B-glucosidase enzyme to remove the glucose molecule from the geniposide and convert it into genipin; (C) Organic solvents Take and purify the genipin crystal. The mixed solution of genip0Side and genipin from the vibrator in step (B) is added to another solution to extract the radon from the aqueous phase. Genginin (Roll: Mai L and then concentrate the extract to obtain a high-purity crystalline genin (genipin). 2. According to the scope of the patent application of the first "Manipulation method of Genipin", the solvent used to remove the fat from Gardenia fruit in step (A), is an aerodynamic model "3. According to the natural delivery method described in item 1 of the scope of patent application The manufacturing method of the combination agent Genipin, in this step (A), the solvent used to remove the fat from the fruit is n-hexane. -16- This paper uses the Chinese National Standard (CNS) ) A4 specification (210/297 cm) I * 1— -Mil Ι · Β · _ t— i nn flm nn nfl ^ i points i (Please read the precautions on the back before filling this page) ABCD 460523 VI. Application Patent Scope 4. Manufacturing method of natural cross-linking agent genipin according to item 1 of the scope of patent application In this step (B), the extracted yin geniposide crystalline powder is dissolved in a buffer solution, and the extracted yin geniposide crystalline 5 powder is further filtered and Drying procedures. 5_ According to the manufacturing method of natural cross-linking agent Genipin described in item 1 of the scope of application for patents, the buffer solution in step (B) is acetic acid. 6_ According to the manufacturing method of the natural cross-linking agent Genipin 10 (genipin) described in item 5 of the scope of the patent application, wherein the pH of the acetic acid is between 4.0 and 6.0. 7. According to the manufacturing method of the natural cross-linking agent Genipin described in item I of the applied patent garden, the buffer solution in step (B) is phosphoric acid. 15 8. According to the method for manufacturing the natural cross-linking agent Genipin described in item 7 of the applied patent garden, the pH value of the phosphoric acid is between 4.0 and 6.0. 9. According to the manufacturing method of natural cross-linking agent genipin described in item 1 of the scope of the patent application, the step (C) is used to extract the 栀 20 genipin in the aqueous phase. Solvents are those of the B-chain. 10. According to the manufacturing method of the natural cross-linking agent genipin described in item 1 of the scope of the patent application, the step (C) is used to extract the solvent in the aqueous phase to genipin, Department of chloroform. -17- This paper size applies to Chinese national standards (CNS > Α4 size (mm) i ^ n · II— I- III l ^^ il jn Please read the precautions for rain before filling this page), 1T Printed by the Ministry of Economic Affairs' Smart Money 4P?