TWI782493B - A method for extracting chondroitin sulfate - Google Patents

Abstract

The present invention provides a method for extracting chondroitin sulfate and includes the following steps: providing a cartilage powder; providing a digestion solution containing guanidine-HCl; mixing the cartilage powder and the digestion solution to obtain a cartilage powder digestion solution; extracting the supernatant of the cartilage powder digestion solution; mixing the supernatant with multiple proteases to form an enzyme reaction solution; inactivating the protease in the enzyme reaction solution and decomposing the guanidine-HCl; precipitating a chondroitin sulfate from the enzyme reaction solution. The present invention prevents salt generation and increases the yield while reducing extraction costs and saving procedures, and is also more conducive to the subsequent separation of type 4 and type 6 chondroitin.

Description

A kind of extraction method of chondroitin sulfate

The invention relates to a method for extracting chondroitin sulfate, in particular to a method for extracting chondroitin sulfate from sturgeon arowana cartilage.

Chondroitin is a polysaccharide composed of glucosaminoglycans, one of the constituents of cartilage tissue, which can keep chondrocytes sufficient water. Another experiment confirmed that taking chondroitin extracted from fish cartilage not only has anti-oxidation effect, but also inhibits lipid peroxidation, thus reducing the incidence of cardiovascular diseases in humans.

Most of the fish chondroitin products on the market are extracted from shark cartilage, but it is becoming more and more difficult to obtain shark cartilage, so it is urgent to find alternative sources of material. With the advancement of fish farming technology, sturgeon arowana has become a gradually popularized cultured fish. The fish bones of Arowana account for about 30% of the total fish weight, and are also rich in chondroitin, which is regarded as one of the main sources of chondroitin extraction in the future.

According to the different covalent positions of SO ₃ H in the chemical structure of chondroitin, chondroitin is often divided into type 4 chondroitin sulfate (chondroitin-4-sulfate, CS-4) which is sulfurized at the C4 position and type 6 chondroitin sulfate which is sulfurized at the C6 position. Two types of chondroitin sulfate (chondroitin-4-sulfate, CS-6). Recent studies have pointed out that different types of chondroitin sulfate have different biochemical mechanisms, resulting in different physiological effects. CS-4 plays an important role in the process of cartilage calcification, and has high anti-inflammatory and anti-oxidative effects when taken. CS-6 is related to the surface integrity of the joint.

In the current experimental technique, the chondroitin sulfate mixed extract of type 4 and type 6 can be obtained by conventional extraction method. Please refer to Figure 7. In the HPLC chromatogram, the chondroitin sulfate produced by the conventional extraction method will only have one peak (between 2.5 and 3 minutes). This peak contains the superimposition of the two peaks of chondroitin sulfate of type 4 and type 6. If only one type of chondroitin is needed, add chondroitinase A (Chondroitinase A) to decompose type 4 chondroitin sulfate, or add chondroitinase C (Chondroitinase C) to decompose type 6 Chondroitin sulfate, finally purify and collect the remaining another type of chondroitin sulfate.

However, in order to collect one of the chondroitin sulfate forms in the traditional method, another chondroitin sulfate form that has been decomposed is bound to be wasted, and the separated forms 4 and 6 cannot be obtained at the same time at low cost and efficiently. type of chondroitin sulfate. In addition, the traditional method uses sodium hydroxide or hydrochloric acid as a strong acid and strong base. During the extraction process, acid-base neutralization must be carried out. In this reaction, a large amount of salt waste liquid will be produced. If the waste liquid is discharged directly without special treatment, it will be harmful to the environment. The environment is harmful, and if there is residual strong acid and strong alkali, it will be more polluting

In view of this, the present invention proposes an extraction method of chondroitin sulfate, which avoids the production of salts while reducing extraction costs and saving procedures, and is conducive to the subsequent effective purification of type 4 and type 6 chondroitin , and even adjust the extraction ratio of Type 4 and Type 6.

The chondroitin sulfate extraction method provided by the present invention comprises the following steps: providing cartilage powder; providing a digestion solution, which contains Guanidine-HCl; mixing the cartilage powder and the digestion solution to obtain a cartilage powder digestion solution; extracting supernatant of cartilage powder digestion solution; mix The supernatant and multiple proteases form an enzyme reaction solution; the proteases in the enzyme reaction solution are inactivated; and chondroitin sulfate is separated from the enzyme reaction solution.

Wherein, in the step of providing cartilage powder, the following sub-steps are further included: providing a sturgeon cartilage; contacting the sturgeon cartilage with liquid nitrogen to embrittle the sturgeon cartilage; and pulverizing the sturgeon cartilage to obtain the sturgeon cartilage Fish cartilage powder.

Wherein, the digestion solution contains 0.5-2M guanidine hydrochloride and 0.2-1.0mM ethylenediaminetetraacetic acid (EDTA).

In particular, in the step of mixing the cartilage powder and the digestion solution, the further step is: mixing the cartilage powder and the digestion solution to obtain the cartilage powder digestion solution, and the mixing ratio of the cartilage powder to the digestion solution is between 25% and 50% w/v.

Wherein, in the step of extracting the digestion supernatant of the cartilage powder digestion liquid, the following sub-steps are further included: Ultrasonic vibration of the cartilage powder digestion liquid for 40-90 minutes in an environment of 50-65°C; centrifugation at a speed of 8000-12000rpm Cartilage powder digestion solution for 8-12 minutes; and the digestion supernatant from the upper layer of the cartilage powder digestion solution.

Wherein, the protease contains a Bacillus subtilis alkaline protease (Alcalase), and the final concentration of the Bacillus subtilis alkaline protease in the enzyme reaction liquid is between 0.5-3.0% v/w. The protease also includes a papain (Papain) and a flavor protease (Flavourzyme), and in the step of mixing the digestion supernatant and multiple proteases, the following sub-steps are further included: mixing the digestion supernatant and Bacillus subtilis alkaline Protease, papain and flavor protease, the final concentration of papain is between 2~10% w/w, the final concentration of flavor protease is between 0.2~1.0% v/w; adjust the acid-base of the enzyme reaction solution The value is between 6.8~7.2; and the enzyme reaction solution is stirred in an environment of 50~60°C for 40~90 minutes.

Wherein, in the step of inactivating the protease in the enzyme reaction solution, the further step is: placing the enzyme reaction solution in an environment above 90° C. for 10 minutes to inactivate the protease in the enzyme reaction solution.

Wherein, in the step of precipitating chondroitin sulfate from the enzyme reaction solution, the following sub-steps are further included: performing a rough extraction procedure to obtain a crude extract from the enzyme reaction solution, and redissolving the crude extract to obtain a solution; and using high performance liquid chromatography (HPLC) to purify a Type 4 chondroitin sulfate solution and a Type 6 chondroitin sulfate solution from the back solution respectively.

Another step of separating out chondroitin sulfate from the enzyme reaction solution further includes the following sub-steps: cooling the enzyme reaction solution; mixing the enzyme reaction solution with 95% ethanol to form a solution to be separated, and the volume of 95% ethanol is the enzyme reaction solution 400~600% of the volume; stir the solution to be precipitated at 2~6°C for at least 8 hours and produce a crude extract; centrifuge the solution to be precipitated at 8000~12000rpm for 8~12 minutes and collect the crude extract; Dissolving the dialysis precipitate in double distilled water (ddH2O) to obtain a back solution; using high performance liquid chromatography to purify a type 4 chondroitin sulfate solution and/or a type 6 chondroitin sulfate solution from the back solution and freeze and dry the 4th type chondroitin sulfate solution and/or the 6th type chondroitin sulfate solution, to obtain a 4th type chondroitin sulfate powder and/or a 6th type chondroitin sulfate powder, type 4 Chondroitin sulfate powder contains type 4 chondroitin sulfate, and type 6 chondroitin sulfate powder contains type 6 chondroitin sulfate.

In summary, the present invention provides a method for separating and extracting chondroitin sulfate, which improves the yield while reducing extraction costs and saving procedures, and avoids the generation of salts. More importantly, the product is easy to separate type 4 and type 6 chondroitin at the same time, and the ratio of type 4 and type 6 in the final product can even be adjusted by adding enzymes during the extraction process.

Compared with the conventional technology, chondroitin sulfate type 4 and type 6 with overlapping peaks mixed in the extraction product must be further selectively decomposed by chondroitinase; the technology provided by the present invention is to add guanidine hydrochloride in the process Sodium hydroxide or hydrochloric acid is replaced, and the precipitated chondroitin sulfate can be easily separated into type 4 and type 6 by high performance liquid chromatography, without adding chondroitinase, and the yield is also greatly improved. In addition, when sodium hydroxide or hydrochloric acid is used to react, salts will be produced. If the salts are not removed, the solution will be toxic, and the waste liquid will be highly polluting; while the guanidine hydrochloride solution is only weakly acidic. Harmless to the environment and non-toxic to organisms. Therefore, in comparison, the extraction method of the present invention saves the cost of the subsequent process, improves the yield, reduces the toxicity of the product, reduces additional waste liquid, and also obtains different types of chondroitin separated without wasting any type Chondroitin.

The advantages and spirit of the present invention can be further understood through the following detailed description of the invention and the accompanying drawings.

S1~S7: steps

S11~S13, S41~S43, S51~S53, S71~S79: sub-steps

Fig. 1 is a flowchart showing the steps of the extraction method of chondroitin sulfate of the present invention.

Fig. 2 is a flowchart showing the sub-steps of providing cartilage powder in the present invention.

Fig. 3 is a flowchart showing the sub-steps of extracting the digested supernatant in the present invention.

Fig. 4 is a flowchart showing the sub-steps of forming the enzyme reaction solution in the present invention.

Fig. 5 is a flow chart showing the sub-steps of separating out chondroitin from the mixed solution in the present invention.

Fig. 6 is a flow chart showing another sub-step of separating out chondroitin from the mixed solution in the present invention.

Fig. 7 is the HPLC analysis spectrum of the chondroitin extracted by conventional extraction methods.

Fig. 8 is a Fourier transform infrared spectrum analysis diagram of the product in an embodiment of the present invention.

Fig. 9 is an ultraviolet-visible light analysis spectrogram of the product in an embodiment of the present invention.

Fig. 10A is a spectrum formed after high performance liquid chromatography of the products of experimental group A in an embodiment.

Fig. 10B is a spectrum formed after high performance liquid chromatography of the product of experimental group B in an example.

Fig. 10C is a spectrum formed after high performance liquid chromatography of the product of experimental group C in an example.

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and examples.

See Figure 1. Fig. 1 is a flowchart showing the steps of the extraction method of chondroitin sulfate of the present invention. The preparation method of the sturgeon cartilage extract of the present invention comprises the following steps. Step S1, providing cartilage powder; step S2, providing a digestion solution, which contains Guanidine-HCl; step S3, mixing cartilage powder and digestion solution to obtain a cartilage powder digestion solution; step S4, extracting cartilage powder The digestion supernatant of the digestive juice; step S5, mixing the digestion supernatant and a plurality of proteases to form an enzyme reaction solution; step S6, inactivating the protease in the enzyme reaction solution; step S7, separating from the enzyme reaction solution Chondroitin monosulfate. There is no absolute sequence relationship between step S1 and step S2.

In the prior art, the digestion solution in step S2 is usually sodium hydroxide (NaOH), hydrochloric acid (HCl), sulfuric acid (H2SO4) plus acetone (acetone), to remove grease, minerals, and hard bone proteins in the cartilage powder and other elements. However, after the digestion solution in the conventional method reacts, salts will be produced. If the salts are not removed, the final product will be toxic, and it is difficult to ensure that the salts are completely removed in practice; and the removal of waste liquid has Highly polluting and unfriendly to the environment. After the extraction of chondroitin sulfate, both type 4 chondroitin sulfate and type 6 chondroitin sulfate are mixed in the extracted product. Due to the close separation time of the two on the chromatographic column and the interference of other compounds between the two, the two present a single peak on the chromatogram, and the extracted products cannot be easily separated by high performance liquid chromatography (HPLC). The existing technology must continue to react the extracted product with chondroitinase to decompose the two types of Only one of them can obtain another type of chondroitin sulfate, chondroitinase is expensive and the purification steps are cumbersome.

The digestion solution in step S2 of the present invention is mainly guanidine hydrochloride, without adding sodium hydroxide or hydrochloric acid, and the reaction process will not produce salts. Therefore, the final product has no toxicity, and the waste liquid has no pollution problem. What was even more unexpected was that when the final product was subjected to high performance liquid chromatography without the action of chondroitinase, a double peak would appear on the chromatogram, which could be easily separated by using the delay time difference of high performance liquid chromatography.

Please refer to Figure 1 and Figure 2. Fig. 2 is a flowchart showing the sub-steps of providing cartilage powder in the present invention. In the step S1 of providing cartilage powder, the following sub-steps are further included: sub-step S11, providing a sturgeon arowana cartilage; sub-step S12, contacting the decomposed arowana cartilage with liquid nitrogen to embrittle the sturgeon arowana cartilage; sub-step S13, And crush Arowana Cartilage to get Arowana Cartilage Meal. In the prior art, most of the methods only use grinding to destroy the cartilage of sturgeon arowana, but it takes a long time, and the components exposed to the air are easy to deteriorate. In this method, liquid nitrogen is first used to embrittle the cartilage, and the pulverization time will be accelerated by at least two times, and the yield of the rear end can be improved.

In the digestion solution in step S2, the appropriate concentration of guanidine hydrochloride is between 0.5-2M. In addition, 0.2-1.0 mM ethylenediaminetetraacetic acid (EDTA) was added in step S2. EDTA can be neutralized with slightly acidic guanidine hydrochloride to stabilize the pH value of the solution. And EDTA can chelate the metal ions in the cartilage powder, and in step S7, EDTA is removed together with the metal ions.

In the step S3 of mixing the cartilage powder and the digestion solution, the further step is: step S30, mixing the cartilage powder and the digestion solution to obtain the cartilage powder digestion solution, the mixing ratio of the cartilage powder to the digestion solution is between 25-50% w/v between. According to the purity of the cartilage powder and the concentration of guanidine hydrochloride and EDTA in the digestive solution, the mixing ratio can be adjusted appropriately.

Please refer to Figure 1 and Figure 3. Fig. 3 is a flowchart showing the sub-steps of extracting the digested supernatant in the present invention. In the step S4 of extracting the digestion supernatant of the cartilage powder digestion liquid, the following sub-steps are further included: sub-step S41, ultrasonic vibration of the cartilage powder digestion liquid in an environment of 50-65 °C for 40-90 minutes; sub-step S42, with 8000 Centrifuge the cartilage powder digestion solution at ~12000rpm for 8-12 minutes; sub-step S43, extract the digestion supernatant from the upper layer of the cartilage powder digestion solution. Ultrasonic vibration can physically destroy the bone structure and prevent microbubbles from adhering to the cartilage powder and hindering the digestive reaction. If only stirring is used, the reaction time may be extended to 15 hours. The time of digestion reaction can be properly adjusted according to the intensity and frequency of ultrasonic vibration, ambient temperature, and the mixing ratio of digestive juice. The sub-step S42 of centrifuging the cartilage powder digestion liquid and the sub-step S43 of extracting the supernatant can remove bone residue or other incompletely decomposed structures, and retain the target product. Substep S42 and substep S43 can be repeated to collect more digestion supernatant.

Please refer to Figure 1 and Figure 4. Fig. 4 is a flowchart showing the sub-steps of forming the enzyme reaction solution in the present invention. Wherein, the type of protease includes a subtilis alkaline protease (Alcalase), a papain (Papain) and a flavor protease (Flavourzyme), and in the step of mixing the digestion supernatant and a plurality of proteases, further includes the following Sub-step: Sub-step S41, mixing the digestion supernatant with Bacillus subtilis alkaline protease, papain and flavor protease, the final concentration of Bacillus subtilis alkaline protease in the enzyme reaction liquid is between 0.5-3.0% v/w, The final concentration of papain is between 2~10% w/w, the final concentration of flavor protease is between 0.2~1.0% v/w; sub-step S42, adjust the pH value of the enzyme reaction solution to 6.8~ Between 7.2; sub-step S43, stirring the enzyme reaction solution in an environment of 50-60°C for 40-90 minutes.

For example, the final concentration of each protease in the enzyme reaction solution is: Bacillus subtilis alkaline protease 1.5% v/w, papain 5% w/w, flavor protease 0.5% w/w, adjust acid-base The value was 7, and the enzyme reaction solution was stirred for 60 minutes in an environment of 55°C. Protease acts on the remaining impurities to purify chondroitin sulfate.

In the prior art, this step uses trypsin instead of Bacillus subtilisin for the reaction. In the method of the present invention, adding Bacillus subtilis alkaline protease can greatly increase the yield of CS-4 in the product, and correspondingly reduce the yield of CS-6 in the product. In this way, the Bacillus subtilisin alkaline protease can be selectively added during the extraction process based on the chondroitin type and ratio requirements of the final product.

In the step S6 of inactivating the protease in the enzyme reaction solution, the further step is: step S60, placing the enzyme reaction solution in an environment above 90° C. for 10 minutes to inactivate the protease in the enzyme reaction solution.

Please refer to Figure 1 and Figure 5. Fig. 5 is a flow chart showing the sub-steps of separating out chondroitin from the mixed solution in the present invention. In the step S7 of separating out chondroitin sulfate from the enzyme reaction solution, the following sub-steps are further included: sub-step S71, a rough extraction procedure is carried out to obtain a crude extract from the enzyme reaction solution, and the crude extract is redissolved to Obtaining a back solution; and sub-step S72 , using high performance liquid chromatography (HPLC) to purify a Type 4 chondroitin sulfate solution and a Type 6 chondroitin sulfate solution from the back solution. In sub-step S71, the crude extract can be dissolved back in pure water or a solution containing a small amount of salt, such as 0.5-2.5% sodium chloride aqueous solution. The crude extraction procedure in sub-step S71 can be performed by various techniques, such as solution dialysis with a dialysis membrane with a molecular weight of 6,000-8,000 MWCO. Since the digestion solution in step S2 contains guanidine hydrochloride, both type 4 chondroitin sulfate (CS-4) and type 6 chondroitin sulfate (CS-6) will be significantly Separate, and then use the delay time difference between the two to collect the outflowing type 4 chondroitin sulfate solution and type 6 chondroitin sulfate solution respectively.

See Figure 1 and Figure 6. Fig. 6 is a flow chart showing another sub-step of separating out chondroitin from the mixed solution in the present invention. In another step of separating out chondroitin sulfate from the enzyme reaction solution, the following sub-steps are further included: sub-step S73, cooling the enzyme reaction solution; sub-step S74, mixing the enzyme reaction solution and 95% ethanol to form a solution to be separated, The volume of 95% ethanol is 400-600% of the volume of the enzyme reaction solution; sub-step S75, stir the solution to be precipitated at 2-6°C for at least 8 hours and produce a crude extract; sub-step S76, at 8000-12000rpm Centrifuge the solution at a high speed for 8-12 minutes and collect the crude extract; sub-step S77, redissolve the dialyzed precipitate in twice distilled water (ddH2O) to obtain a solution; sub-step S78, use high performance liquid chromatography Purify a type 4 chondroitin sulfate solution and/or a type 6 chondroitin sulfate solution from the back solution; substep S79, freeze and dry the type 4 chondroitin sulfate solution and/or type 6 chondroitin sulfate solution to obtain a Type 4 chondroitin sulfate powder and/or a Type 6 chondroitin sulfate powder, the Type 4 chondroitin sulfate powder containing Type 4 chondroitin sulfate, and the Type 6 chondroitin sulfate powder containing Type 6 type chondroitin sulfate.

In sub-step S74, 70% or other concentration of ethanol can also be used, but the corresponding volume of ethanol must be increased, and ethanol can further take away the impurities of non-target products in the enzyme reaction solution. In sub-step S75, it is better to stir the solution to be precipitated overnight in a refrigerated environment, and the crude extract will gradually precipitate out. In sub-step S76, centrifugation may be performed multiple times to remove alcohol, residual guanidine hydrochloride and ethylenediaminetetraacetic acid and other substances. In sub-step S77, the crude extract can be dissolved back in pure water or a solution containing a small amount of salt, such as 0.5-2.5% sodium chloride aqueous solution. After the crude extract was redissolved, it was further filtered through a 0.22um filter. Since the digestion solution in step S2 contains guanidine hydrochloride, both type 4 chondroitin sulfate (CS-4) and type 6 chondroitin sulfate (CS-6) will be significantly Separate, and then use the delay time difference between the two to collect the outflowing type 4 chondroitin sulfate solution and type 6 chondroitin sulfate solution respectively. By sub-step S79, the 4th type chondroitin sulfate solution or the 6th type sulfuric acid The chondroitin solution reforms into a powder state with relatively stable properties.

In the following examples, the benefits of the method of the present invention will be illustrated by means of experimental chart results.

The extraction and preparation process of chondroitin sulfate in this example is to cook the sturgeon arowana, remove the fish meat, and only keep the cartilage. Wash the cartilage with clean water to remove oil initially, and then dry it to remove moisture. Use a bean grinder to crush the cartilage initially, then add the crushed cartilage to liquid nitrogen to make it embrittled, and then grind the crushed cartilage into sturgeon cartilage powder.

Prepare 2M guanidine hydrochloride and 1mM ethylenediaminetetraacetic acid in ddH2O, and mix them at a volume ratio of 1:1 to form a digestion solution. The final concentration of guanidine hydrochloride in the digestion solution was 1 M, and the final concentration of EDTA was 0.5 mM. Mix the sturgeon cartilage powder with the digestion solution at a ratio of 33% w/v to form the cartilage powder digestion solution.

The cartilage powder digestion solution was placed in an ultrasonic oscillator (Branson 3510), and reacted by shaking at 60°C for 1 hour. After the reaction was completed, the cartilage powder digestion solution was centrifuged at 10000 rpm for 10 minutes, and the digestion supernatant was removed. Then add Bacillus subtilis alkaline protease (Alcalase), papain (Papain) and flavor protease (Flavourzyme) into the digestion supernatant respectively to form an enzyme reaction solution. The final concentration of Bacillus subtilisin alkaline protease was 1.5% v/w, papain final concentration was 5% w/w, and flavor protease final concentration was 0.5% v/w. In addition, an experimental group in which Bacillus subtilis alkaline protease was not added to the enzyme reaction solution was used as a comparison. Adjust the pH value of the enzyme reaction solution to 7.0, and stir and react at a temperature range of 50-60° C. for 1 hour.

Bacillus subtilis alkaline protease (Alcalase) is selected from Sigma's P4860, the specific activity (specific activity) is at least 2.4U/g, and it is in a liquid state. Papain was purchased from Shunyi Chemicals in powder form. Flavor protease is a protease isolated from Koji oryzae, selected from Sigma's P6110, with a specific activity of at least 500U/g, in liquid state. However, the protease mentioned above may vary from manufacturer to manufacturer purity or specific activity. Therefore, if proteases provided by different manufacturers are used in this method, and a ratio outside the concentration range of the present invention is used, but the specific activity or purity ratio contributed by the protease is still the same as the present invention, it should still constitute the principle of equivalence without departing from the scope of the present invention.

After the reaction of the enzyme reaction solution, heat to 100°C for 10 minutes to inactivate the protease. After cooling the enzyme reaction solution, add 95% ethanol 5 times the volume of the enzyme reaction solution to form a solution to be precipitated, and stir and precipitate in a refrigerator at 4°C overnight.

The next day, the solution to be precipitated was centrifuged at 10,000 rpm for 10 minutes to remove ethanol, residual guanidine hydrochloride and ethylenediaminetetraacetic acid, and the precipitated crude extract was retained. After redissolving the crude extract in ddH2O, filter it with a 0.22um filter to obtain a chondroitin sulfate solution. At this time, at least one of component analysis experiments, separation of type 4 chondroitin sulfate and type 6 chondroitin sulfate by HPLC, and lyophilization of the solution for several days using a freeze dryer can be performed, and there is no restriction on the order of combination. The above is the extraction and preparation process of chondroitin sulfate in this embodiment.

The component analysis experiment of the chondroitin sulfate solution was carried out at this time point as follows. The experimental group is divided into A: commercially available chondroitin product (Sigma C4384), which has been treated with chondroitinase, and used as a chondroitin component standard in this experiment for comparison (CS standard); B: using the present invention The chondroitin sulfate solution product obtained by the method is not added with Bacillus subtilis alkaline protease (Alcalase negative); C: the chondroitin sulfate solution product obtained by using the method of the present invention, the process is added with Bacillus subtilis alkaline protease (Alcalase positive).

The first analysis is Fourier Transform Infrared Spectroscopy (FTIR) analysis. See Figure 8. Fig. 8 is the Fourier transform infrared spectrum analysis figure of the product of the method of the present invention. The main purpose of Fourier transform infrared spectroscopic analysis is to preliminarily confirm whether the extracted substance of the present invention is chondroitin sulfate, and there are representative functional groups in different wave number intervals, and the fingerprint area in the wave number interval of 600~1400cm ^-1 is known The closer the wave types, the higher the similarity between the two functional groups. The characteristic peaks at 857cm ^-1 and 825cm ^-1 are the characteristic peaks of the sulfur functional groups at C4 and C6 respectively. In Figure 8, experimental group B and experimental group C have similar functional group performances compared with experimental group A as a standard product, and have the characteristics of expressing CS-4 and CS-6 at 857cm ^-1 and 825cm ^-1 respectively It can be preliminarily determined that the extracted substance is mainly chondroitin sulfate. This picture is to split the three stacked results up and down for easy viewing.

Chondroitin sulfate is a polysaccharide with uronic acid in its structure. The second analysis is the total polysaccharide content analysis (Phenol-sulfuric Assay), the purpose is to detect the total polysaccharide content contained in the extract. The third analysis is uronic acid content analysis (Carbazole Assay), the purpose is to detect the content of uronic acid contained in the extract. The results of the two analyzes are shown in the table below.

Table 1. Contents of total polysaccharides and uronic acid in chondroitin from different experimental groups

The total polysaccharide content in the chondroitin products produced by two kinds of extraction methods (B experimental group, C experimental group) in the present invention reaches 94.80% and 93.10% respectively, relative to the 84.45% of the commercially available products of A experimental group, it means that this Invented extraction method can obtain higher total polysaccharide content. The content of uronic acid in the chondroitin products produced by the extraction method of experimental group C in the present invention reaches 71.12%, which is higher than 58.53% of the commercially available products of experimental group A.

The fourth analysis is UV-Visible Spectrometer Analysis. This analysis uses UV light to irradiate the target, and observes the absorption peak excited by it to determine the similarity and concentration of the substance. In this test, it can be more specific to detect sulfated cartilage chondroitin sulfate content, and can further calculate the chondroitin sulfate yield of the extraction method.

See Figure 9. Fig. 9 is the ultraviolet-visible light analysis spectrogram of the product of the method of the present invention. In this analysis result, the peak positions of the three experimental groups are all in similar positions, indicating that the similarity is high. Only the peak position of experimental group C (Alcalse+) is to the right, which should be due to more CS-4 components; the peak position of experimental group B (Alcalse-) is to the left, which should be due to more CS-6 components; experimental group A (CS Standard) peak is approximately in the middle. According to the concentration converted according to the area occupied by the wave peak, the concentration of the experimental group C is the highest, followed by the experimental group A, and the experimental group B is lower.

The fifth analysis was high performance liquid chromatography (HPLC). High performance liquid chromatography utilizes the interaction between the mobile phase and the stationary phase. When the mixture enters the system, the characteristics of different components will determine the speed of the substance flowing in the chromatography column, and then use each The difference in the rate of movement of components is used to map or separate.

Please refer to Figure 7, Figures 10A, 10B, 10C. 10A, 10B, and 10C are the spectra formed after high performance liquid chromatography of experimental group A, experimental group B, and experimental group C in this embodiment, respectively. There are three peaks in each of the three spectra, which appear around 1.1 minutes, 1.3 minutes, and 1.6 minutes. The peak separated at 1.1 minutes represents the component of CS-0, which has no sulfur functional group and is chondroitin which has no effect. The peak separated at 1.3 minutes represents the composition of CS-6, and the type 6 chondroitin sulfate can be separated by collection at this time; the peak separated at 1.6 minutes represents the composition of CS-4, and the type 4 chondroitin sulfate can be separated by collection at this time white.

In the commercially available standard product of experimental group A in FIG. 10A , the peak of CS-4 in the product treated with chondroitinase is very high, indicating that the content of CS-4 is high. And compared with the map of the product without chondroitinase treatment by the traditional method in Fig. 7, CS-4 in Fig. 10A is clearly separated from CS-0 and CS-6. However, the peak of CS-0 in experimental group A was also very high and almost swallowed the peak of CS-6, indicating that the CS-6 content of its output was relatively high. Low.

Experimental group B in FIG. 10B is a group that uses the extraction method of the present invention and does not add Bacillus subtilis alkaline protease (Alcalase negative). The peak of CS-6 in the spectrum is very high, indicating that the content of CS-6 is high. The relative CS-4 peak is low, but there is almost no CS-0 peak. Compared with the spectrum of the product of the traditional method without chondroitinase treatment in Figure 7, the method of the experimental group B of the present invention is also without chondroitinase treatment, but the CS-4 peak and the CS-6 peak are separated by 0.3 minutes. The trough can effectively use the delay time to collect Type 4 Chondroitin Sulfate and Type 6 Chondroitin Sulfate respectively.

Experimental group C in FIG. 10C is a group using the extraction method of the present invention and adding Bacillus subtilis alkaline protease (Alcalase positive). The peak of CS-4 in the spectrum is very high, indicating that the content of CS-4 is high. The relative CS-6 peak is lower, but still higher than the CS-0 peak. Compared with the spectrum of the product of the traditional method without chondroitinase treatment in Figure 7, the C experimental group method of the present invention is also without chondroitinase treatment, but the interval between the CS-4 peak and the CS-6 peak is 0.3 minutes. The trough can effectively use the delay time to collect Type 4 Chondroitin Sulfate and Type 6 Chondroitin Sulfate respectively.

The chondroitin content can be deduced from the fourth analysis and the fifth analysis, followed by the yield calculation. Yield is calculated by dividing the actual extracted chondroitin content per gram of cartilage by the theoretical chondroitin content per gram of cartilage (7%). By converting the chondroitin content actually extracted from each gram of cartilage in the experimental group B and the experimental group C, the conversion yield was 80.23% for the experimental group B and 86.42% for the experimental group C. Since commercially available products do not indicate the yield of their production process, the data of their chondroitin yield were obtained from previous relevant research literature for comparison. The following is the yield comparison table of C experimental group and previous literature.

Table 2. Comparison table of chondroitin yield in different literatures

It is known from Table 2 that most of the products obtained by the previous literature process are rich in type-6 chondroitin sulfate (CS-6), while the proportion of type-4 chondroitin sulfate (CS-4) rarely exceeds 59%. And, the productive rate in previous literature is lower than 40%, far lower than 86.42% of the method of the present invention. In other words, compared with the previous literature, the method of the present invention not only has a very high yield, but also can obtain a higher proportion of type IV chondroitin sulfate.

In addition, the protein concentration of the method of the present invention is close to zero (no detection), indicating that there is almost no protein in the ingredients, and the above extraction process has successfully removed unnecessary proteins, which helps to improve the purity of the extract.

In summary, the present invention provides a method for separating and extracting chondroitin sulfate, which increases the yield (up to 80% by reducing the extraction cost (chondroitinase consumable cost) and saving procedures (without waiting for the action of chondroitinase). above), and avoid generating salts (no hydrochloric acid or sodium hydroxide). In addition, the product is easy to separate type 4 and type 6 chondroitin in high performance liquid chromatography (the difference between the two is 0.3 minutes); even the enzyme (subtilis alkaline protease) added during the extraction process can be used , and then adjust the ratio of type 4 chondroitin and type 6 chondroitin in the final product (the ratio of type 4 chondroitin increases significantly after adding).

Compared with the conventional technology, the extraction product is mixed with Type 4 and Type 6 chondroitin sulfate with overlapping peaks, which must be further selectively decomposed by chondroitinase; the technology provided by the present invention, In the process, guanidine hydrochloride was added instead of sodium hydroxide or hydrochloric acid, and the precipitated chondroitin sulfate can be easily separated into type 4 and type 6 by high performance liquid chromatography, without adding chondroitinase, and the yield is also low. raised dramatically. In addition, when sodium hydroxide or hydrochloric acid is used to react, salts will be produced. If the salts are not removed, the solution will be toxic, and the waste liquid will be highly polluting; while the guanidine hydrochloride solution is only weakly acidic. Harmless to the environment and non-toxic to organisms. Therefore, in comparison, the extraction method of the present invention saves the cost of the subsequent process, improves the yield, reduces the toxicity of the product, reduces additional waste liquid, and also obtains different types of chondroitin separated without wasting any type Chondroitin.

Through the above detailed description of the preferred embodiments, it is hoped that the characteristics and spirit of the present invention can be described more clearly, and the scope of the present invention is not limited by the preferred embodiments disclosed above. On the contrary, the intention is to cover various changes and equivalent arrangements within the scope of the patent application for the present invention.

S1~S7: steps

Claims (2)

A method for extracting chondroitin sulfate, comprising the steps of: providing a cartilage powder; providing a digestion solution, which contains 1M guanidine hydrochloride (Guanidine-HCl) and 0.5mM ethylenediaminetetraacetic acid (EDTA); Mix the cartilage powder and the digestion solution at a ratio of 33% w/v to obtain a cartilage powder digestion solution; ultrasonically shake the cartilage powder digestion solution at 60°C for 60 minutes; centrifuge the cartilage powder digestion solution at 10,000rpm for 10 minutes minutes; extract one of the digestion supernatant of the cartilage powder digestion solution; mix the digestion supernatant with 1.5% v/w of Bacillus subtilis alkaline protease (Alcalase), 5% w/w of papain (Papain) and 0.5 % v/w flavor protease (Flavourzyme) to form an enzyme reaction solution; adjust the pH value of the enzyme reaction solution to 7.0; stir the enzyme reaction solution at 55°C for 60 minutes; place the enzyme reaction solution at 90°C In the above environment for 10 minutes, inactivate the proteases in the enzyme reaction solution; cool the enzyme reaction solution; mix the enzyme reaction solution and ethanol to form a solution to be precipitated; stir the solution to be precipitated at 2~6°C The solution was at least 8 hours and a crude extract was produced; the solution to be precipitated was centrifuged at a speed of 10000 rpm for 10 minutes and the crude extract was collected; Back-dissolving the dialyzed precipitate in double distilled water (ddH2O) to obtain a back solution; using high performance liquid chromatography to purify a Type 4 chondroitin sulfate solution and/or a Type 6 chondroitin sulfate solution from the back solution Type chondroitin sulfate solution; and freezing and drying the Type 4 chondroitin sulfate solution and/or the Type 6 chondroitin sulfate solution to obtain a Type 4 chondroitin sulfate powder and/or a Type 6 chondroitin sulfate powder , the type 4 chondroitin sulfate powder contains a type 4 chondroitin sulfate, and the type 6 chondroitin sulfate powder contains a type 6 chondroitin sulfate. The extraction method as described in item 1 of the scope of the patent application, wherein in the step of providing the cartilage powder, the following sub-steps are further included: providing a sturgeon cartilage; contacting the sturgeon cartilage with a liquid nitrogen to make the sturgeon cartilage embrittlement of fish cartilage; and pulverizing the sturgeon cartilage to obtain the cartilage powder.

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