TWI382085B - The use of chitin to produce pethlosan (piceatannol) method - Google Patents

Description

Method for producing piceatannol using chitin

The present invention relates to a method for simultaneously producing piceatannol and resveratrol, and more particularly to a method for producing spruce and resveratrol by using chitin to induce groundnut callus.

Plants are a source of many herbs or chemicals that humans use to fight disease. Some plants are infected with pathogens or other environmental stimuli to produce phytoalexin, while piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene) and resveratrol ( Resveratrol, trans-3, 5, 4'-trihydroxystilbene) are plant defensins and low molecular weight polyphenolic compounds based on stilbene. The two compounds mentioned above have various health benefits such as anti-oxidation, inhibition of platelet aggregation, prevention of myocardial stiffness, cerebral embolism, protection against hypoxic heart, inhibition of cancer cell proliferation, anti-inflammatory and expansion of arterial vessels and improvement of microcirculation. . However, until now, it has been found that plants containing spruce are rare and low in content, and even in the case of high acacia content, they are only in the range of μg. Since the content is too low, it is difficult to directly extract spruceol from plants. In addition, high-purity resveratrol is also commercially expensive, so how to obtain a reasonable price and easy source of spruceol and resveratrol is A big topic.

Related studies have shown that derivatives of plant or fungal cell walls (enzymes, cell wall fragments) induce plant-derived plant defensins. When a fungus infects a plant, the cell wall is the first structure that comes into contact with the plant. The main component of many fungal cell walls is chitin, and it has been documented that when infected by a pathogen, the plant produces some enzymes such as chitinase. And other pathogenesis-related proteins (PR proteins), these enzymes are simultaneously activated and directly resistant to pathogen infection. When the plant recognizes pathogenic bacteria, the plant-derived chitinase decomposes the pathogenic cell wall. Chitin is released and converted to chitosan by deacetylation. Deacetylated chitin induces different defense responses in many plants, such as pea pods producing plant defensins, leaves of rice seedlings producing pathogenicity-related proteins, and lignification of injured wheat leaves inhibits fungal growth and infection. The addition of chitin to the medium of Drosophyllum lusitanicum Link induces the production of plumbagin.

In other words, if chitin is a fungal infection of the main component of the cell wall, the plant will produce a pathogenic protein such as chitinase. The plant will decompose the fungal cell wall by chitinase, and the chitin of the fungal cell wall will It has been converted to chitosan, and studies have found that deacetylated chitin induces different defense responses of many plants, such as the production of plant defensins.

The aforementioned related studies show that chitosan is directly related to the production of plant defensins, and in the literature currently using chitin as the source of induction, chitin is also required to be dissolved in acid to make chitin pass the deacetylate. The action is converted into chitosan, which is then added to the medium to induce the formation of plant defensins or to investigate the antibacterial activity. However, there is currently no literature on the direct use of chitin to induce callus to produce plant defensins, especially the callus that induces groundnuts to produce spruce and resveratrol.

Accordingly, the present invention is directed to solving the above-described disadvantages of the prior art. In order to solve the problems of the prior art, the object of the present invention is to provide a method for simultaneously producing piceatannol and resveratrol, in particular to use chitin to induce the production of groundnut callus. Method of spruceol and resveratrol.

Another object of the present invention is to mass produce a method of providing spruceol and resveratrol using tissue culture.

For the purpose of the foregoing, the present invention uses chitin to produce spruceol and resveratrol. The method of the present invention comprises: taking a hypocotyl of a groundnut as a culture, forming a wound on the implant; Adding a suspension solution of chitin to a callus formed by the wound; and finely pulverizing and filtering the callus to obtain spruceol and resveratrol; wherein the hypocotyl of the groundnut is from a seed after de-pod Deionized water washing, and 1% sodium hypochlorite plus soil temperature 20 emulsifier (TWEEN 20), washed and incubated for 2 minutes with ultrasonic vibration; then a wound was cut on the implant and the implant was placed Dark culture is carried out at room temperature in a medium containing 0.1-0.3 mg/L naphthaleneacetic acid (1-naphthylacetic acid) and 0.05-0.08 mg/L benzylamino purine to form callus, and The callus is further finely ground and filtered to obtain spruceol and resveratrol.

The present invention directly uses chitin to induce callus without first dissolving chitin in an acid, converting it to deacetylation to chitosan, and then inducing callus. The tissue produces a stilbene-like compound.

The method of the invention is easy to operate, chitin and groundnut are convenient as plant materials for inducement and cell culture, can reduce environmental impact, and obtain high concentration of spruceol and resveratrol, wherein the concentration of chitin suspension aqueous solution and spruce The yield of alcohol (piceatannol) shows a positive dose dependency relationship; and the present invention can start to produce spruceol and resveratrol at the 4th hour after adding the aqueous solution of chitin suspension, which is much higher than the rate of spruceol produced in the rest of the literature, so The present invention provides a method for efficiently producing spruceol and resveratrol.

The above and other objects and advantages of the present invention will be described in detail with reference to the accompanying drawings and claims. However, it is to be understood that the appended drawings are purely illustrative of the spirit of the invention and are not to be construed as limiting the scope of the invention. For a definition of the scope of the invention, please refer to the attached patent application.

In order to confirm that the present invention can produce piceatannol and resveratrol, the present invention analyzes the concentration of 0.01, 0.02, 0.04 g/mL chitin suspension by high performance liquid chromatography (HPLC). The aqueous solution treated callus was used to detect the effect of different concentrations of chitin suspension aqueous solution to induce the production of spruceol and resveratrol. In addition, the effect of the length of the callus induced by the aqueous suspension of chitin on the amount of spruceol and resveratrol was also evaluated. From these test results, it was confirmed that the present invention can produce the effects of spruceol and resveratrol using a suspension aqueous solution of chitin. The foregoing embodiment is described in detail as follows:

Example 1 Induction of callus from groundnut

Groundnut seeds are provided by Yunlin Farmhouse No. 9 Tainan. After de-pod, the seeds were washed with water, and then sterilized by ultrasonic wave with 2% sodium hypochlorite and two drops of soil temperature 20 emulsifier (TWEEN 20) in an aseptic table, and then aseptically sown in MS (Murashige and Skoog, 1962) is the basic medium for the basic salt; add 30g/L sucrose and 2.5g/L crystal agar to the basal medium. Before adding the agar to the agar, adjust the pH to 5.8±0.3 with sodium hydroxide and hydrochloric acid. It was sterilized at 120 ° C, 1.05 Kg / cm 2 for 15 minutes, poured into a Petri dish, and cooled for use.

One week after the seeding of the groundnuts, the hypocotyls were taken as the culture body, and cut into a plurality of culture fragments, which were contained in 0.25 mg/L naphthalene acetic acid (1-naphthylacetic acid) and 0.075 mg/L benzylamino purine (6-benzylamino purine). In the basal medium, the callus is induced by the incision and subcultured every four weeks. Since the callus needs to grow in the absence of light and room temperature, the culture dish is placed in a growth chamber with a dark environment of 28 ± 1 ° C in the culture environment together with the culture body.

Example 2 Chitin-induced formation and extraction of stilbene-like compounds in groundnut callus

Chitin (purified from sigma) was first sieved through a 0.22 μm sieve, and then formulated into a suspension aqueous solution of 0.01, 0.02, and 0.04 g/mL, and sterilized and then cooled.

The peanut callus was subcultured for three weeks, and 1 mL of chitin suspension aqueous solution or sterile water (H2O) was dropped on the groundnut callus, and then the culture vessel which was subjected to induction treatment and not subjected to induction treatment (blank) was placed. After darkening at 28±1 °C for 12 hours, 1 g of callus was weighed and added to 100% methanol (MeOH) for grinding. Then, the mixture was filtered by suction for three times, then quantified to 5 mL, and finally filtered through a 0.22 μm filter. .

Example 3 Determination of the content of piceatannol and resveratrol induced by callus by high performance liquid chromatography (HPLC)

The determination of spruceol and resveratrol in the callus of the present invention is analyzed by high performance liquid chromatography (HPLC), and the analysis conditions are L-7100 HPLC pump, L7420 UV, L7485 fluorescence detector. (Hitachi Co. Ltd., Tokyo, Japan), and Mightysil C18 separation column (250 x 4.6 mm, 5 μL). The excitation and emission wavelengths of the fluorescence detector are designed to be 343 and 395 nm, respectively. The concentration of spruceol and resveratrol was calculated from the signal of the fluorescence chromatogram. The mobile phase was acetonitrile/0.1% formic acid deionized water with a mobile phase gradient of 20% acetonitrile, 32% after 20 minutes, and 90% after 10 minutes. Finally, it was maintained at 90% acetonitrile for 5 minutes.

Before quantifying the content of spruce and resveratrol in callus, the standards of spruceol and resveratrol were diluted to 100 ppb, 100 ppb, 250 ppb, 500 ppb, and 1000 ppb, respectively. A standard curve was prepared using peak area and concentration after HPLC analysis. Standard curve of spruceol: y=427.0x-189.0, correlation coefficient (R2) is 1.0; standard curve of resveratrol: y=1308.6x-8116.4, correlation coefficient (R2) is 0.9997. The content of spruce and resveratrol in the callus was calculated from this standard curve.

Example 4 Determination of spruce and resveratrol produced by different concentrations of chitin-induced callus

The content of spruceol and resveratrol produced was determined after inducing groundnut callus with different concentrations of chitin suspension aqueous solution: 0.01, 0.02, 0.04 g/mL for 12 hours. The results are shown in Table 1.

It can be seen from Table 1 that the content of spruceol increases with the concentration of aqueous suspension of chitin, and the content of spruceol is 582.64±169.19, 1056.27±379.66, 2362.15±771.33 ng/g (fresh weight) respectively; the content of resveratrol is The induction content of chitin suspension aqueous solution concentration was 0.04g/mL, the highest was 0.01g/mL, the lowest was 0.02g/mL, and the resveratrol content was 10090.78±758.46ng/g and 6266.35±4165.63ng/ g, 3587.54±2028.83 ng/g (fresh weight); no liquid (blank) or dripping sterile water (H2O) in the control group, no spruceol and resveratrol were detected. Callus was induced by high-dose chitin (0.04g/mL), which produced the best effect of spruceol and resveratrol. The results showed that the aqueous suspension of chitin could induce the production of spruce and white from the groundnut callus. Resveratrol, and the concentration of chitin is high, and the induction effect is better.

Example 5 Determination of the content of spruce and resveratrol in chitin-induced callus at different times

The groundnut callus was induced by a fixed concentration (0.01 g/mL) of chitin suspension aqueous solution. The content of spruce and resveratrol produced by callus was analyzed at different times. The results are shown in Table 2.

It can be seen from Table 2 that the groundnut callus produced spruceol and resveratrol 4 hours after the induction of the aqueous suspension of chitin, and with reference to the first figure, the content of spruce decreased at the 8th hour. The 24-hour content was 2361.76±269.75 ng/g (fresh weight), and the content decreased to 948.16±94.13 ng/g (fresh weight) at 72 hours, and no spruceol was detected until 120 hours.

As can be seen from the second figure, the change in resveratrol content gradually increased from 4 hours to 24 hours, reaching a maximum content of 4593.06±669.80 ng/g (fresh weight), and then decreasing to a temperature of 507.26±63.94 ng in the 120th hour. g (fresh weight). The results showed that after the chitin suspension induced the callus of the groundnut, spruceol and resveratrol were detected, and the content of spruceol was higher than that of resveratrol, while spruceol and Resveratrol reached the highest level at 24 hours after induction.

In summary, the direct use of chitin in the method of the present invention can effectively induce the production of spruceol and resveratrol from the groundnut callus, and the yield of both will increase as the concentration of chitin increases.

On the other hand, the method of the present invention can culture callus using a general medium at room temperature, so that the demand for mass production of spruceol and resveratrol can be provided.

However, the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and all modifications and changes may be made without departing from the spirit and scope of the invention. It should be covered by the scope of the invention.

In the first figure, the aqueous suspension of chitin (0.01g/mL) induced the callus of groundnut, and the content of spruceol changed at different times.

The second figure is an aqueous suspension of chitin (0.01g/mL) to induce callus of groundnut, and the content of resveratrol changes in different time.

Claims (16)

A method for producing piceatannol using chitin, comprising: (1) taking a hypocotyl of a groundnut as a culture, forming a wound on the implant; and (2) forming a wound in the wound Adding a suspension of chitin to the callus; and (3) finely pulverizing and filtering the callus to obtain spruceol. According to the method of claim 1, the hypocotyls of the groundnut are washed and cultured from a post-pod seed. According to the method of claim 2, the method of washing the seed after de-poding is followed by deionized water washing, and 1% sodium hypochlorite plus soil temperature 20 emulsifier (TWEEN 20), and then ultrasonically oscillated for 2 minutes. According to the method of claim 1 or 2, wherein the cultivation system is placed in a medium containing 1-naphthylacetic acid and 6-benzylamino purine for dark culture to form the Injured tissue. According to the method of claim 4, the concentration of naphthaleneacetic acid is 0.1-0.3 mg/L, and the concentration of benzylaminopurine is 0.05-0.08 mg/L. The method of claim 1, wherein the wound is formed by cutting. According to the method of claim 1, wherein the concentration of the chitin suspension aqueous solution is 0.01 to 0.05 mg/L. According to the method of claim 7, wherein the chitin suspension The concentration of the floating aqueous solution and the yield of picatinannol showed a positive dose dependence relationship. The method of claim 1, wherein the wound callus begins to produce piceatannol at the 4th hour after the addition of the chitin suspension aqueous solution. According to the method of claim 9, wherein the chitin suspension aqueous solution concentration is 0.01 mg/L. According to the method of claim 7, wherein the chitin is sieved through a sieve before being formulated into an aqueous solution. According to the method of claim 1, the step (2) further comprises a treatment step of placing the callus treated with the aqueous suspension of chitin in a dark room for a predetermined period of time. The method of claim 12, wherein the darkroom temperature is room temperature. According to the method of claim 13, wherein the darkroom temperature is from 27 ° C to 29 ° C. The method of claim 1, wherein the finely pulverizing step is to grind the callus with 100% methanol (MeOH). According to the method of claim 1, wherein the filtering step is sequentially filtering for suction filtration and filtration.