TWI627279B - Method for cultivating plant callus rich in stilbene - Google Patents

Abstract

A method for cultivating plant callus rich in stilbene, which comprises the following Step: (a) activating step: making a mouth at the acute angle of the seeds of the pine plant, and placing the seed in the activation solution to activate the seed embryo in the seed; (b) stress treatment step: after activation The embryo is taken out from the seed and a wound gap is created at the hypocotyl; (c) a culture step: the embryo obtained in the above-mentioned stress treatment step (b) is placed in a medium for tissue culture, the wound The callus is formed by cell proliferation in the notch; and the callus contains at least one of catechin, proanthocyanidins, stilbene-like compounds, and mixtures thereof.

Description

Method for cultivating plant callus rich in stilbene

The present invention relates to a process for producing stilbene-like, and more particularly to a process for producing stilbene-like plants from plant callus.

Callus is widely recognized for its advantages of mass production by means of tissue culture. Among them, the production of plant secondary metabolites is most valued by food and industry, such as liquid crystal polymerization induced by plant bodies. Material: p- hydroxybenzoic acid is superior to the Kolbe-Schmitt process used in industry (McQualter et al., 2005). In addition, callus can be further produced by a suitable induction method as a compound commonly referred to as phytoalexins, in which stilbenoids (Hammerschmidt and Dann, 1999) are particularly affected. Attention. Such compounds not only play an important role in the plant defense system, but also can adjust a variety of pathological mechanisms in the human body, such as anti-inflammatory, anti-oxidation, anti-viral, prevent aging and cardiovascular diseases, reduce obesity and neuroprotection (Aggarwal And Shishodia, 2006; Losa, 2003; Ray et al., 1999; Rice-Evans et al., 1995), considered a natural antioxidant with dual medical and health benefits.

Furthermore, because stilbenoids are rich in physiological activity but rare in content, even with the tissue culture technology that can be mass-produced, it is still difficult to meet their needs. Therefore, the current research mostly uses adversity to improve callus. The type and content of the stilbene compound produced, for example, Fritzemeier et al. (1983) induced the stilbene-like compound by the UV-C (ultraviolet-C) stimulation to induce the groundnut callus; Ku et al. (2005) utilized UV. Stimulate the groundnut callus to increase the yield of resveratrol and discover the novel stilbene compound piceatannol.

According to the previous literature, Pinus genus has a variety of high-equivalent antioxidant active substances, which can be regarded as potential raw materials for emerging health care products. However, due to many limitations such as its growth environment, it is difficult to obtain a large amount of samples and limit its application space. To this end, considering the maintenance of the ecological environment and the concept of the development of emerging health products, it is important to develop the meristematic characteristics cultivated by the plant parts, and the mass-produced callus.

In summary, the present inventors used the embryo of Pinus genus as a basis, and hoped to establish a culture system of pine plant callus by means of tissue culture, thereby achieving mass production for development of a follow-up health product. Research and mass production. And with appropriate induction methods, explore the changes in the content of stilbene in callus, as the basis for the development of follow-up health products.

That is, according to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, which comprises the steps of: (a) an activation step: making an incision at an acute angle of a seed of a Pinus genus plant, And placing the seed in an activation solution to activate the embryo in the seed; (b) stress processing step: removing the activated embryo from the seed and creating a wound gap at the hypocotyl; (c) Culture step: the embryos obtained in the above-mentioned stress treatment step (b) are placed in a medium for tissue culture, and the wounds are formed into callus due to cell proliferation.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus, which is an aqueous hydrogen peroxide solution, can be provided.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided. The concentration of the above aqueous hydrogen peroxide solution is not particularly limited, and is generally in the range of 0.5 to 3%; preferably A range of 0.5 to 2%; particularly preferably in the range of 0.5 to 1.5%; most preferably 1%.

According to another embodiment of the present invention, a stilbene-rich plant callus culture method can be provided, and the activation time in the activation step (a) is not particularly limited, and is generally 1 to 5 days, preferably. It is 3 days.

According to one aspect of the present invention, a method for culturing a stilbene-rich plant callus can be provided, wherein the medium comprises a solid medium or a liquid medium.

According to one aspect of the present invention, a method for culturing a stilbene-rich plant callus can be provided, wherein the solid medium is a solid medium (LPG (leaf protoplasts glutamine) (Anderson, 1990).

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the liquid medium is composed of sucrose, 6-Benzylaminopurine, 2,4-dichlorobenzene. It consists of at least one of 2,4-dichlorophenoxyacetic acid, Glutamine, Sigma type agar, and mixtures thereof.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the concentration of sucrose in the liquid medium is not particularly limited, and is generally in the range of 1 to 10%; Preferably, it is in the range of 1 to 8%; more preferably in the range of 1 to 6%; particularly preferably in the range of 2 to 5%; most preferably 3%.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the concentration of 6-Benzylaminopurine in the liquid medium is not particularly limited, and is generally From 2μM to 10μM The range; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably in the range of 3 to 5 μM.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein a concentration of 2,4-dichlorophenoxyacetic acid in the liquid medium is It is not particularly limited and is generally in the range of 2 μM to 10 μM; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably 3 to 5 μM. The scope.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the concentration of the glutamic acid (Glutamine) in the liquid medium is not particularly limited, and is generally 2 μM to A range of 20 μM; preferably in the range of 4 to 18 μM; more preferably in the range of 6 to 16 μM; particularly preferably in the range of 8 to 14 μM; most preferably in the range of 8 to 12 μM.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the pH of the liquid medium is not particularly limited, and is generally in the range of 5.0 to 7.0; preferably Range of 5.0 to 6.5; optimally in the range of 5.5 to 6.5.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the pine plant is five-leaf pine, red pine, mountain pine, thorn pine, long-leaf pine, huashan pine, and five needles. Pine, big fruit pine, Korean pine, two-leaf pine, masson pine, or white pine; preferably pine, pine, pine, pine, pine, pine, pine, pine, pine or pine; Jiawei is a five-leaf pine, long-leaf pine, Huashan pine, five-pin pine, two-leaf pine, masson pine, or white pine; special is five-leaf pine, huashan pine, five-pin pine, masson pine, or white pine; the best is five-leaf pine, Huashan pine, Or masson pine.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, which further comprises an inducing step of adding glucose water to the culture. The callus obtained in the step (c) is irradiated with ultraviolet light for a suitable period of time, and then cultured in a dark room.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, wherein the ultraviolet light has a wavelength in the range of 220 nm to 350 nm; preferably in the range of 220 nm to 330 nm; Preferably, it is in the range of 240 nm to 310 nm; more preferably in the range of 240 nm to 290 nm; most preferably in the range of 240 nm to 280 nm.

According to one aspect of the present invention, a method for cultivating a stilbene-rich plant callus can be provided, and the above-mentioned appropriate time is generally in the range of 10 to 60 minutes; preferably in the range of 10 to 50 minutes; more preferably A range of 10 to 40 minutes; preferably a range of 20 to 40 minutes.

In addition, the present invention can provide a stilbene-rich plant callus obtained by the above-described culture method, and the callus contains catechin, proanthocyanidins, stilbene-like compounds, And at least one of the mixtures thereof.

According to another embodiment of the present invention, a stilbene-rich plant callus obtained by the culture method described above may be provided, and the catechin content of the plant callus is 3.0 to The range of 4.7 mg/Kg plant callus dry weight; preferably from 3.06 to 4.3 mg/Kg of plant callus dry weight; more preferably from 3.20 to 4.16.

According to another embodiment of the present invention, a stilbene-rich plant callus can be provided, which is obtained by the culture method described above, and the proanthocyanidin content in the plant callus is 1.1 to 2.5 mg. /Kg plant callus dry weight range; preferably in the range of 1.17 to 2.19; more preferably in the range of 1.43 to 1.93.

According to another embodiment of the present invention, a stilbene-rich plant callus can be provided, which is obtained by the above-described culture method, and the stilbene content in the plant callus is 0.2 to The range of 0.8 mg/Kg plant callus dry weight; preferably from 0.35 to 0.65 mg/Kg of plant callus dry weight; more preferably from 0.44 to 0.62.

Moreover, the present invention can provide a method for preparing stilbene, which comprises the following steps:

(a) activating step: making a mouth at an acute angle of the seeds of the pine plant, and placing the seed in an activating solution to activate the seed embryo in the seed;

(b) a stress treatment step: removing the activated embryo from the seed and creating a wound gap at the hypocotyl of the embryo;

(c) culturing step: the embryo obtained in the above-mentioned stress treatment step (b) is cultured in a medium in which callus is formed due to cell proliferation.

(d) Extraction step: The callus obtained by the above induction step (c) is subjected to extraction to obtain a stilbene-like compound.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the extracting step (d) is carried out by chopping the callus obtained in the inducing step (c), adding it to methanol, and then grinding. The clear filtrate was collected by filtration through a 0.22 μm pore size filter.

According to another embodiment of the present invention, a method for producing a stilbene-like styrene may be provided, wherein the activation liquid is an aqueous hydrogen peroxide solution.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided. The concentration of the above aqueous hydrogen peroxide solution is not particularly limited, and is generally in the range of 0.5 to 3%; preferably 0.5 to 2%. Range; particularly preferably in the range of 0.5 to 1.5%; optimally 1%.

According to another embodiment of the present invention, a method for producing stilbene-like styrene may be provided, and the activation time in the above activation step (a) is not particularly limited, and is usually 1 to 5 days, preferably 3 days.

According to another embodiment of the present invention, a method for producing stilbene-like can be provided, wherein the medium comprises a solid medium or a liquid medium.

According to another embodiment of the present invention, a method for preparing a stilbene-like styrene may be provided, wherein the solid medium is an LPG solid medium.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the liquid medium is composed of sucrose, 6-Benzylaminopurine, 2,4-dichlorophenoxyacetic acid (2, 4-dichlorophenoxyacetic acid, at least one of Glutamine, Sigma type agar, and mixtures thereof.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the concentration of sucrose in the liquid medium is not particularly limited, and is generally in the range of 1 to 10%; preferably at 1 to A range of 8%; more preferably in the range of 1 to 6%; particularly preferably in the range of 2 to 5%; most preferably 3%.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the concentration of 6-Benzylaminopurine in the liquid medium is not particularly limited, and is generally from 2 μM to 10 μM. The range; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably in the range of 3 to 5 μM.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein a concentration of 2,4-dichlorophenoxyacetic acid in the liquid medium is not particularly limited. It is generally in the range of 2 μM to 10 μM; preferably in the range of 2 to 8 μM; more preferably in the range of 2 to 6 μM; particularly preferably in the range of 2 to 5 μM; most preferably in the range of 3 to 5 μM.

According to another embodiment of the present invention, a method for preparing stilbene-like styrene may be provided, wherein the concentration of glutamic acid in the liquid medium is not particularly limited, and is generally in the range of 2 μM to 20 μM; Preferably, it is in the range of 4 to 18 μM; more preferably in the range of 6 to 16 μM; particularly preferably in the range of 8 to 14 μM; most preferably in the range of 8 to 12 μM.

According to another embodiment of the present invention, a method for preparing a stilbene-like syrup may be provided, wherein the pH of the liquid medium is not particularly limited, and is generally in the range of 5.0 to 7.0; preferably in the range of 5.0 to 6.5. The best is in the range of 5.5 to 6.5.

According to another embodiment of the present invention, a method for preparing stilbene-like stilbene can be provided, wherein the pine plant is Pinus sylvestris, Pinus koraiensis, Pinus koraiensis, Pinus sylvestris, Pinus sylvestris, Pinus sylvestris var. , Korean pine, pine, pine, or white pine; preferably pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine, pine Ye Song, Huashan Pine, Wuzheng Pine, Pinus sylvestris, Pinus massoniana, or Pinus bungeana; Tejia Pine, Pinus armandi, Pinus sylvestris, Pinus massoniana, or Pinus bungeana; the best is Pinus bungeana, Pinus armandi, or Pinus massoniana.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the cultivation process of plant callus in the relevant embodiment of the present invention.

2 is a schematic diagram of an on-line electrochemical antioxidant activity analysis parallel electrospray mass spectrometer in accordance with an embodiment of the present invention.

Figure 3 is a diagram showing the UV absorption spectrum and electrochemical scanning spectrum of an on-line electrochemical antioxidant activity analysis system in accordance with an embodiment of the present invention.

Fig. 4 is a view showing the fluorescence spectrum of the plant callus in the relevant embodiment of the present invention after being irradiated with ultraviolet light for a different period of time.

Hereinafter, the embodiments of the present invention will be described in more detail with reference to the specific embodiments, so that the spirit and content of the present invention are more complete and easy to understand; It is to be understood by those skilled in the art that the present invention is not limited to the examples, and other equivalent or equivalent functions and steps can be used to achieve the invention.

"Embodiment 1"

Take 25 capsules of Taiwanese Pinus armandii seeds, and make an incision that can be observed by the naked eye at the acute angle of the seed, as shown in Figure 1a. Next, transfer the incisioned seeds to 150 ml of 1% hydrogen peroxide. An aqueous solution (aqueous hydrogen peroxide solution is required to be activated daily to activate the embryo) to activate the growth activity of the embryo, as shown in Figure 1b. After the activation treatment, the embryo part was obviously observed to expand, and the growth activity of the embryo was successfully activated and the cells began to divide and grow. After three days, the seeds were removed from the seed coat and then sterilized with 20% bleach for 3 minutes and then sterilized with a 70% aqueous solution of ethanol for one minute. Next, after washing three times with sterile water, a notch was made at the hypocotyl, and the embryos were taken out using a scalpel and placed on a medium, as shown in Fig. 1c, and then subcultured every 4 weeks.

The above medium is a solid medium based on LPG and added with 3% sucrose, 4.4 μM of 6-Benzylaminopurine, and 4.5 μM of 2,4-dichlorophenoxyacetic acid. And 10 μM of glutamic acid (Glutamine), the pH of the medium was adjusted to 5.8, then 0.6% sigma type agar was added, and placed in an autoclave at a pressure of 1.1 Kg/cm ² , 121 Sterilize at a temperature of °C for 15 minutes.

After 3 days of culture, the new cell tissue can be observed in the wound gap of the embryo, as shown in Figure 1d; after a week of culture, the proliferation and enlargement of the cells can be clearly observed, as shown in Figure 1e. Shown.

Then, the newborn cell tissue was removed and transferred to other culture dishes. After 6 months of subculture, the callus culture of Pinus armandii was performed, and the resulting callus was as shown in Fig. 1f.

Next, 1.0 g of the callus was weighed and placed in a mortar, 2 ml of a 60% aqueous methanol solution was added and ground, and the extract was taken out as a dropper, and then left in 1 ml of a 60% aqueous methanol solution. After the sample in the mortar was washed out, the extract was placed in a 20 ml sample vial for 10 minutes to be clarified, and then the supernatant was filtered through a 0.22 μm filter. The filtrate was collected and quantified to 5 mL in a quantitative bottle.

Then, the obtained filtrate is subjected to structural analysis and activity analysis of antioxidant substances; as shown in FIG. 2, the experimental instrument configuration is divided into two parts, and after the sample is injected, the mobile phase is pushed by the HPLC pump to drive the sample into the chromatography column ( Agilent Poroshell 120 PFP 4.6*150mm, 2.7μm) was separated and the sample was separated by a column and passed through a 200:1 splitter (QuickSplitTM) for splitting.

The first part enters the ultraviolet-visible light detector to detect the absorption value and directly enters the electrochemical detector (Agilent HPLC 1200 Series instrument (Agilent Technologies, Waldbronn, Germary), G1314B UV-vis detector After detecting the absorption value at 280 nm, it enters the electrochemical detector, and the oxidizing voltage is applied to the oxidizing voltage of 0.7 V by the amperometric method.

In the second part, the flow washing liquid is injected into an electrospray ionization-ion trap mass spectrometer (Finnigan LCQ advantage Electrospray ion-trap mass spectrometer (Thermal, San Jose, CA, USA)). The mass spectrometry conditions were: detection in the negative mode, the sheath gas flow rate was 30 arb, the auxiliary gas flow rate was 0 arb, the spray voltage was 3.25 kV, and the capillary temperature (capillary temp) It is 220 °C. Two or more mass spectra were performed by collision induced dissociation (CID) to generate structural identifiable ion fragments for structural identification.

The mobile phase used consisted of mobile phase A containing an aqueous solution of 2.5 mM ammonium acetate and mobile phase B also containing 2.5 mM ammonium acetate in methanol; when the gradient was 0 min, The mobile phase B was 2%. After 5 min, the mobile phase B rose to 1% with an increase of 1% per minute in 85 min, after which the mobile phase B was maintained at 87% for 5 min.

The above analysis method can directly analyze the structure of the component having antioxidant activity, and then compare the literature and the database to determine the structure thereof, and the obtained UV absorption spectrum and electrochemical scanning spectrum are shown in FIG. 3; Eight kinds of substances with antioxidant activity were found in the extract of callus of Pinus armandii, which is numbered 1-8 in Figure 3.

Next, the identification results and their antioxidant activity capacities were ranked according to the retention time (R.t.) in the chromatographic conditions, and are recorded in Table 1.

The results showed that eight kinds of antioxidant active substances were found in the extracts of callus of Pinus armandii, including two catechins, epigallocatechin (epigallocatechin). 1) With catechin (No. 5), since catechins have been proven to have good physiological activity in the past literature, they have greatly contributed to the promotion of human health, indicating that the callus is becoming health care. The raw materials of the products have their feasibility.

Similarly, in the analysis of the extract, the proanthocyanidin compounds synthesized by catechin were also found to be Proanthocyanidin trimer isomer-1 and -2 (Nos. 3 and 6) and B-type proanthocyanidinisomer-1 and - 2 (Nos. 2 and 4), proanthocyanidins can be used to supplement vitamin C and E in addition to its high water solubility and strong antioxidant activity.

Furthermore, in addition to the observation of the presence of catechin and its polymer proanthocyanidins, two stilbene-like compounds were also compared from the map, which are derivatives of resveratrol, after alignment It is known that these two stilbene-like compounds are Resveratrol-hexose (No. 7) and Methoxyresveratrol-hexose (No. 8), respectively.

Then, the contents of the above eight active substances were converted into standard solvents, respectively, and are reported in Table 2.

It can be seen from the above results that the callus of Pinus armandii has not only the common antioxidant substance catechin, but also the proanthocyanidin and stilbene-like compounds which are very beneficial to the physiological activity of human body, and because of its mass production. Advantages, if properly managed and systematically managed, will contribute to the improvement of the production and quality of Huashan Pine related health care products.

<<Example 2-4》

The callus of Pinus armandii seeds was cultured in the same manner as in Example 1 except that the subculture time of Example 2-4 was 1 month, and 2 ml of 20 g/L aqueous glucose solution was added to the callus under aseptic conditions. The medium was irradiated with a laminar flow ultraviolet lamp at a wavelength of 254 nm for 30 minutes, and then taken out in a dark room at room temperature for 12, 24, and 96 hours, and then the callus was taken out for extraction. .

1 g of calli was placed in a mortar, 2 ml of 60% aqueous methanol solution was added and ground, and the extract was taken out as a dropper, and then dissolved in 1 ml of 60% methanol. After washing the sample remaining in the mortar, the extract was placed in a 20 ml sample vial for 10 minutes to be clarified, and then the supernatant was filtered through a 0.22 μm filter.

After the filtrate was collected, the quantitative flask was quantified to 5 mL and the content of the stilbene compound was analyzed by a flow detector with a flow detector gradient analysis. The mobile phase combination used was an aqueous solution containing 0.01% formic acid (mobile phase A). ) with a solution of 0.01% formic acid in cyanide (mobile phase B). When the gradient used was 0 min, the mobile phase B was 20%, rose to 32% in 20 min, and then moved to 90% in 10 min, and the mobile phase B was maintained at 90% for 10 min.

It can be observed from the fluorescence spectrum of Fig. 4 that the peak area of resveratrol-hexose does not change significantly with the increase of the standing time, but the wave front area of another compound methoxyresveratrol-hexose will become longer with the standing time. And increase.

Next, the results are integrated and statistically analyzed, and the results are recorded in Table 3.

It can be seen from the results in Table 3 that there is no significant difference in the integrated area of Resveratrol-hexose after standing for different time, which means that the content of the compound does not change, indicating that the cause of callus formation of Resveratrol-hexose is not ultraviolet light. The factors are either pathogens or other chemicals. However, with the increase of the rest time after illumination, the peak integration area of another type of stilbene compound Methoxyresveratrol-hexose also increased, and reached the maximum value after 96 hours. From this result, it is known that ultraviolet light can effectively induce Huashan. Pine embryo callus The tissue produces Methoxyresveratrol-hexose, which in turn increases its content for mass production purposes.

It is to be understood that the above-described embodiments and examples are merely illustrative, and various modifications may be made by those skilled in the art. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.

Claims (14)

A method for cultivating plant callus rich in stilbene, comprising the steps of: (a) activating step: making a mouth at an acute angle of a seed of a pine plant, and placing the seed at a concentration of 0.5% Up to 3% aqueous hydrogen peroxide to activate the embryo in the seed; (b) stress treatment step: removing the activated embryo from the seed and creating a wound gap at the hypocotyl; (c) culturing step: the embryo obtained in the above-mentioned stress treatment step (b) is placed in a medium for tissue culture, and the wound is notched to form callus due to cell proliferation. The culture method according to claim 1, wherein the medium comprises a solid medium or a liquid medium. The culture method according to claim 2, wherein the liquid medium is made of at least one of sucrose, 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, glutamic acid, agar and a mixture thereof. composition. The culture method according to claim 1, wherein the pine plant is five-leaf pine, red pine, mountain pine, thorn pine, long-leaf pine, huashan pine, five-pin pine, big fruit pine, red pine, two pine, and masson pine. Or white pine. The culture method according to claim 1, further comprising an inducing step of adding glucose water to the callus obtained in the culturing step (c), irradiating with ultraviolet light for a suitable period of time, and then culturing in a dark room. The culture method according to claim 5, wherein the ultraviolet light has a wavelength in the range of 220 to 350 nm. The culture method according to any one of claims 1 to 6, wherein the callus contains at least one of a catechin compound, a proanthocyanidin compound, a stilbene, and a mixture thereof. The culture method according to claim 7, wherein the catechin compound is contained in the plant callus in an amount of from 3.0 to 4.7 mg/kg of the plant callus dry weight. The culture method according to claim 7, wherein the content of the proanthocyanidin compound in the plant callus is in the range of 1.1 to 2.5 mg/kg of the dry weight of the plant callus. The culture method according to claim 7, wherein the stilbene content in the plant callus is in the range of 0.2 to 0.8 mg/kg of the plant callus dry weight. A method for preparing stilbene-like styrene, comprising the steps of: (a) activating step: making an incision at an acute angle of a seed of a pine plant, and placing the seed in an aqueous hydrogen peroxide solution having a concentration of 0.5% to 3%. In order to activate the seed embryo in the seed; (b) the stress treatment step: taking the activated embryo from the seed and creating a wound gap at the hypocotyl; (c) culturing step: the above-mentioned stress processing step The embryos obtained in (b) are placed in a medium for tissue culture, and the wounds are formed into callus due to cell proliferation. (d) Extraction step: The callus obtained by the above induction step (c) is subjected to extraction to obtain a stilbene-like compound. The method for producing stilbene according to claim 11, wherein the medium comprises a solid medium or a liquid medium. The method for preparing stilbene according to claim 12, wherein the liquid medium is made of sucrose, 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, glutamic acid, agar and the other And consisting of at least one of the mixtures. The preparation method of stilbene according to claim 11, wherein the pine plant is five-leaf pine, red pine, mountain pine, thorn pine, long-leaf pine, huashan pine, five-pin pine, big fruit pine, red pine, two leaves Pine, masson pine, or white pine.