KR102096348B1 - Method for producing ginsenoside from adventitious root of Gynostemma pentaphyllum - Google Patents

Abstract

The present invention relates to a method for producing ginsenosides from an adventitious root derived from Gynostemma pentaphyllum . Specifically, forming a film root from the outside of the stone; And comprising the step of separating ginsenosides from the root of the film, relates to a method for producing ginsenosides from the roots derived from off-sold, using the roots derived from off-solds that have not been used to produce ginsenosides so far By producing cenoside, it can be useful industrially.

Description

Method for producing ginsenoside from adventitious root of Gynostemma pentaphyllum}

The present invention relates to a method for producing ginsenosides from an adventitious root derived from Gynostemma pentaphyllum . Specifically, forming a film root from the outside of the stone; And separating ginsenosides from the roots of the membrane, a method for producing ginsenosides from roots derived from stones.

Gynostemma pentaphyllum is also called chlorophyll or vine, and is a vine perennial plant of Cucurbitaceae that grows in mountains or forests.Slim stems are vines and are wrapped around other plants or objects. Ascendant, with 3 to 9 (mostly 5 to 7) palm-shaped leaves (Blumert and Liu 2003). In general, it is possible to make tea using the above-ground portion mainly as a medicinal portion, and other portions can be used by slicing. In the private sector, rhizomes or outposts are used for chronic bronchitis. On the other hand, in the wild, outgrowth is affected by cold weather, which can be successfully grown in greenhouses, but is not too dry and requires warm temperatures (Razmovski-Naumovski et al., 2005).

In addition to stones, it contains about 100 types of dammarane saponin, i.e., gypenoside or ginosaponin, which can be used in various ways such as lowering cholesterol, strengthening immunity, anti-tumor, antioxidant, and anti-diabetic effects. It is known as an active ingredient with biological and clinical effects (Yuin et al. 2004). Due to these biological effects, it has received considerable attention as an alternative to / replenishing ginseng ( Panax ginseng ) (Razmovski-Naumovski et al. 2005). Specifically, Zipenoside I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XIII, XIV, XV, XVI, XVII, XIX, XXII, XXIII, XXV, XXVIII, XXXVIII, XLIX, LV, LVIII, LXII, LXIII, LXXV, ginsenosides Rb1, Rb2, F2, Rc, Rf, Rg3, malonyl-Rb1, malonyl-Rd were found to be the main components of the stone (Kao et al. 2008; Tang and Eisenbrand 2011).

Meanwhile, the ginsenoside biosynthetic pathway is only partially known. The biosynthetic pathway is isopentenyl diphosphate (isopentenyl) by the action of IPP isomerase (IPI), GPP synthetase (GPS), FPP synthase (FPS), squalene synthetase (SS) and squalene apoxidase (SE) Until a series of condensation reactions of diphosphate) and DMADP (dimethylallyldiphosphate) occur and oxidosqualene is synthesized, it is known to share some of them along with other triterpene pathways (Ajikumar et al. Science, 330, 70 -74. 2010; Ro et al. Nature, 440, 940-943. 2006; Sun et al. BMC genomics, 11, 262, 2010).

Plant tissue culture technology is known to play an important role in the preservation and proliferation of germplasm (Iankova et al., 2001, Bhatia et al., 2002). Root culture is considered to be a promising method for producing secondary metabolites of plants due to its genetic / biochemical stability, rapid growth rate, and the ability to synthesize secondary products at a level similar to that of the original plant (Sevo´). n & Oksman-Caldentey 2002). In this regard, the extracellular hairy root was established by a leaf disc method through co-culture with Ahgrobacterium rhizogenes (Chang et al. 2005), and stem propagation ) Method was established by the tissue culture method (Liu and Wu, 2000), but there have been no reports of extracellular membrane root culture.

Against this background, the present inventors have developed an extra-membrane root culture method that can be used for biosynthesis of ginsenosides. Specifically, the present inventors are squalene synthase (GpSS), squalene epoxidase (GpSE), and protopanaxadiol (PPD) type ginsenosides, protopas in the extracellular membrane roots induced by the culture method of the present invention. It was confirmed that the ginsenoside-related gene, which contains the sugar transfer enzyme GpUGT23, which delivers sugars to naxatriol (protopanaxatriol, PPT) type ginsenoside, expresses ginsenosides in extracellular membrane induced by the culture method. , Specifically, it was confirmed that it can be used for the production of ginsenoside Rd to complete the present invention.

Journal of the Korean Ginseng Society vol 8, 2 pp. 172-177 1226-8453 KCI (Dec. 1984)

The object of the present invention is to form a membrane root (adventitious root) from a stone ( Gynostemma pentaphyllum ); And separating ginsenosides from the roots of the membrane, to provide a method for producing ginsenosides from roots derived from stones.

The present invention solves the above problems, and to achieve the object of the present invention, provides a method for producing ginsenosides from a membrane root derived from stone.

Hereinafter, the present invention will be described in detail.

In the present invention, a method for producing ginsenosides from a membrane root derived from a stone, comprising: forming a membrane root from a stone; And separating ginsenosides from the roots of the membrane.

In addition, the formation of membrane roots from the stone outside, culturing the stone outside to induce callus; And inducing membrane roots by culturing the callus.

The present invention is characterized in that it is intended to first use membrane roots in producing ginsenosides from stones. Specifically, the callus was derived from the stone, and the membrane roots were cultured through a process of inducing the membrane root, from which various ginsenosides, including ginsenoside Rd, can be produced separately, and in particular, compared to other sites other than stone It was confirmed that the senoside Rd can be produced at a remarkably high level, and thus it can be confirmed that it can be used industrially.

The term " Gynostemma pentaphyllum " in the present invention is also referred to as Childamdam ( 七葉膽) or vine tea, refers to a vine perennial plant of Cucurbitaceae growing in a mountain or forest, but is not limited thereto. Gynostemma may contain all plants. The stone can be purchased and used commercially, or can be used that is collected or cultivated in nature.

Stems other than stones extend sideways, have white hairs on nodes, grow tangled, but sometimes crawl up with tendrils. The leaves are alternate and have 3 to 9 (mostly 5 to 7) small leaves, and are narrow egg-shaped oval or narrow egg-shaped. The small leaf at the end is 4 ~ 8cm long and 2 ~ 3cm long with a small petiole, has a sharp tip, fine hairs on the front leaf vein, and serrates on the edge. As a medicinal part, it is possible to make tea using the above-ground part, and other parts can be chopped and used. It has anti-inflammatory, detoxifying, expectorant, and subcutaneous effects, and in the private sector, rhizomes or outposts are used for chronic bronchitis. In addition to stones, it contains about 100 types of dammarane saponin, i.e., gypenoside or ginosaponin, which have various effects such as lowering cholesterol, strengthening immunity, anti-tumor, antioxidant, and anti-diabetic effects. It is known as an active ingredient with biological and clinical effects (Yuin et al. 2004).

In one embodiment of the present invention, seeds of plants of various genus Gynostemma collected in various regions in Korea are grown at 22 to 24 ° C. in 16 hours of light / 8 hours of dark conditions, and stem extracts of each plant are used. By comparing the level of ginsenoside or gifenoside. Among the seeds, four plants of the genus Gynostemma (2258-1, 2258-2, 3083-1, 3083-2) were selected and used for the analysis of metabolites.

In the present invention, in order to cultivate the extracellular membrane root, it may include the step of incubating the extracellular to induce callus and culturing.

In the present invention, the term "callus (callus)" refers to a special tissue or cell mass that occurs when a plant is wounded, also known as epidermal tissue, connective tissue, callus, and cultured under appropriate conditions to cause fission proliferation. It means an amorphous cell mass. It is possible to make any part of a plant into callus, it has no differentiation function and is dedifferentiated, but it can grow indefinitely as it is when transplanted and cultured regularly. In addition, when cultured under certain conditions, it can re-differentiate to shoots, roots, pears, or even complete plants. I can do it.

What is used for the induction of callus may be all or a part of the extracellular stone, but is not limited thereto, preferably, the callus can be derived from the extracellular stem region. In order to induce and cultivate callus, it is not limited thereto, and may include a process of sterilizing explants of plants using ethanol or sodium hypochlorite solution, etc. It may include the process of inducing and transferring to another medium after a period of time. In addition, in the process of culturing callus, it is known that factors such as culture temperature, light, gas supply, nitrogen source, composition of inorganic salt, concentration of agar, and growth regulator of plants are influenced. In the present invention, the callus may be a callus derived from a stem other than a stone.

The medium for inducing the callus in the present invention may be a nutritional basic medium such as MS (Murashige and Skoog), B5 (Gamberg), N6 (Gupfa and Durzan) and White, and the medium is an energy source, vitamin and plant growth Modulators, and the like, but are not limited thereto. The energy source is preferably a saccharide, more preferably sucrose, and the vitamin preferably includes, but is not limited to, nicotinic acid, thiamine and pyridoxine. The plant growth regulator may be one or more auxins selected from the group consisting of indoleacetic acid (IAA), indolebutyric acid (IBA), naphthalene acetic acid (NAA) and 2,4-D (2,4-dichlorophenoxyacetic acid). , 0.01-5 ppm, preferably 0.1-5ppm of auxin to treat the callus, but is not limited thereto.

In the present invention, in order to induce the callus for cultivation of extra-membranous roots, extra-stem stems can be used as explants.

In the present invention, the term "explant (explant)" refers to a fragment excised from the tissue or organ of a plant used to start cultivation in a test tube or medium, mainly in animal embryology and experimental morphology, etc. It refers to a cell group, tissue piece, organ, etc. that are cultured in vitro by separating them. In the present invention, the explant may be one using a stem other than a stone.

In one embodiment of the present invention, callus was derived from the stem using an extra-stem stem as an explant. Specifically, the surface of the explant was sterilized with 70% ethanol for 1 minute, and then sterilized with 1% sodium hypochlorite for 10 minutes. Thereafter, the explants were immersed in sterile distilled water 3 to 5 times. After sterilization, the explants were cut to a length of 1 cm, a half strength MS medium (2 mg / L α-naphthaline acetate acid (1-NAA) at 25 ° C., 16 hours light / 8 hours dark conditions), 3% sucrose, 0.8% agar). As a result, callus was induced at the wound site of the stem after 7-10 days of culture, and after 2 weeks of culture, the whole of the stem explant was enclosed.

Therefore, in the present invention, in the process of inducing callus from the extracellular, it may be to induce the callus by culturing the extracellular 7 to 10 days.

In the present invention, it is possible to use membrane roots derived from callus derived by culturing extracellulars to produce ginsenosides.

The term "adventitious root" in the present invention is also referred to as an irregular root, and refers to a root formed secondary to an organ other than the root of a plant, that is, a stem or a leaf.

In order to induce and culture the membrane root in the present invention, MS (Murashige and Skoog); 1/2 MS, B5 (Gamberg), SH (Schenk and Hild ebrandt), LS (Linsmair-Skoog) or N6 (Gupfa and Durzan) medium may be used, but is not limited thereto.

In one embodiment of the present invention, membrane roots were prepared from callus derived from stems other than stones. Specifically, when callus appeared from the surface of the wound site of the extracellular stem, it was cultured by transferring it to MS (including B5 vitamin) / B5 medium to which 2 mg / l IBA (Indole-3-Butyric Acid) was added. As a result, membrane roots were derived from callus 3 to 4 weeks after cultivation, and 100 ml of MS (including B5 vitamin) with 2 mg / l IBA added in an orbital shaker of 150 rpm at a temperature of 25 ° C. and dark condition at 25 ° C. It was preserved by passage in / B5 medium every 4 weeks. The growth of the roots was confirmed by measuring the relative fresh weight (FW) of the roots. As a result of measuring the raw weight of the membrane roots, it was confirmed that the value increased by 2.5 times or more after 2 and 3 weeks of liquid culture.

Accordingly, in the present invention, in the process of inducing membrane roots from the callus, the callus may be incubated for 3 to 4 weeks to induce membrane roots.

In addition, in the present invention, after inducing the membrane roots, it may further include the step of subculture to maintain the membrane roots, which may be subcultured about every 4 weeks.

When the callus is derived from the stone as described above and the root of the membrane is derived therefrom, finally the membrane root derived from the stone is formed, and for the purpose of the present invention, the membrane root can be extracted to separate ginsenoside, that is, saponin. .

In the present invention, the term "saponin" (Saponin), refers to a substance composed of several cyclic compounds in the non-sugar portion of the glycosides widely present in the plant system. In the present invention, the term saponin can be used as a term that includes both ginsenosides and gifenosides.

In particular, triterpene saponin, a saponin component contained in ginseng or red ginseng as a main bioactive component, has a different chemical structure from saponins found in other plants, so to differentiate these ginseng saponins from other plant-based saponins (Ginseng) Glycoside means "Ginsenoside." The ginsenosides are protopanaxadiol-type (PPD type) ginsenosides, protopanaxatriol-type (PPT type) ginsenosides and oleanoline according to the structure of aglycone (aglycone) Oleanolic acid-type ginsenosides can be classified into three types. These three groups, again, of sugar moieties (sugar moieties (aglycones)) attached by a glycosicid bond to the 3, 6, and 20 carbon positions of the ring in the compound structure. Sorted by location and number. Currently, more than 40 ginsenosides have been isolated, most of which are PPD type ginsenosides. Ginsenosides of PPD type include Rb1, Rb2, Rb3, Rc, Rd, Ra, Rh2, Gypenoside XVII, Compound O (Compound O), Compound Mc1 (Compound Mc1), F2, Compound Y (Compound Y), Compound Mc (Compound Mc), Rg3, Compound K (Coumpound K). Ginsenosides of the PPT type include Re, Rg1, Rf, Rg2, Rh1, F1, and the like. The type of ginsenosides produced by the ginsenoside production method of the present invention is not limited, but preferably ginsenosides Ro, Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2 days May be, more preferably ginsenoside Rd, and in the present invention, the term ginsenoside may be used interchangeably with zeppenoside.

In the present invention, the term "jiphenoside" is a triterpene saponin-based compound, and the phenonoside separated from the stone reaches about 100 kinds, and contains phyllodulcin, flavonoid, and cartin, which are aromatic components. The main physiological activities include antioxidant activity, vascular endothelial cell protection, and cardiovascular function improvement. According to a recent study, Zyphenoside activates T-cells and regulates immunity such as neuroprotective. It is known to have an antioxidant effect. In particular, it has been known that zipenoside is similar to ginsenoside, which is known to be contained only in ginseng, in structure and physiological activity, and its content is also 2-3 times higher than ginseng.

In the present invention, it may include the step of separating ginsenosides from the extract by extracting the root of the stone derived from the stone.

In the present invention, the term "extract" may be obtained by extraction with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, 1,3-butylene glycol, and ethyl acetate, but is not limited thereto. Does not work. Specifically, in the present invention, the extraction of the saponin component is a step of freezing the roots derived from the stone; Grinding the frozen membrane roots and extracting with an extraction solvent to obtain an extract; And concentrating the extract. In the extraction of the saponin component, it is possible to purchase and use a commercially available product other than stone, or can be used without limitation, such as being harvested or cultivated in nature.

The step of obtaining an extract by extracting with the extraction solvent may use an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited thereto. The extraction solvent may be extracted by adding 1 to 10 times to the amount of membrane roots derived from stones, and specifically, it may be extracted by adding 2 to 3 times, but is not limited thereto. The extraction temperature may be 20 ° C to 100 ° C, specifically 60 ° C to 80 ° C, and more specifically 65 ° C, but is not limited thereto. The extraction time may be 10 minutes to 1 hour, specifically 20 minutes to 40 minutes, and more specifically 25 minutes, but is not limited thereto. The number of extraction times may be 1 to 5 times, and specifically, it may be repeatedly extracted 3 to 4 times, but is not limited thereto. In addition, the step of obtaining the extract may include separation of the saponin component by resin adsorption, but is not limited thereto.

The step of concentrating the extract may further include a process of concentrating the extract under reduced pressure, preferably a rotary evaporator, but is not limited thereto.

In one embodiment of the present invention, the membrane root sample outside the stone is frozen with liquid nitrogen, ground with a mortar and pestle, and extracted with 70% ethanol in a constant temperature water bath at 65 ° C for 25 minutes, and then ultrasonic for 5 minutes in an ultrasonic bath. Treatment. The treated ethanol extract was diluted 7-fold and saponin was extracted by performing a Diaion® HP-20 column. The saponins (ginsenoside and phenenoside) adsorbed on a Diaion® HP-20 column were eluted with 100% ethanol, dried and concentrated using a rotary evaporator to obtain a concentrated compound.

Therefore, in the present invention, it may include the step of producing ginsenosides from the root extract derived from the stone.

In addition, the ginsenoside is not limited thereto, but may be preferably ginsenoside Ro, Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, and more preferably ginsenoside Rd.

In one embodiment of the present invention, a thin film with a silica gel 60 plate using CHCl ₃ : CH ₃ OH: H ₂ O (65:35:10, v / v / v, low phase) as a developing solvent for the saponin component Chromatography (TLC) analysis was performed. As a result, it was confirmed that ginsenosides F1, Rg1 and Re are produced from 4 seeds (2258-1, 2258-2, 3083-1, 3083-2) selected from seeds of various genus Gynostemma , or 2258-1 or It was confirmed that 3083-1 and 3083-2 seeds produced more ginsenosides F1, Rg1 and Re compared to 2258-2 (FIG. 4).

In addition, in one embodiment of the present invention, in order to confirm whether ginsenosides are produced from extra-membrane roots through quantitative analysis of the saponin component, a ginsenoside Rd standard, a membrane root extract and a ginsenoside Rd are added. High performance liquid chromatography (HPLC) was performed on a C18 column (5 μm, 4.6 × 250 mm) using water and acetonitrile as the mobile phase for the membrane root extract, and each ginsenoside was compared with a reference ginsenoside sample. As a result, it was confirmed that a peak corresponding to ginsenoside Rd was detected in the chromatogram of the extracellular membrane root extract (B in FIG. 5), from which the extracellular membrane root extract was used for the production of ginsenoside including ginsenoside Rd. It can be used.

In addition, in an embodiment of the present invention, as a result of quantitatively comparing and analyzing the amount of ginsenoside Rd produced from leaves, stems, and non-stone roots, the leaves, stems, and leaves other than stones When the extract was used, it was confirmed that the content of ginsenoside Rd produced was significantly higher (C in FIG. 7).

On the other hand, in the present invention, the culture of the root-derived membrane root may be to express the ginsenoside biosynthesis gene. The ginsenoside biosynthesis gene is not limited thereto, but may include squalene synthase (GpSS), squalene epoxidase (GpSE), and glycosyltransferase (GpUGT23).

In the present invention, the term, "GpSS ( Gynostemma pentaphyllum squalene synthase)" is squalene synthase, "GpSE ( Gynostemma pentaphyllum squalene epoxidase)" is squalene epoxidase, "GpUGT23" is Uridine diphosphate (UDP) -sugar transferase (UGT, UDP -glycosyltransferase), and these enzymes are known to be involved in the biosynthetic pathway of ginsenosides.

In one embodiment of the present invention, total RNA was extracted from the membrane root of the stone using a Spectrum Plant Total RNA Kit (Sigma-Aldrich). CDNA was synthesized by performing RT-PCR using 1000 ng of total RNA as a template, ₂₀ mM oligo (dT) primer and 5 U / µl M-MLV reverse transcriptase (Promega).

Quantitative real-time PCR was performed using 100ng cDNA using Topreal ™ qPCR 2X PreMIX (SYX Green with high ROX) (Enzynomics). At this time, primers specific to each gene (GpSS, GpSE, and GpUGT23) shown in Table 1 were used, and temperature and time conditions were 15 minutes at 95 ° C, 20 seconds at 95 ° C, 20 seconds at 53 ° C, 54 cycles were performed at 72 ° C. for 20 seconds. The detection of fluorescent materials for quantitative analysis was performed at the last step of each cycle, and the relative amount of gene transcription level was measured using the Bio-Rad CFX connect Real-Time PCR Detection System, and comparative to normalize the measured data It was calculated using the cycle threshold (CT) method. To evaluate the relative expression level of the gene, the expression level of the actin gene was used as an internal reference.

As a result, the GpUGT23 gene, which has the activity of delivering glucose to both squalene synthase (GpSS), squalene epoxidase (GpSE), PPD and PPT-type ginsenosides, related to ginsenoside biosynthesis, is found in extracellular membrane root cultures. It was confirmed that the expression (A in Figure 5). From this, it was confirmed that the extract of the root of the stone can be used for the production of ginsenosides including ginsenoside Rd.

The present invention can be used industrially by producing ginsenosides using a membrane root derived from a stone that has not been used to produce ginsenosides so far.

1 is a view showing the process of culturing the membrane roots from the stone.
Specifically, A of FIG. 1 shows the overall process of cultivating the extracellular-derived membrane root, FIG. 1 B shows the callus derived from the side of the extracellular stem, and FIG. 1 C shows the membrane root formed from the induced callus 1, D of FIG. 1 shows the growth of membrane roots in the medium, and FIG. 1E shows the liquid culture of the membrane roots.
Figure 2, in the callus induction medium, shows the callus formed in the entire periphery of the stem after 2 weeks of culture, the white bar corresponds to a length of 1cm.
Figure 3 shows the growth of extracellular-derived membrane roots after 4 weeks of cultivation in a storage medium to which 2 mg / l IBA (Indole-3-Butyric Acid) was added.
Figure 4 shows the results of performing a thin layer chormatography (TLC) using stem extracts of four different Gynostemma genus plants.
5A shows the relative expression levels of squalene synthase (GpSS), squalene epoxidase (GpSE), and glycosyltransferase (GpUGT23) genes for actin in the dermal root-derived membrane root culture system.
FIG. 5B shows HPLC chromatograms of ginsenoside Rd standards, extra-derived membrane root extract and Rd-added extra-derived membrane root extract.
Figure 6 shows the HPLC chromatogram of the ginsenoside Rd standard and extra-leaves leaf extract, stem extract, and membrane root extract, A in FIG. 6 is leaf extract, B in FIG. 6 is stem extract, and C in FIG. 6 is membrane root The results of the HPLC chromatogram of the extract are shown.
7A and B show HPLC chromatograms of the ginsenoside Rd standard and extra-leaves leaf extract, stem extract and membrane root extract, and FIG. 7C shows the ginsenosides produced from extra-leaves leaves, stem and membrane root extracts It is a quantitative comparison of the amount of Rd (mg / gFW).

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention and the scope of the present invention is not limited by them.

Example 1: Wild Gynostemma  Selection of genus plants

It is reported that each Gynostemma genus has a significantly different composition (Takemoto et al. 1983), and the seeds of various Gynostemma genus plants collected from various regions in Korea have a light / hour of 16 hours at a temperature of 22 to 24 ° C. It was grown in dark conditions, and the levels of ginsenosides and gifenosides were compared using the extract of the stem. Among the seeds, four lines of genus Gynostemma (2258-1, 2258-2, 3083-1, 3083-2) were selected and used for the analysis of metabolites.

Example 2: Induction of callus

To induce callus for membrane root culture, Gynostemma pentaphyllum Makino stem was used as explant. The surface of the explant was sterilized with 70% ethanol for 1 minute, and then sterilized with 1% sodium chlorite for 10 minutes. Thereafter, the explants were immersed in sterile distilled water 3 to 5 times. After sterilization, the explants were cut to a length of 1 cm, a half strength MS medium (2 mg / L α-naphthaline acetate acid (1-NAA) at 25 ° C., 16 hours light / 8 hours dark conditions), 3% sucrose, 0.8% agar).

As a result, callus was induced 7-10 days after culture at the wound site (FIG. 1B), and after 2 weeks of culture, the entire stem explant was enclosed (FIG. 2).

Example 3: Measurement of induction and growth of membrane roots

In order to induce membrane roots from the callus of Example 2, when the callus appeared on the stem surface, it was transferred and cultured in MS (including B5 vitamin) / B5 medium with 2 mg / l of IBA (Indole-3-Butyric Acid) added. .

As a result, the membrane roots were derived from callus 3 to 4 weeks after cultivation (FIG. 3), and the induced membrane roots were added to 100 ml of MS (2 mg / l IBA in an orbital shaker at 150 rpm at a temperature of 25 ° C. and dark conditions). B5 vitamins) / B5 medium and preserved by subculture every 4 weeks.

Meanwhile, the growth of the membrane roots was confirmed by measuring the relative fresh weight (FW) of the membrane roots. As a result of measuring the fresh weight of the membrane roots, it was confirmed that the value increased 2.5 times or more after 2 and 3 weeks of liquid culture.

Example 4: Extraction of saponin component

In order to extract saponin from the induced roots of Example 3, fresh Gynostemma root samples were frozen with liquid nitrogen, ground in a mortar and pestle, extracted with 70% ethanol in a constant temperature water bath at 65 ° C for 25 minutes, and then an ultrasonic bath ( ultrasonic bath) for 5 minutes. The treated ethanol extract was diluted 7-fold and saponin was extracted by performing a Diaion® HP-20 column. The saponins (ginsenoside and phenenoside) adsorbed on the Diaion® HP-20 column were eluted with 100% ethanol and concentrated to dryness with a rotary evaporator to obtain a concentrated compound. The compound was dissolved in methanol and used for component analysis.

Example 5: TLC analysis of extracted saponins

To develop and separate the saponin component extracted in Example 4, a silica gel 60 plate using CHCl ₃ : CH ₃ OH: H ₂ O (65:35:10, v / v / v, low phase) as a developing solvent Thin membrane chromatography (TLC) analysis was performed. Spots appearing on TLC plates were detected by spraying 10% (v / v) H ₂ SO ₄ and heating at 110 ° C. for 10 minutes.

As a result of analyzing the spot on the TLC plate, it was confirmed that ginsenosides F1, Rg1 and Re were produced from the selected 4 seeds of the genus Gynostemma (2258-1, 2258-2, 3083-1, 3083-2), and 2258 Compared to -1 or 2258-2, it was confirmed that the 3083-1 and 3083-2 lines produced more ginsenosides F1, Rg1 and Re (FIG. 4).

Example 6: HPLC analysis of extracted saponins

In order to determine whether ginsenosides are produced from extra-membrane roots through quantitative analysis of the saponin component extracted in Example 4, the ginsenoside Rd standard, the membrane root extract and the ginsenoside Rd-added root extract HPLC separation was performed on a C18 column (5 μm, 4.6 × 250 mm) using water and acetonitrile as mobile phases. The time and ratio of water (A) and acetonitrile (B) were B 65% for 8 minutes, B 90% for 12 minutes, B 100% for 50 minutes, B 32% for 5 minutes. At this time, the flow rate of the mobile phase was 1.0 ml / min, and ginsenoside was observed at a wavelength of 203 nm. Each ginsenoside was compared to a reference ginsenoside sample (ginsenoside Rd).

As a result of analyzing the chromatogram obtained by performing the HPLC, it was confirmed that a peak corresponding to ginsenoside Rd was detected in the chromatogram of the extra-membrane root extract (FIG. 5B). From this, it was confirmed that the extract of the root of the stone can be used for the production of ginsenosides including ginsenoside Rd.

Example 7: Analysis of the expression of genes related to ginsenosides and gifenosides through RT-PCR and real-time quantitative real-time PCR analysis of RNA extracted from extracellular membrane roots

To determine whether the cultures of extracellular membrane roots express ginsenoside and gifenoside related genes such as squalene synthase (GpSS), squalene epoxidase (GpSE) and glycosyltransferase (GpUGT23), extracellular membrane roots Total RNA was extracted from RT-PCR of RNA and real-time quantitative real-time PCR analysis was performed.

Specifically, total RNA was extracted from extra-membrane roots using a Spectrum Plant Total RNA Kit (Sigma-Aldrich). CDNA was synthesized by performing RT-PCR using 1000 ng of total RNA as a template, ₂₀ mM oligo (dT) primer and 5 U / µl M-MLV reverse transcriptase (Promega).

Quantitative real-time PCR was performed using 100 ng of cDNA in a 20 μl reaction volume using Topreal ™ qPCR 2X PreMIX (SYX Green with high ROX) (Enzynomics). At this time, primers specific to each gene (GpSS, GpSE, and GpUGT23) shown in Table 1 were used, and temperature and time conditions were 15 minutes at 95 ° C, 20 seconds at 95 ° C, 20 seconds at 53 ° C, 54 cycles were performed at 72 ° C. for 20 seconds. The detection of fluorescent materials for quantitative analysis was performed at the last step of each cycle, and the relative amount of gene transcription level was measured using the Bio-Rad CFX connect Real-Time PCR Detection System, and comparative to normalize the measured data It was calculated using the cycle threshold (CT) method. To evaluate the relative expression level of the gene, the expression level of the actin gene was used as an internal reference.

[Table 1]

As a result, the GpUGT23 gene, which has the activity of delivering glucose to both squalene synthase (GpSS), squalene epoxidase (GpSE), PPD and PPT-type ginsenosides, related to ginsenoside biosynthesis, is found in extracellular membrane root cultures. It was confirmed to be expressed. Specifically, compared to the actin gene used as an internal reference, GpSS showed a relative expression level of about 0.65, GpSE about 0.25, and GpUGT23 about 0.53 (FIG. 5A).

From this, it was confirmed that the ginsenoside biosynthesis gene is expressed at a significant level in the extracellular membrane root culture, and thus the extracellular membrane root can be used for the production of ginsenoside.

Example 8: Quantitative comparison and analysis of HPLC of ginsenoside Rd and HPLC of extract of leaves, stems, or roots of leaves other than stone

In order to quantitatively compare and analyze the amount of ginsenoside Rd produced from other parts such as leaves and stems other than stones and the roots of stones, ginsenoside Rd standards, leaves, stems, leaves of roots and extracts of ginsenosides Rd HPLC was performed in the same manner as in Example 4 for the leaves, stems, and membrane root extracts to which is added, and the resulting chromatogram was analyzed.

As a result, it was confirmed that the content of ginsenoside Rd produced compared to the leaf or stem extract was significantly higher in the extract of the root of the stone beside the stone (FIG. 6). In addition, as a result of quantitative analysis of the ginsenoside Rd content produced, it is possible to produce ginsenoside Rd of about 0.09 mg / gFW in the case of the membrane root extract, while it is possible to produce about 0.04 mg / gFW, which is half of the stem extract, , In the case of leaf extract, it was confirmed that ginsenoside Rd was hardly produced (FIG. 7C).

That is, this is a result showing that the method of the present invention using a membrane root derived from doldon compared to other parts other than doldol can be usefully used to produce ginsenoside Rd at a significantly higher level.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the detailed description and equivalent concepts thereof.

Claims (12)

Forming an adventitious root from a Gynostemma pentaphyllum stem; And
A method of producing ginsenosides from a membrane root derived from a stone, comprising the step of separating ginsenosides from the membrane roots,
The formation of membrane roots from the extra-stem stems,
Inducing a callus by culturing an extracellular stem; And
Including the step of inducing the roots by culturing the callus,
Wherein the ginsenoside is ginsenoside Rd.
delete The method according to claim 1, wherein the extracellular for cultivation is an extracellular explant prepared by excising extracellular tissue or organs.
delete The method of claim 1, wherein the extracellular stem is cultured for 7 to 10 days to induce callus.
The method of claim 1, wherein the callus is incubated for 3 to 4 weeks to induce membrane roots, and ginsenosides are produced from membrane roots derived from stones.
According to claim 1, After inducing the membrane roots, further comprising the step of subculture to maintain the membrane roots, a method of producing ginsenosides from the membrane roots derived from the stone.
The method of claim 1, wherein the ginsenoside further comprises any one or more selected from the group consisting of ginsenosides Ro, Rb1, Rb2, Rc, Re, Rg1, Rg2, Rg3, Rh1, Rh2. A method of producing ginsenosides from a derived root.
delete The method of claim 1, wherein the ginsenoside is to extract and separate membrane roots from the roots, producing ginsenosides from membrane roots derived from stones.
The method of claim 1, wherein the culture of the extracellular-derived membrane root expresses a gene of ginsenoside biosynthesis, and the production of ginsenoside from the extra-derived membrane root.
12. The method of claim 11, wherein the ginsenoside biosynthesis gene is squalene synthase (GpSS), squalene epoxidase (GpSE), and glycosyltransferase (GpUGT23), which produces ginsenosides from membrane roots derived from stones. Way.

Images