CN115725620B - Method for synthesizing panax japonicus saponins in pseudo-ginseng cells - Google Patents
Method for synthesizing panax japonicus saponins in pseudo-ginseng cells Download PDFInfo
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Abstract
The invention discloses a method for synthesizing panax japonicus saponins in pseudo-ginseng cells, which comprises the following steps of constructingPnDSGene RNAi expression vector and method for introducing the same into pseudo-ginseng cells to obtain synthetic panax japonicus soapThe nucleotide sequence of the RNAi fragment of the notoginseng cell of the glycoside is shown as SEQ ID NO. 1; the method can inhibit the first key enzyme gene related to dammarane type triterpene saponin synthesis branch in Notoginseng radix saponin biosynthesis pathway from reverse regulation point of viewPnDSThe expression of the dammarane type triterpenoid saponin is reduced, so that the pseudo-ginseng cells are promoted to synthesize the oleanane type saponin which is not originally contained, namely the panax japonicus saponin.
Description
Technical Field
The invention belongs to the technical field of saponin synthesis, and particularly relates to a method for synthesizing panax japonicus saponins in pseudo-ginseng cells.
Background
Panax (Panax) belonging to Araliaceae (Araliaceae) comprises various medicinal plants, and Ginseng radix (Panax ginseng), radix Panacis Quinquefolii (Panax quinquefolius), notoginseng radix (Panax notoginseng) and Panax japonicum (Panax japonicum) are most widely used. The main active components of the compound are triterpene saponins, and the compound has various pharmacological effects of preventing and treating cardiovascular and cerebrovascular diseases, resisting fatigue and oxidation, enhancing immunity, protecting liver and the like. The biological synthesis paths of the triterpene saponin of the ginseng medicinal plant are basically the same in the initial stage and the framework construction stage, and the main difference of the saponin synthesis among the species is that after the 2, 3-oxidation squalene is formed, the 2, 3-oxidation squalene is taken as a common precursor, and two independent triterpene saponin synthesis branches respectively appear. 2, 3-oxidation squalene enters a dammarane type saponin synthesis branch through the catalysis of dammarane glycol synthetase (DS); catalyzed by beta-amyrin synthase (beta-AS), enters the synthesis branch of oleanane-type saponin.
The triterpene saponin separated from the ginseng is mainly dammarane type saponin, the ginseng, the American ginseng and the rhizoma panacis majoris all contain dammarane type and oleanane type triterpene saponin, but the radix notoginseng only contains dammarane type saponin, and the oleanane type saponin can not be synthesized, which is the characteristic of the radix notoginseng which is different from other ginseng species. It is known that the panax japonicus saponins IV and the panax japonicus saponins IVa belong to oleanane type saponins, and the pseudo-ginseng under the natural state does not have the capability of synthesizing the panax japonicus saponins IV and the panax japonicus saponins IVa.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synthesizing panax japonicus saponins in pseudo-ginseng cells, which comprises the steps of constructing a PnDS gene RNAi expression vector, transferring the RNAi expression vector into pseudo-ginseng cells, inhibiting the expression of a first key enzyme PnDS gene of a dammarane type triterpenoid saponins synthesis branch in pseudo-ginseng, weakening the metabolic flux of the dammarane type triterpenoid saponins synthesis branch, and promoting the pseudo-ginseng cells to synthesize the panax japonicus saponins, wherein the nucleotide sequence of an RNAi fragment is shown as SEQ ID NO. 1;
the invention aims at realizing the following technical scheme:
1. extracting total RNA from root of notoginseng, synthesizing notoginseng cDNA by reverse transcription, using synthesized first-strand cDNA as template, amplifying PnDS gene interference fragment by PCR, in which the nucleotide sequence of gene PnDS is shown in SEQ ID NO. 1;
2. constructing a PnDS gene RNAi interference vector pHelshag-PnDS, transforming agrobacterium, and screening out positive monoclonal by PCR;
3. introducing pHellsagate-PnDS into pseudo-ginseng cells for expression by using an agrobacterium-mediated genetic transformation method, and screening positive transgenic cell lines by antibiotic screening and qRT-PCR;
4. extracting saponin from transgenic cells and non-transgenic cell lines of Notoginseng radix, and analyzing the difference of the type and content of saponin between transgenic cells and non-transgenic cell lines.
The invention has the advantages and technical effects that:
the invention provides a new method for producing the panax japonicus saponin IVa and the panax japonicus saponin IV, which are main active components of the panax japonicus, belong to typical oleanane type saponins and are not contained in pseudo-ginseng medicinal materials. The invention synthesizes the panax japonicus saponin IVa and the panax japonicus saponin IV in the pseudo-ginseng cells by a gene regulation method, realizes the heterologous expression of the saponins among seeds, has simple method and easy operation, and has the potential of industrial production and market popularization and application.
Drawings
FIG. 1 shows the result of RNAi fragment sequence amplification;
FIG. 2 shows the amplification product of RNAi fragments with attB recombination sites, wherein 1: RNAi fragment, M: marker dl2501;
FIG. 3 is an identification electrophoresis pattern of transfer of pHellshag-PnDS vector into E.coli DH 5. Alpha. Wherein 1-8: bacterial liquid sample, M: marker dl2501, +: pHellsagate-PnDS plasmid, -: a negative control;
FIG. 4 is a diagram showing the identification electrophoresis of transfer of pHellagate-PnDS vector into Agrobacterium LBA4404, wherein 1-5: a bacterial liquid sample; m: marker dl2502; negative control;
FIG. 5 is an electrophoretogram of PCR detection of transgenic pseudo-ginseng cells, wherein 1-6: transgenic pseudo-ginseng cell line, M: marker dl2502;
FIG. 6 shows the results of real-time fluorescence quantitative PCR detection of the relative expression levels of the PnDS genes and 5 genes related to the synthesis of panax japonicus saponin IVa and panax japonicus saponin IV in transgenic pseudo-ginseng cells, wherein WT is a wild type cell line, and T1-T4 are transgenic pseudo-ginseng cell lines of 4 PnDS genes;
FIG. 7 is a graph showing the content of saponins in transgenic Notoginseng radix cell line, wherein WT is wild type Notoginseng radix cell line, and T1-T4 are transgenic Notoginseng radix cell line.
Detailed Description
The present invention will be described in further detail by way of examples, but the scope of the invention is not limited to the above description, and the methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional commercial reagents or reagents prepared by conventional methods unless otherwise specified.
Example 1: cloning of RNAi fragment of PnDS Gene
(1) Extraction of Panax notoginseng RNA
The invention adopts an improved guanidine isothiocyanate method to extract total RNA of pseudo-ginseng callus, and the specific operation is as follows: and (3) carrying out RNase removal treatment on the mortar and the mortar rod, carrying out high-temperature dry heat sterilization treatment, and cooling to room temperature. Weighing a proper amount of pseudo-ginseng callus cells, placing the pseudo-ginseng callus cells into a treated grinding bowl, grinding the pseudo-ginseng callus cells into powder by utilizing liquid nitrogen, adding 10% (w/v) of precooled RNA extraction buffer solution and 1.0% (w/v) of beta-mercaptoethanol, and fully grinding. Transferring 1.0mL of grinding fluid into a 2mL centrifuge tube, adding 500 mu L of RNA to extract phenol, 100 mu L of chloroform and 1/10 volume of 2M sodium acetate solution (pH 4.0), shaking vigorously and mixing, standing on ice for 5min, centrifuging at 4 ℃ for 12500g for 15min, slowly sucking the supernatant, transferring into a new 2mL centrifuge tube, adding 1:1 equal volume of RNA to extract phenol/chloroform, shaking vigorously and mixing, standing on ice for 5min, centrifuging at 4 ℃ for 12500g for 15min (repeating the steps until the middle layer almost disappears). Slowly sucking the supernatant, transferring to a new 2mL centrifuge tube, adding equal volume of chloroform, shaking vigorously, mixing, standing on ice for 5min, centrifuging at 4 ℃ for 12500g for 15min, slowly sucking the supernatant, transferring to a new 1.5mL centrifuge tube, adding 1/10 volume of 3M sodium acetate solution (pH of 5.2), adding equal volume of isopropanol, slowly reversing, mixing, standing at-20 ℃ for 1.5h, centrifuging at 4 ℃ for 25min, and centrifuging at 12500 g; the supernatant was discarded, the pellet was washed with 75% ethanol solution by pipetting, after two pipetting washes, the liquid was blotted off using a pipetting gun and air-dried on a super clean bench until ethanol was evaporated thoroughly, and then 15-30. Mu.L of RNae water was added to dissolve the RNA pellet. The total RNA integrity of the extracted pseudo-ginseng callus cells is detected by agarose gel electrophoresis, and then the concentration and purity of the extracted RNA are detected by an ultraviolet spectrophotometer.
(2) Synthesis of first strand cDNA
Selecting better quality RNA, synthesizing cDNA first strand by using GoScript reverse transcription system of Promega company, firstly Oligo (dT) 15 1.0 mu L of total RNA5.0 mu g and 10 mu L of nucleic-free Water are added, fully and gently mixed, and the mixture is placed in a Water bath kettle at 70 ℃ for Water bath for 5min for pre-denaturation, then placed in an ice bath for 5min immediately, and then the following reagents are added: nuclease-free Water 1.6 mu L, goScript TM 5×Reaction Buffer 4.0μL、PCR Nucleotide Mix 1.0μL、MgCl 2 (25mM)2.0μL、RecombinantRibonuclease Inhibitor 0.4μL、GoScript TM Reverse Transcriptase 1.0.0 μl, mixing, centrifuging, annealing at 25deg.C for 5min, extending in a water bath at 42deg.C for 90min, standing in a water bath at 70deg.C for 15min, and stopping reverse transcriptase activity to obtain Notoginseng radix cDNA first strand.
(3) Synthesis of RNAi fragments
The specific primer PnDS was designed based on the full-length sequence of the Notoginseng radix PnDS gene (KJ 804174.1) by PrimerPremier5.0 F :5'-TCCCCTTATCATTGCCCT-3' and PnDS R :5'-GCTTTTTCCCCATTTCCT-3' performing high-fidelity PCR amplification under the action of DNA polymerase Ex Taq by taking the first strand of the obtained pseudo-ginseng cDNA as a template, wherein the PCR reaction conditions are as follows: 98 ℃ for 3min;98 ℃, 10s,60 ℃, 15s,72 ℃, 15s,35cycles;72 ℃ for 5min; separating the obtained amplified product by agarose gel electrophoresis of 1%, and recovering the fragment of the amplified product to be the 503 th-1246 th nucleotide sequence of the gene PnDS;
the target fragment is recovered and then connected with pGEM T-easy vector, transferred into escherichia coli DH5 alpha, single colony on a flat plate is randomly selected, bacterial liquid PCR amplification is carried out, and positive clone is detected. Sequencing the monoclonal which is detected as positive by PCR (polymerase chain reaction) of bacterial liquid, comparing the sequenced sequence with the pseudo-ginseng PnDS gene sequence by Blast software in NCBI, wherein the result is shown in figure 1, the nucleotide sequence is shown as SEQ ID NO. 1, the comparison result is correct, inoculating the escherichia coli monoclonal with correct result into a liquid LB culture medium containing spectinomycin for expansion culture, culturing for 15h at 37 ℃ under 200rpm, and extracting plasmid (T-PnDS) by using a SanPrep column type plasmid extraction kit.
Example 2: construction of RNAi expression vector for PnDS Gene
(1) Cloning of RNAi fragments with attB recombination sites
According to the technical principle of gateway TM, a primer BP-C is designed F (5’-GGGGACAAGTTTGTACAAAAAAGCAGG CTTCCCCTTATCATTGCCCT-3') and BP-C R (5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGCTTTTTCCCCATTTCCT-3') (underlined is attB recombination site), the adaptor was added to both ends of RNAi fragment by PCR means,the target fragment and the carrier are subjected to homologous recombination reaction, and the extracted plasmid is used as a template, BP-C F /BP-C R Carrying out attB-PCR amplification by using Ex Taq as a primer, wherein the PCR reaction conditions are the same as those in the step (3) of the example 1, and both sides of the RNAi fragment are connected with attB recombination sites after the amplification; the PCR products were recovered with the gel, and the results are shown in FIG. 2;
(2) Construction of RNAi expression vector for PnDS Gene
According toBP Clonase TM II Enzyme Mix kit instruction manual RNAi vector construction, the reaction system is set as follows: attB-PCR gel recovery product 0.15. Mu.g, pHellsag 2 vector plasmid 0.15. Mu.g, BP Clonase TM II Enzyme Mix 2.00 μ L, TE buffer (ph=8.0) was made up to 10 μl; sequentially adding the solutions, blowing and mixing uniformly by a pipetting gun, placing in a water bath at 25 ℃ for water bath, reacting for 4 hours, then adding 1 mu L of proteinase K into the reaction solution, water-bathing in the water bath at 37 ℃ for 10 minutes, stopping the reaction, converting the reaction product into competent cells of escherichia coli, picking up monoclonal, performing amplification culture on the monoclonal, and then extracting plasmids (pHellgate-PnDS); the resulting plasmid was subjected to single cleavage with restriction enzymes XbaI and XhoI, respectively, in the following manner: pHellsag-PnDS plasmid 5.0. Mu.g, 10 XBuffer 2.0. Mu.L, restriction enzyme XbaI/XhoI 1.5. Mu. L, ddH 2 Supplementing O water to 20 mu L;
placing the enzyme digestion system in a water bath kettle at 37 ℃ for 5 hours of enzyme digestion reaction, detecting enzyme digestion products by 1% agarose gel electrophoresis after the reaction is finished, and judging that the construction of the RNAi expression vector pHellsag-PnDS of the PnDS genes is successful according to the sizes of the enzyme digestion products of the two enzymes; the vector was then transferred into competent cells of Agrobacterium LBA4404 by liquid nitrogen freeze thawing, and empty vector pHelflag 2 was also transferred into competent cells of LBA4404, as an empty vector control, and the result was shown in FIG. 4, from which it can be seen that the amplified band was about 750bp, indicating that RNAi fragment of PnDS gene had been successfully transformed into Agrobacterium.
Example 3: agrobacterium tumefaciens mediated genetic transformation of Notoginseng radix
1. Pseudo-ginseng cell preculture
(1) Collecting stem and leaf of Notoginseng radix, and culturing with Notoginseng radix callus culture medium (MS culture medium +2, 4-D2 mg/L +KT1mg/L, culture medium pH 5.6) at 25+ -1deg.C for 28 days in dark place to obtain Notoginseng radix callus; performing secondary culture on the obtained pseudo-ginseng callus by adopting an MS solid culture medium containing 2, 4-D2 mg/L, KT mg/L for 15 days to obtain pseudo-ginseng secondary culture cells;
(2) Selecting a pseudo-ginseng callus cell with good growth state, transferring the pseudo-ginseng callus cell onto a pseudo-ginseng callus cell preculture medium (acetosyringone is added to the pseudo-ginseng callus culture medium to 40 mg/L), spreading and covering the whole surface of the culture medium, and performing dark culture for 3 days at 25 ℃;
(2) Notoginseng cell infection
100-200 mu L of activated LBA4404 agrobacterium tumefaciens bacteria liquid carrying PnDS gene RNAi expression vector plasmid pHellshag-PnDS is sucked and coated on LB solid medium plates containing 50mg/L kanamycin and 25mg/L rifampicin (the step can be carried out simultaneously with the preculture of pseudo-ginseng callus cells), and the plates are inversely cultured for 2-3 days in a 28 ℃ incubator until the plates are full of a layer of thick bacteria. Scraping the grown bacterial colony with a certain size with inoculating needle, culturing in MGL liquid culture medium containing 40mg/L acetosyringone at 28deg.C under shaking with 200rpm shaker until bacterial liquid OD 600 0.6-0.8, inoculating into the above radix Notoginseng callus cells pre-cultured for 3 days, completely immersing the radix Notoginseng callus cells in the bacterial liquid, and shake culturing at 25deg.C on a shaking table at 105rpm for 20min;
(3) Cell harvesting and co-cultivation
After infection, suction filtration is carried out on the pseudo-ginseng callus cells by using a Buchner funnel to remove bacterial liquid, then residual bacterial liquid on the surfaces of the pseudo-ginseng callus cells is sucked and dried by using sterile filter paper, then the pseudo-ginseng callus cells are transferred to pseudo-ginseng co-culture medium (the direct contact of agrobacterium with the culture medium is prevented, and the overgrowth of the agrobacterium is caused) with the surface of the pseudo-ginseng callus cells, and the pseudo-ginseng co-culture medium and the pseudo-ginseng callus cell pre-culture medium are co-cultured for 3 days under the dark condition of 25 ℃.
(4) Degerming culture
Transferring the pseudo-ginseng callus cells into a sterilized beaker after co-culture is finished, washing the pseudo-ginseng callus cells with sterile water containing 400mg/L of cephalosporin for 5-6 times to fully remove agrobacterium, carrying out suction filtration on the pseudo-ginseng callus cells with a Buchner funnel after washing is finished to remove liquid, and then sucking residual liquid on the surfaces of the pseudo-ginseng callus cells with sterile filter paper. Transfer the pseudo-ginseng callus cells into a degerming culture medium (cephalosporin and kanamycin are added to the pseudo-ginseng callus culture medium to a final concentration of 400mg/L and 50 mg/L), and perform degerming culture for 15 days under the dark condition at 25 ℃.
(5) Screening culture and subculture
After 15 days of degerming culture, transferring the pseudo-ginseng callus cells to a pseudo-ginseng callus screening culture medium (kanamycin is added to the pseudo-ginseng callus culture medium to a final concentration of 50 mg/L), carrying out subculture once about 35 days (the subculture period can be adjusted according to the growth condition), and carrying out 4-5 times of subculture screening to obtain the PNDS gene RNAi transgenic pseudo-ginseng cell line with kanamycin resistance.
Example 4: detection of PnDS Gene in transgenic pseudo-ginseng cell and Gene expression level related to Synthesis of Panax japonicus saponins
(1) Transgenic pseudo-ginseng cell line genome DNA level detection
The modified CTAB method is adopted to extract genome DNA of a transgenic pseudo-ginseng cell line, upstream and downstream primers npt-F (5'-CTCTGATGCCGCCGTGTT-3') and npt-R (5'-CCCTGATGCTCTTCGTCCA-3') are designed according to kanamycin npt II resistance gene sequence on T-DNA in pHellsagge 2 vector, PCR detection is carried out by taking the extracted pseudo-ginseng genome DNA as a template, positive transgenic pseudo-ginseng cells are screened, and a specific strip with the size of about 430bp is amplified in the graph as shown in figure 5, and accords with the expected size, so that the six pseudo-ginseng transgenic cell lines are all imported into exogenous DNA and integrated onto genome DNA for stable inheritance, and the PnDS gene RNAi transgenic cell line is obtained through preliminary determination.
(2) Fluorescent quantitative PCR (polymerase chain reaction) detection of transgenic pseudo-ginseng cells
Extracting total RNA of positive transgenic pseudo-ginseng cell strains and non-transgenic pseudo-ginseng cell strains, and reversely transcribing the total RNA into cDNA, wherein the specific operation steps are the same as in the example 1;
primers for fluorescent quantitative PCR were designed based on the 18S rRNA gene (accession number: D85171.1), the beta-amyrin synthase (beta-AS) gene (accession number: KP 658156), the oleanolic acid synthase (Oleanic acid synthase, CYP716A52v 2) gene (accession number: JX 036032.1), the oleanolic acid glucuronyl transferase (oleanolic acid glucuronosyltra nsferase, OAGT) gene (accession number: MH 819287.1), the PjmUGT1 gene and the PjmUGT2 gene: 18S-F:5'-GGGGAGTATGGTCGCAAGG-3',18S-R:5'-CAGAACAT CTAAGGGCATCACAG-3'; beta-AS-F: 5'-GTATTCCCTGTAGAGCATCGCAT-3' beta-AS-R: 5'-GGCACAGGCGTTGTTTTCAC-3'; CYP716A52v2-F:5'-AGGAGCAAATGGAGATAGTGA-3', CYP716A52v2-R:5'-GATTGAGAAACCGTTGTAGG-3'; OAGT-F:5'-GCATAATCTCGGACA AGTAC-3', OAGT-R:5'-AAAGGTTGGGAGTCTGAAGT-3'; pjmUGT1-F:5'-TCACATAAA TCCGATGGTCC-3', pjmUGT1-R:5'-AGAAATCCCTGAAATCCTCC-3'; pjmUGT2-F:5'-GC ATTCTCCCTTTGTTTCAG-3', pjmUGT2-R:5'-CGACTTGCCTCACTCTTCCT-3'. The cDNA synthesized by reverse transcription was diluted 5-fold, i.e., 20. Mu.L of cDNA was diluted to 100. Mu.L. Using the cDNA after dilution as template, using the primer for fluorescence quantitative PCR, and performing fluorescence quantitative PCR according toqPCRMaster Mix instructions were run and each gene corresponding to each sample was tested 3 times in duplicate. Utilization 2 -ΔΔCt The method processes fluorescence quantitative data, analyzes and detects the relative expression quantity of PnDS genes in a transgenic pseudo-ginseng cell line relative to a non-transgenic pseudo-ginseng cell line, and the result is shown in figure 6, and shows that the expression quantity of key enzyme PnDS genes in four selected T1-T4 transgenic pseudo-ginseng cell lines is obviously lower than that of a common pseudo-ginseng cell line, and the relative expression quantity of genes beta-AS, CYP716A52v2, OAGT, pjmUGT1 and PjmUGT2 related to the synthesis of the panax japonicus saponins IV is obviously higher than that of the common pseudo-ginseng cell line.
Example 5: detection of saponin content in transgenic pseudo-ginseng cells
(1) Preparation of sample solutions
Collecting callus cells of the transgenic pseudo-ginseng cell line and the common pseudo-ginseng cell line in the step (1) of example 4, respectively marking the callus cells, then placing the callus cells in a 55 ℃ oven for drying (about 12 hours), fully grinding the callus cells into powder after drying, respectively weighing 0.5g of transgenic pseudo-ginseng cell line powder and common pseudo-ginseng cell line powder, respectively placing the transgenic pseudo-ginseng cell line powder and the common pseudo-ginseng cell line powder in a 50mL centrifuge tube, respectively adding 50mL of 70% methanol solution into the centrifuge tube, soaking the transgenic pseudo-ginseng cell line powder and the common pseudo-ginseng cell line powder overnight, carrying out crushing treatment (60W, ultrasonic for 4s and intermittent for 2 s) by utilizing ultrasonic waves for 1.5-2.0 hours until the pseudo-ginseng cell line powder is fully crushed, transferring the pseudo-ginseng cell line powder into a centrifuge for centrifugation at 4000rpm for 30 minutes after ultrasonic waves are finished, collecting supernatant, placing the supernatant in the oven for drying at 50-55 ℃, dissolving the supernatant in 10mL distilled water, and extracting the supernatant with water saturated n-butanol for 3 times. Collecting the extract, placing the extract in a 50-55 ℃ oven for drying, dissolving with 70% methanol solution after drying, fixing the volume to 25mL, and filtering with a 0.45 mu M microporous filter membrane to obtain the saponin solution.
(2) High performance liquid chromatography
Detecting the type and the content of the saponins in the transgenic pseudo-ginseng cells by utilizing HPLC, wherein the detection conditions of the HPLC are as follows: the column was a Waters-XTerra-MS-C18 (5 μm,250 mm. Times.4.6 mm, USA). The mobile phase is water and acetonitrile. Gradient elution: 0-20min,20% acetonitrile; 20-30min,20% -35% acetonitrile; 30-40min,35% acetonitrile; 40-50min,35-40% acetonitrile; 50-60min,40-100% acetonitrile. The flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is set to 203nm; the results are shown in FIG. 7, and the results show that the transgenic pseudo-ginseng cells contain oleanane-type saponins (panax japonicus saponins IVa and panax japonicus saponins IV), while the unmodified wild pseudo-ginseng cells do not produce oleanane-type saponins (panax japonicus saponins IVa and panax japonicus saponins IV).
Claims (1)
1. A method for synthesizing panax japonicus saponins in pseudo-ginseng cells is characterized in that: transferring RNAi fragments of the interference gene PnDS into pseudo-ginseng cells to obtain pseudo-ginseng cells for synthesizing the panax japonicus saponins, wherein the nucleotide sequence of the RNAi fragments of the interference gene PnDS is shown as SEQ ID NO. 1;
the transgenic Notoginseng radix cell can synthesize Panax japonicus saponin IV and Panax japonicus saponin IVa.
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