|Year : 2019 | Volume
| Issue : 4 | Page : 236-242
Purification and identification of a novel protein isolated from Panax quinquefolium and evaluation of its In vitro antioxidant properties
Xue-Hui Li1, Yang Yang1, Zhao Yu1, Ning Xu2, Xiao-Lei Tang2, Xue-Yuan Bai1, Si-Ming Wang1, Hao-Yuan Chen2, Da-Qing Zhao1, Li Liu2, Bin Qi2
1 Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, China
2 College of Pharmacy, Changchun University of Chinese Medicine, Changchun, China
|Date of Submission||02-Mar-2019|
|Date of Decision||02-Apr-2019|
|Date of Acceptance||22-Apr-2019|
|Date of Web Publication||03-Dec-2019|
Prof. Bin Qi
College of Pharmacy, Changchun University of Chinese Medicine, Changchun, Jilin, 130117
Prof. Li Liu
College of Pharmacy, Changchun University of Chinese Medicine, Changchun, Jilin,130117
Source of Support: None, Conflict of Interest: None
Objective: A novel protein was first purified from Panax quinquefolius L. (AGNP), and in vitro antioxidant activities of AGNP were first studied in this work. Methods: AGNP was purified by Ion-exchange chromatography and Gel-filtration chromatography. The chemical characterizations of AGNP were tested by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure gel-filtration chromatography, MALDI-TOF-MS and HPLC. In vitro antioxidant effects were tested in simple antioxidant assay including 2,2-diphenylpicrylhydrazyl radical scavenging, superoxide radical (O2−) scavenging, hydroxyl radical (OH) scavenging, and ferric-reducing ability. Results: The results showed which the content of AGNP measured by Bradford method was 2.42 mg/mL and the subunit molecular weight of AGNP measured by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was 15 kD. The AGNP molecular weight was15, 114 Da both of SDS-PAGE and mass spectrum purity. The result of high-pressure gel-filtration chromatography demonstrated that the molecular weight of AGNP was 31,086 Da, which implied that AGNP was a homodimer. The in vitro Antioxidant results indicated that AGNP had obvious effects to remove the free radicals in vitro. Conclusion: In conclusion, AGNP had more powerful antioxidant capacity and it can be used as an effective natural antioxidant to alleviate oxidative stress.
Keywords: In vitro antioxidant properties, novel protein, Panax quinquefolium
|How to cite this article:|
Li XH, Yang Y, Yu Z, Xu N, Tang XL, Bai XY, Wang SM, Chen HY, Zhao DQ, Liu L, Qi B. Purification and identification of a novel protein isolated from Panax quinquefolium and evaluation of its In vitro antioxidant properties. World J Tradit Chin Med 2019;5:236-42
|How to cite this URL:|
Li XH, Yang Y, Yu Z, Xu N, Tang XL, Bai XY, Wang SM, Chen HY, Zhao DQ, Liu L, Qi B. Purification and identification of a novel protein isolated from Panax quinquefolium and evaluation of its In vitro antioxidant properties. World J Tradit Chin Med [serial online] 2019 [cited 2020 Apr 2];5:236-42. Available from: http://www.wjtcm.net/text.asp?2019/5/4/236/271960
| Introduction|| |
Ginseng is a main traditional Chinese medicine in oriental countries being part of the Araliaceae family. It has been used for thousands of years with mysterious powers as one of the popular selling herbs in the orient. Both the common varieties are ginseng and American ginseng. Compared with Asian ginseng, American ginseng has a long history of use and a large number of studies. However, the study of its constituents is less extensive. Panax quinquefolium is primarily herb in both eastern US and Canada and transplanted in China as a kind of herb just like Asian ginseng.
P. quinquefolium contains a good deal of active ingredients such as phenolic compounds, polysaccharides, essential oil, fatty acids, ginsenosides, peptidoglycans, and nitrogen-containing compounds. Ginsenoside is generally believed to be the main active ingredient. The triterpene saponins belong to a family of steroids with a four transring rigid steroid skeleton. At least 40 saponins have been identified and isolated from ginseng until now. Modern researches show that American ginseng has a large number of pharmacological properties including hypoglycemic activities, neuroprotective effects, anticancer, antioxidant, immunomodulation, and so on. During the recent 10 years, Panax ginseng and Panax quinquefolium have been put to use in Asia countries as the dietary supplementation, especially in Korea and China, which may rich in antioxidant components that can persuasively defend against the molecular effects of free radicals and reactive oxygen species (ROS) and lipid peroxidation. It has been reported that ginseng extract shows good antioxidant effect including free radical scavenger, protecting low density lipoprotein from oxidization, and inhibiting lipid peroxidation. Moreover, American ginseng can prevent Fe-EDTA and H2O2 caused plasmid DNA strand scissions on account of the water-soluble and lipid-soluble antioxidant effect of the ingredients. Ginsenosides have been regarded as the main active ingredient in ginseng and American ginseng. Modern studies show that ginsenosides Rh2, Rg2, and Rg3 were prooxidative; R1, Rc, Rd, Re, Rb1, Rg1, Rh1, and Rb3 served as antioxidants, and Rc guarded human erythrocytes mainly against hemin-induced hemolysis., Nevertheless, fewer studies have been reported for the rest of constituents for the biological activities of American ginseng and ginseng. It is reported that proteins purified from ginseng and American ginseng have multifarious biological activities, including antiviral, anti-fatigue, and antifungal.,,,,, In the study of our laboratory, we find that proteins purified from ginseng may possess the monocyte–macrophage function, cell-mediated immunity, and the weight of immune organs in rats. Ginseng proteins may increase the ability of antifatigue and antihypoxia. The proteins purified from ginseng have the therapeutic effects on experimental hyperlipidemia in mice. Moreover, we observe that ginseng proteins have the protective effect of against radiation damage in rats.
In the research, we purified a novel protein from P. quinquefolium and studied its characteristic initially with high-pressure gel-filtration chromatography (HPGFC) and matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) method.
Our study focused onin vitro antioxidant activities of a novel protein purified from P. quinquefolium with simple antioxidant assay including 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging, superoxide radical (O2−) scavenging, hydroxyl radical (OH) scavenging, and ferric-reducing ability. Moreover, the purpose to investigate the antioxidant activitiesin vitro is to make clear the potential mechanism of P. quinquefolium nutritious properties.
| Materials and Methods|| |
Plant material and extraction
The roots of 4-year-old fresh Panax quinquefolius were purchased from Changbai Mountains. Roots of American ginseng were homogenized with 0.05 M Tris-HCl buffer solution (pH 7.4) at 4°C for 24 h and then centrifuged. The ammonium sulfate was added to the supernatant to 80% saturation and stirred incessantly. Dialysis was carried out to get rid of the ammonium sulfate, then dissolved in water, and applied to the hollow fiber membrane, and the concentrated solution was lyophilized, and then, American ginseng water-soluble proteins (AGWSPS) were obtained, in which the content of AGWSPS measured by Bradford method was 78% (w/w). The yield of AGWSPS was 4.2%.
Chemicals and reagents
SP Sepharose FF, Sephacryl S-100, and DEAE Sepharose FF were obtained from Amersham Biosciences; trifluoroacetic acid (TFA) (high performance liquid chromatography [HPLC] grade) was purchased from Tedia, and acetonitrile (ACN) (HPLC grade) was purchased from Sigma. The other reagents used were analytical grade.
Purification of the novel protein
AGWSPS were applied to SP Sepharose column (10 mm × 100 mm), which was preequilibrated with acetate buffer (pH 4.5). The column was washed with acetate buffer (pH 4.5) to get rid of the unbound proteins, after that eluted with 0.3 M NaCl at a flow rate of 1.0 ml/min. The fractions were gathered and concentrated and dialyzed against 0.05 M Tris-HCl buffer (pH 7.4). The proteins were loaded onto a preequilibrated DEAE column (10 mm × 100 mm) and washed with Tris-HCl buffer (pH 7.4) to get rid of the unbound proteins. The target protein was eluted with a linear gradient of 0.2 M NaCl at a flow rate of 1.0 mL/min, and fractions were gathered and monitored at 280 nm to isolate the crude novel protein of American ginseng roots (AGCNP).
The fractions which were separated after ion-exchange chromatography were gathered and then purified employing gel-filtration chromatography. Samples were dissolved in 2 ml of 10 mM sodium phosphate buffer (pH 7.4) to apply to a Sephacryl S-100 h column (10 mm × 600 mm) and washed with 10 mM sodium phosphate buffer (pH 7.4) at a flow rate of 1 ml/min, and the absorbance peaks observed at 280 nm were collected and then lyophilized.
Determination of molecular weight of the novel protein
The novel protein isolated from American ginseng roots (AGNP) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) employing 12.0% separating gel. The novel protein isolated (30 mg each) from American ginseng roots were loaded in each lane. The bands were intuitional by Coomassie brilliant blue R-250 staining when SDS-PAGE was over. Protein contents of AGNP were tested by the Bradford assay employing bovine serum albumin as a standard.
High-pressure gel-filtration chromatography
The ultraviolet spectrum of the AGNP was scanned over a range of 200–800 nm. The proteins were dissolved in sodium phosphate buffer, filtered with filter a 0.22 μm filter, and then analyzed by HPLC (Agilent 1220 series; Agilent Technologies, USA) with TSKgel G2000 SWXL column (7.8 mm × 300 mm, Tosoh). The marker was used as follows: thyroglobulin (669000), ferritin (440000), aldolase (158000), conalbumin (75000), ovalbumin (43000), carbonic anhydrase (29000), ribonuclease A (13700), and aprotinin (6500).
Identification of the novel protein using matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry
The protein molecular weight was determined by MALDI-TOF-MS (Voyager-DE STR, Tosoh). The acid solution A was prepared by ACN, ultrapure water, and TFA at the ratio of 200:100:3 (V/V) and afterward put the sinapic acid in solution A to get the solution B as 100 mg/ml. One microliter proteins were dissolved in 1 μL solution B and stirred constantly to mix uniformly to obtain solution C. One microliter solution C was dried at room temperature and then applied to MALDI-TOF-MS (Voyager-DE STR, Tosoh), corrected by myohemoglobin and cytochrome C.
Amino acid composition analysis of novel protein isolated from American ginseng roots
AGNP were hydrolyzed in vacuum in 6 mol/L HCl for 24 h at 110 ± 1°C for commonly amino acid analysis. For the analysis of cysteine, samples were hydrolyzed in 6 mol/L HCl for 24 h at 110 ± 1°C after peroxidation treatment with formic acid: hydrogen peroxide (10:1). For the analysis of tryptophan, samples were hydrolyzed in 4 mol/L methanesulfonic acid, and then put 4 mol/L NaOH in. Amino acids converted to phenyl isothiocyanate derivatives were analyzed by HPLC (Agilent 1220 series; Agilent Technologies, USA) with WondaSil-C18 (4.6 mm × 150 mm, 5 μm).
Assay of antioxidant activity in vitro
2,2-diphenylpicrylhydrazyl radical scavenging activity
To measure the DPPH radical scavenging effects of the samples, our test was according to previous method with slight modifications. The proteins were diluted in distilled water at different concentrations. After that, 1 mL of sample was added to 0.5 mL of 0.1 mmol/L DPPH solution dissolved in 95% ethanol and then shocked and held for half an hour, in the environment without light at 25 ± 1°C, after that centrifuged at 8000 rpm for half an hour. The absorbance of the resulting solution was tested at 517 nm. Ethanol was added instead of DPPH solution as the control, and distilled water was added taking the place of proteins solution as the blank. All experiments were carried out in triplicate and at least on two separate occasions. The activities to scavenge DPPH radical of samples were calculated by the following formula:
DPPH scavenging activity = [(A0-A1)/A0] × 100%,
A0 is the absorbance of the control at half an hour, and A1 is the absorbance of the sample at half an hour.
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging effects were measured in the light of formerly described method by Mathew and Abraham (2006) with slight amendments. Briefly, 0.1 mL samples at different concentrations were added to 0.3 mL PBS (0.05 M, pH = 7.5), 0.1 mL 2-deoxyribose (60 mM), 0.1 mL FeCl2-EDTA (10 mM), 0.4 ml H2O, and 0.1 ml H2O2(10 mM), and our samples were incubated at 37°C for 60 min. After incubation, 1.0 mL TFA (2.8%) and 1 mL thiobarbituric acid (TBA) (1%) were added the samples incubated at 95°C for 15 min. The proteins were cooled on ice for 5 min, in the next moment centrifuged at 4000 rpm for 15 min at 25°C. The absorbance was measured at 536 nm. In each experiment, the samples without H2O2 were used as blank, and the samples without AGNP were used as control. All tests were implemented in triplicate and at least on two separate occasions. The activity to scavenge OH radical of proteins was calculated by the following equation:
OH scavenging activity = [(A0-A1)/A0] × 100%,
where A0 and A1 are the absorbance values of the control and the tested sample separately.
Superoxide anion radical scavenging activity
Superoxide anion radical scavenging effects were measured according to modified method. Superoxide radicals were generated in the 1, 2, 3-phentriol autoxidation system under alkaline conditions. Briefly, 5.0 mL of 0.05 M Tris-HCl buffer (pH 8.2) was held in water bath at 25°C for 20 min. After that, 1.0 mL of samples at different concentrations and 0.4 mL of 25 mM 1, 2, 3-phentriol were added and incubated at 25°C of 4 min. 0.5 mL of 10 mM hydrochloric acid was added rapidly to stop the reaction. The absorbance of the mixture was tested at 320 nm. All tests were implemented in three times and at least on two separate occasions. The O2− scavenging activity was calculated by making use of the formula:
O2− scavenging effect = [(A0–As)/A0] × 100%,
where A0 is the absorbance without sample and As is absorbance with AGNP samples.
The ferric-reducing ability of ferric-reducing antioxidant power assay
The ferric-reducing antioxidant power (FRAP) assay was implemented in terms of the method with slight modifications. Briefly, 4.5 mL of freshly prepared FRAP reagent was added with 1.0 mL of distilled water and 1.0 mL of samples different concentrations (or distilled water, for the blank), in the next moment warmed at room temperature for 5 min. The absorbance was measured at 593 nm. Trolox was used as a standard with its final concentrations ranging from 0 to 0.20 mM. Results were showed as TEAC (umol/L). All tests were implemented with three replications.
Each data value represents a minimum of three (n = 3) replicate experiments, and all antioxidant assays were performed in triplicates. All data are expressed as mean ± standard deviation. In vivo antioxidant activities, differences between groups were determined by analysis of variance and Student's t-test. P < 0.05 was considered to be statistically significant.
| Results and Discussion|| |
Measurement of protein contents and molecular weight of novel protein isolated from American ginseng roots
Measurement of molecular weight of novel protein isolated from American ginseng roots by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
We determined the subunit molecular mass of the novel protein purified from P. quinquefolius L. in SDS-PAGE. Our results showed that a single band with an apparent molecular mass of about 15000 Da was shown on SDS-PAGE for the desalted proteins [Figure 1]. The content of the novel protein purified from P. quinquefolius L. measured by Bradford method was 2.42 mg/mL.
|Figure 1: Determination of molecular weight of novel protein isolated from American ginseng roots through sodium dodecyl sulfate–polyacrylamide gel electrophoresis. M represents molecular markers. Coomassie brilliant blue staining was used to stain protein moieties of novel protein isolated from American ginseng roots. The numbers represent standard proteins used: (1) rabbit phosphorylase b (97 kDa), (2) bovine serum albumin (66 kDa), (3) rabbit actin (43 kDa), (4) bovine carbonic anhydrase (31 kDa), (5) trypsin inhibitor (20 kDa), and (6) hen egg white lysozyme (14 kDa)|
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High-pressure gel-filtration chromatography of novel protein isolated from American ginseng roots
The wavelength of maximum absorption for the novel protein purified from P. quinquefolius L. was 280 nm [Figure 2]. The elution time of the AGNP was 8.665 min for HPGFC, corresponding to molecular weight of 31,086 Da.
|Figure 2: The ultraviolet spectrum of novel protein isolated from American ginseng roots|
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Identification of novel protein isolated from American ginseng roots by matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry
It shows two primary peaks in MALDI-TOF-MS: peak 1 at 7562.24 (m/z) with double charges and peak 2 at 15,114.70 (m/z) with single charge. It is intended to be the protonated molecular ion. The relative molecular mass of AGNP was identified as 15, 114.70 ± 50 Da for ten different scans [Figure 3]. Our research showed that the molecular weight of the novel protein purified from P. quinquefolius L. was 15,114 Da both of SDS-PAGE and mass spectrometry purity. The consequence of HPGFC demonstrated that the molecular weight of AGNP was 31,086 Da, and every result of above implied that the purified AGNP was a homodimer.
|Figure 3: The mass spectrum for novel protein isolated from American ginseng roots|
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Novel protein isolated from American ginseng roots amino acid composition analysis
Amino acid composition of AGNP is shown in [Table 1]. The top three of AGNP amino acid composition were glycine, threonine, and leucine, but tyrosine and tryptophan were not detected in AGNP. There were eight essential amino acids for people, and the proportion was 40.31%.
In vitro antioxidant properties of novel protein isolated from American ginseng roots
2,2-diphenylpicrylhydrazyl radical scavenging activity
DPPH is considered to be a stable free radical that it can accept an electron or hydrogen to become a stable molecule. DPPH shows maximum absorbance at 517 nm in ethanol. When antioxidants react with DPPH, the number of DPPH will be reduced, and the absorption at 517 nm is proportional to the amount of residual DPPH.
As shown in [Figure 4]a, AGWSPS, AGCNP, and AGNP were found to possess the DPPH radical scavenging effect in a concentration-dependent manner. The DPPH scavenging effect elevated with the increasing quantity of AGWSPS, AGCNP, and AGNP at a range of 0.2–3.0 mg/mL, the scavenging activity ranged from 13.12% to 69.75%, 16.02% to 82.85%, and 19.96% to 95.15%, and the EC50 values were separately 1.52 mg/mL, 1.04 mg/mL, and 0.52 mg/mL severally.
|Figure 4: Antioxidant activity of novel protein isolated from American ginseng roots in vitro. (a) scavenging effects of the novel protein isolated from American ginseng roots on 2,2-diphenylpicrylhydrazyl radical; (b) scavenging effects of the novel protein isolated from American ginseng roots on hydroxyl radical; (c) scavenging effects of the novel protein isolated from American ginseng roots on superoxide radical; (d) the ferric-reducing ability of ferric-reducing antioxidant power; values are means ± standard deviation (n = 3). Vcis a control|
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Because the method of DPPH radical scavenging effect is a simple, rapid, sensitive, and reproducible procedure, it has been widely performed for the measurement of primary antioxidant effect for food materials, plant extracts, and pure antioxidant compounds. In our study, AGWSPS, AGCNP, and AGNP all showed scavenging activities against DPPH radicals, and AGNP showed more powerful effect than the others.
This indicates that AGNP may be a source for the antioxidant compounds.
Hydroxyl radical scavenging activity
Hydroxyl radical which is generated by the Fenton reaction is highly reactive one among the oxygen radical. It may react with various important biological molecules, including lipids, proteins, and nucleic acids. This free radical may cause tissue damage or cell death. Hence, scavenging the hydroxyl radical is significant for antioxidant defense in cell or food systems. In our experiment, hydroxyl radicals are gathered by the Fenton reaction. Hydroxyl radicals may attack 2-deoxy-D-ribose and can lead to the degradation of 2-deoxy-D-ribose. The degradation products may combine with TBA to form a pink compound with the maximum wavelength at 536 nm. The concentration of hydroxyl radical is reflected by the degree of decolorization of the reaction solution. As shown in [Figure 4]b, AGWSPS, AGCNP, and AGNP showed evident scavenging effect on hydroxyl radicals in a concentration-dependent manner. The scavenging rates increased dramatically from 13.67% to 59.20% at the concentration range from 0.2 to 3.0 mg/mL for AGWSPS as well as AGCNP reached 73.02% at 3.0 mg/mL.
The scavenging effects of AGNP are better than the other two proteins, and it increased from 17.77% to 87.79% at the concentration range from 0.2 to 3.0 mg/mL. The EC50 values for AGWSPS, AGCNP, and AGNP were 2.27, 1.40, and 0.72 mg/mL, respectively. Hence, AGNP had a higher level of hydroxyl radical scavenging activity than AGWSPS and AGCNP.
Our results demonstrated that AGNP could be employed as the scavenging agent for protecting hydroxyl radical-induced injury in living organism.
Superoxide anion radical scavenging activity
Superoxide anion radical is a ROS which can be produced by various biological and photochemical reactions. It can react with the hydroxyl radical to cause oxidative injury in DNA, lipids, and proteins. The removing activity of AGWSPS, AGCNP, and AGNP for superoxide anion radicals was studied at various concentrations (from 0.1 to 1.0 mg/mL). Our results showed that the removing activity of AGWSPS, AGCNP, and AGNP for superoxide anion radicals prominently increased with more concentration [Figure 4]c. The scavenging rate of the AGWSPS ranged from 2.98% to 40.89%, AGCNP ranged from 3.98% to 51.04%, and at 1.0 mg/mL, the scavenging rates of AGNP reached 91%. The EC50 values were 1.33 mg/mL, 1.02 mg/mL, and 0.26 mg/mL, respectively. AGNP showed the most powerful scavenging effects among the three proteins.
The superoxide anion radicals may be generated in the human body and may induce injure to the cells and DNA resulting in many diseases.
It was reported that the existence of better levels of electron-donating amino acids could be contribute to higher scavenging superoxide anion radicals. In our research, the contents of negatively charged amino acids in AGNP are much higher than in AGWSPS and AGCNP [Table 1]. These results implied which the ability to donate electrons is a significant contributive structural feature to improve scavenging effect of superoxide anion radicals.
The ferric-reducing ability of FRAP assay
The FRAP is generally applied to confirm the antioxidant effect of plant herbs, which determinates the ability of the samples to reduce ferric complex to the ferrous form. As shown in [Figure 4]d, the TEAC values measured in AGWSPS, AGCNP, and AGNP ranged from 20.76 to 90.71 umol/L, 17.85 to 84.92 umol/L, and 3.04 to 41.01 umol/L; Trolox equivalent in the samples ranged from 0.2 to 3 mg/mL. The values obtained in term of Trolox equivalent were low, but the overall trend was similar to those obtained by DPPH and hydroxyl radical assay methods.
In conclusion, AGNP has better effects to remove the free radicals in vitro. More and more interests focus on identifying antioxidants from natural sources including dietary proteins. Modern researches have demonstrated that proteins may restrain lipid oxidation though multiply pathways such as scavenging free radicals, alteration of the physical properties of food systems, inactivation of ROS, reduction of hydroperoxides, and chelation of prooxidative transition metals. It is reported that the peculiar amino acid sequence of proteins contributed to the antioxidant effect, including Met, His, Lys, Tyr, Trp, and Gly, was generally accepted as antioxidants., The aromatic amino acid residues are primary targets for oxidation, such as phenylalanine residues, tyrosine residues, and so on. The amino acid composition of AGNP is indicated that AGNP were rich in Asp, Ser, Lys, Glu, and Leu, which might be vital in antioxidant effect. This might be a reason which AGNP had a good activity of scavenging the free radicals.
| Conclusions|| |
Our study reports the antioxidant activity of AGNP for the first time. The results in this study indicated the antioxidant potential in AGNP. AGNP had the strong scavenging effects on superoxide radical, DPPH radical, hydroxyl radical, and the ferric-reducing capacitiesin vitro tests. It is concluded that our study might contribute to a rational basis for the application of the antioxidant proteins isolated from American ginseng as suitable candidate for exploring functional foods or drugs for the therapy of diseases associated with oxidative stress.
The authors are grateful for the financial support from the Jilin Provincial Key Laboratory of Biomacromolecules of Chinese Medicine, Local Standard Project of Chinese Medicinal Materials in Jilin Province (No. PZ-2016-03 and No. JLPZGF-2018-004), The Science and Technology Development Plan Project of Jilin Province (No. 20190101010JH, No. 20190304095YY), The National Key Research and Development Program of China (2017YFC1702106), and the National Traditional Chinese Medicine Standardization Project of China (No. ZYBZH-C-JL-22 and No. ZYBZH-C-HEB-11).
Financial support and sponsorship
We have checked the financial support taken from acknowledgment carefully.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]