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Abstract

The methods of extraction, separation, and purification of zizyphusine, spinosin, and 6′′′-feruloylspinosin from Zizyphi Spinosi Semen (ZSS) were investigated. From 31.2 g defatted ZSS, 47.7 mg zizyphusine, 57.8 mg spinosin, and 80.5 mg 6′′′-feruloylspinosin were obtained after ultrasonic extraction, purification with macroporous resin, separation by flash chromatography, and purification by high-pressure preparative chromatography. The purities of zizyphusine, spinosin, and 6′′′-feruloylspinosin were 98.6%, 98.2%, and 99.0%, respectively, and their yields 85.0%, 82.8%, and 83.0%. The methods provide stable samples of these compounds for further study of their physiological function and application.

Keywords

zizyphusine spinosin 6′′′-feruloylspinosin separation

Zizyphi Spinosi Semen (ZSS), the mature seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae), are used for the treatment of insomnia and anxiety.¹ The seeds are rich in pharmacologically active components, such as saponins, ﬂavones, and alkaloids, which have antioxidant,^2,3 hypnotic, sedation, anti-inflammatory, and antiarrhythmic properties, as well as prevent myocardial ischemia and lower blood pressure.⁴

Many studies have proved that aging and some diseases (such as cancer, diabetes, and cardiovascular disease) are closely related to the increase in the number of free radicals and their induced oxidation reactions.^5,6 Many natural products derived from plants have the potential to be used as antioxidants to remove free radicals in the body.⁷ Thus, in recent years, research on the antioxidant activity of natural products has developed rapidly.^8,9 Recent studies have found that spinosin could be beneficial to treat learning and memory deficits in patients with Alzheimer’s disease via multitargets.¹⁰ Both spinosin and 6′′′-feruloylspinosin were found to increase the transcription level of GABA_Aα₁, GABA_Aα₅, and GABA_BR₁ mRNA significantly.¹¹ Most studies were focused only on the identification of different natural antioxidants. However, if there were no specific directions on the separation and purification of these antioxidants, it was difficult to study their biological activities and physiological functions if insufficient antioxidant had been applied. Therefore, it is necessary to study the methods of extraction, separation, and purification of active components according to their properties.^12

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Papers have reported the presence of zizyphusine (C₂₀H₂₄NO₄), spinosin (C₂₈H₃₂O₁₅), and 6′′′-feruloylspinosin (C₃₈H₄₀O₁₈) in ZSS,^16,17 but these previous studies have only carried out preliminary detection and identification of flavonoids in extracts by LC-mass spectrometry (MS).⁷ Therefore, it was difficult to attribute the cumulative observed effects to an individual molecular component, which was detrimental to further research and clinical applications. Our previous study demonstrated that these 3 compounds, especially 6′′′-feruloylspinosin and zizyphusine, have significant antioxidant activity.¹⁸ In this current study, effective and reproducible methods for the simultaneous extraction and purification of zizyphusine, spinosin, and 6′′′-feruloylspinosin were established using ultrasonic extraction and purification through macroporous resin column chromatography, flash chromatography, and high-pressure preparative chromatography. The methods can provide stable samples for further study of the physiological function and application of zizyphusine, spinosin, and 6′′′-feruloylspinosin from ZSS.

Materials and Methods

Materials and Reagents

ZSS (harvested in 2016 in Hebei Province, China) was purchased from Medicinal Materials Market in Anguo (Hebei Province, China).

SEPABEADS SP825 macroporous resin, produced by Mitsubishi Chemical Co. (Japan), had the physical properties as follows: nonpolar, 1000 m²/g of surface area, 57 Å of average pore diameter, 200 to 600 nm particle diameter, 58% moisture.

Standards of zizyphusine, spinosin, and 6′′′-feruloylspinosin (≥98%) were purchased from Shun Bo Bioengineering Co., Ltd (Shanghai, China). HPLC-grade methanol was produced by Fisher Scientific Co. (USA). Pure and ultra-pure water was prepared using a Millipore-Q purification system (USA). Other reagents of analytical grade were purchased from Beijing Beihua Fine Chemicals Co., Ltd (Beijing, China).

Extraction of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin From ZSS

ZSS (50 g) was oven dried at 80°C and ground into 40 mesh powder (selected by sieve), 41.05 g of which was defatted by refluxing with light petroleum (60°C-90°C) for 8 hours in a Soxhlet extractor and then air-dried. The defatted powder (31.20 g) was extracted with 70% (v/v) ethanol solution (936 mL × 3) using a set of circulation supersonic extraction equipment (CTXNW-2B, Beijing Hongxianglong Biotechnology Developing Co., Ltd, China) for 120 minutes under constant mechanical agitation (1200 rpm) and at a frequency of 59 kHz. At the same time, another supersonic way of multistage shaped charge type ran at 750 W and worked for 3 seconds with ‘‘power on’’ and 3 seconds with ‘‘power off’’ in turn to keep the temperature of the extraction solvent at 40°C. After centrifugation, the supernatant was condensed to quarter volume by rotary evaporation under vacuum at 40°C. The ethanol concentration of the extraction was controlled at 35% (v/v). The concentrations of zizyphusine, spinosin, and 6′′′-feruloylspinosin were about 0.068, 0.16, and 0.096 mg/mL, respectively. The condensed extract solution was stored in a dark place at 4°C. The recoveries of the extraction were all above 95% for zizyphusine, spinosin, and 6′′′-feruloylspinosin.

Purification of Extract With Macroporous Resin

The macroporous resin was pretreated with 1 mol/L HCl and 1 mol/L NaOH successively to remove the monomers and porogenic agents. The resin was dried at 60°C under vacuum. About 500 g macroporous resin was soaked in 95% ethanol for 12 hours and subsequently washed with pure water thoroughly before use. A glass column (300 mm × 35 mm) was wet-packed with 200 g pretreated SEPABEADS SP825 macroporous resin. The bed volume (BV) of the resin was 288.5 mL.

Before sample loading, the resin was conditioned with 5 BV 35% (v/v) ethanol. Then the condensed extract solution (14 500 mL) was applied onto the column at a flow rate of 10 mL/min. After reaching adsorptive saturation, the resin was first washed with 4 BV distilled water and then eluted with ethanol solution. The eluent consisted of water (A) and ethanol (B) in different ratios and the flow rate was 5 mL/min. The gradient elution modes were as follows: 0 to 40 minutes, 35% B; 40 to 80 minutes, 45% B; 80 to 160 minutes, 55% B. The eluent was screened by an online UV detector at 270 nm for reference and all collected in tubes (50 mL ×60). The gradient elution curves were obtained by off-line HPLC-diode array detector (DAD) determination of eluent in each tube. The eluents of 45% and 55% ethanol were evaporated to dryness under vacuum at 40°C, and the obtained dried extracts contained spinosin and 6′′′-feruloylspinosin, respectively. The sample solution through the column from 600 to 3790 mL and the eluent of 35% ethanol were combined together and evaporated to dryness under vacuum at 50°C to obtain the dried extract, which mainly contained zizyphusine. The contents of zizyphusine, spinosin, and 6′′′-feruloylspinosin were measured by HPLC.

Separation by Flash Chromatography

The flash preparative RP-LC apparatus was made up of a flash-150 binary-gradient pump (SSI, Washington, NY), a tee with an injection port, a UV 201 detector (SSI), a BSZ-40 fraction collector (Shanghai Hu Xi Analysis Instrument Factory Co., Ltd, Shanghai, China), and a glass chromatographic column (400 mm × 35 mm, EYELA, Tokyo, Japan) wet packed artificially with particles of RP C₁₈ silica gel (50 mm, 12 nm, Sunchrom ODS-A).

About 2000 mg of dried phytochemical extract was dissolved in 60 mL methanol and applied onto the glass chromatographic column, which was eluted with a solvent system consisting of water (A) and methanol (B). The gradient elution mode was as follows: 0 to 10 minutes, 5% to 25% B; 10 to 25 minutes, 25% to 30% B; 25 to 85 minutes, 30% to 40% B; 85 to 130 minutes, 40% B (v/v, binary gradient). The flow rate was 30 mL/min. The eluent was screened by an online UV detector at 280 nm for reference and all collected in tubes (60 mL ×720). All eluted fractions were determined off-line by HPLC-DAD and pooled according to the results from the chromatograms. As a result, the extract was fractionated into 4 parts, which contained impurity (fraction A), zizyphusine (fraction B), spinosin (fraction C), and 6′′′-feruloylspinosin (fraction D), respectively. The organic solvent in fractions B, C, and D was removed by vacuum drying at 40°C. The remaining liquid was dried by lyophilization. The contents of the 3 compounds in each fraction were detected by HPLC.

Purification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

The high-performance preparative RP-LC apparatus was made up of 2 series Ⅲ high-pressure pumps (SSI), a 7725i 6-way valve, a Model 201 UV detector (SSI), a BSZ-40 fraction collector (Shanghai Hu Xi Analysis Instrument Factory Co., Ltd), a workstation software (ver. 6.14 SP1), and a YMC-Pack ODS-AQ C₁₈ prep-column (250 mm × I.D. 20 mm, S-5 μm, 12 nm) or Grace adsorbsphere XL C₁₈ prep-column (250 mm × I.D. 22 mm, S-5 μm, 12 nm).

Twenty-five milligrams of freeze-dried fraction B or C obtained from flash chromatography, which mainly and separately contained zizyphusine (B) and spinosin (C), was resolubilized in methanol and applied onto a YMC-Pack ODS-AQ C₁₈ prep-column. The mobile phase consisted of water (A) and methanol (B). The gradient elution mode was as follows: 0 to 5 minutes, 5% to 25% B; 5 to 25 minutes, 25% to 30% B; 25 to 45 minutes, 30% to 50% B (v/v, binary gradient). The flow rate was 10 mL/min, and the eluent was screened by an online UV detector at 280 nm for reference. The eluted solutions were collected in tubes (12 mL ×50). Then the eluted fraction in each tube was detected off-line by HPLC-DAD and pooled according to the chromatograms. Fractions containing zizyphusine, spinosin, or 6′′′-feruloylspinosin were combined. Methanol was evaporated under vacuum at 40°C. The remaining liquid was dried by lyophilization. The obtained zizyphusine, spinosin, and 6′′′-feruloylspinosin had purities of >98%, as detected by HPLC-DAD.

Fraction D, mainly containing 6′′′-feruloylspinosin, was separated on a Grace adsorb sphere XL C₁₈ prep-column. The mobile phase consisted of water (A) and methanol (B), and the gradient elution mode was 0 to 15 minutes, 10% to 38% B; 15 to 75 minutes, 38% to 43% B; 75 to 115 minutes, 43% to 50% B (v/v, binary gradient). The flow rate was 12 mL/min. The other processes of purification were the same as for fractions B and C. Spinosin and 6′′′-feruloylspinosin were also obtained with purity of >98% in this separation step.

Chromatographic Analysis of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

Off-line analysis of zizyphusine, spinosin, and 6′′′-feruloylspinosin in the prep-LC (middle-pressure or high-pressure) fractions was carried out by HPLC on a Shimadzu 20-AD UFLC system composed of dual LC-20AD UFLC pumps with a degasser, a CTO-20A thermostated column compartment, an SPD-M20A DAD, an SCL-10A controller, and Class-VP workstation software (ver. 6.14 SP1).

Sample analysis was performed with a guard column (10 L × 4.6 mm, Shim-pack GVP-ODS C₁₈) and a YMC C₁₈ column (250 L × 4.6 mm, I.D., S-5 μm, 12 nm, ODS-AQ) at a column temperature of 40°C. The mobile phase consisted of 0.5% (v/v) acetic acid aqueous solution (A) and 0.5% (v/v) acetic acid MeOH solution (B). The gradient elution mode was as follows: 0 to 5 minutes, 10% to 38% B; 5 to 15 minutes, 38% to 42% B; 15 to 18 minutes, 42% to 10% B; 18 to 23 minutes, 10% B. The flow rate was 1.0 mL/min, the oven temperature 40°C, and injection volume 10 µL. All samples were filtered through a 0.45 µm membrane before HPLC analysis.

MS and Nuclear Magnetic Resonance (NMR)

MS and NMR spectra were obtained at the Center of Analysis, Beijing University of Chemical Technology. A Waters (Milford, MA) Quattro Premier Micromass 70-VSE mass spectrometer was used with the selected ion monitoring mode for the pseudomolecular negative ion [M-H]^- of zizyphusine, spinosin, and 6′′′-feroylspinosin. The ion source temperature was 100°C, the desolvation temperature 350°C, the desolvation gas flow 602 L/h, and the cone gas flow 45 L/h. NMR spectra were performed in CDCl₃ using a Bruker high-resolution AV600 NMR spectrometer at 600 MHz (Billerica, MA).

Statistical Analysis

All statistical analyses were performed using SPSS 12.0 for Windows software.

Results and Discussion

Purification With SP825 Resin

The dynamic breakthrough curves of zizyphusine, spinosin, and 6′′′-feruloylspinosin on an SP825 resin column are shown in Figure 1. The resin had almost no adsorption for the alkaloid zizyphusine. The dynamic adsorption capacities of the 2 flavonoids, spinosin and 6′′′-feruloylspinosin, on the resin were 7.93 and 17.87 mg/g, respectively. Therefore, the characteristic of SP825 resin can be used to purify simultaneously zizyphusine, spinosin, and 6′′′-feruloylspinosin from the extract of ZSS. As shown in the dynamic breakthrough curves of Figure 1, the extract solution was loaded onto an SP825 macroporous resin column for adsorbing spinosin and 6′′′-feruloylspinosin, and the solution through the column from 600 to 3790 mL which mainly contained zizyphusine was collected at the same time. In order to avoid the loss of zizyphusine, the sample solution after loading from 600 to 3790 mL and the 35% ethanol eluent were collected and combined in the practical operation. Therefore, the part mainly containing zizyphusine was obtained after vacuum concentrating and freeze drying. The results for zizyphusine, spinosin, and 6′′′-feruloylspinosin eluted by 35%, 45%, and 55% ethanol solution successively are shown in Table 1, and the elution curves in Figure 1. The recovery of the purification with SP825 resin was above 96%.

Figure 1

Dynamic breakthrough curves and gradient elution curves of flavones in chromatography column packed with SP825 resin.

Table 1

Comparison of the Results of Different Elution Modes.

Sample loading phase						A	F₁		F₂
	Concentration of compounds in SS (mg/mL)					0.068	0.163		0.096
	Volume of SS (mL)					14 500
	Mass of compounds absorbed on resin (mg)					40.8	618.0		1392.7
	Content of compounds in solid of SS (%)					0.64	1.23		1.07
Eluting phase	Elution mode	Solid mass(mg)	Content of compounds in solid (%)			Desorption yield(%)			Multiple of purification
			A	F₁	F₂	A	F₁	F₂	A	F₁	F₂
	35%	2016.8	1.73	2.03	1.39	85.50	6.62	2.01	2.57	1.65	1.30
	45%	3675.2	0.10	10.92	18.90	9.00	64.94	49.88	0.27	8.88	17.66
	55%	2993.4	0	5.17	20.91	0	25.04	44.94	-	4.20	19.54

SS, sample solution; A, zizyphusine; F₁, spinosin; F₂, 6'''-feruloylspinosin.

Preliminary Fractionation by Flash Chromatography

Zizyphusine, spinosin, and 6′′′-feruloylspinosin all had obvious ultraviolet absorption at 270 and 330 nm. In addition, zizyphusine had obvious ultraviolet absorption at 220 nm. These 3 compounds can be detected online using a UV detector during the fractionation. Since the extract of ZSS consisted of complex components, it usually took a few steps to separate the monomers from other compounds. Flash preparative chromatography can be used to fractionate the extract into several fractions, each of which mainly contains one or several components, and then every fraction can be further separated in other ways. The extract of ZSS was fractionated in a prep-column packed with RP C₁₈ medium and the preparative chromatogram is shown in Figure 2(a). As shown, the extract was fractionated into 4 fractions; fractions B, C, and D mainly contained zizyphusine, spinosin, and 6′′′-feruloylspinosin, respectively, and the recoveries for the 3 compounds were all above 95%.

Figure 2

(a) Elution profile of the phytochemical extract on a flash chromatography RPC column obtained with a UV detector at 280 nm (online). Solid line: UV signal at 280 nm; dashed line: gradient curve of MeOH; the fractions that were collected are indicated by the different areas marked with A, B, C, D; chromatography conditions: a glass column (400 mm × 35 mm, 50 mm, 12 nm, Sunchrom ODS-A); flow rate: 30 mL/min; solvent system: ultra-pure water (A) and methanol (B); elution mode: 0 to 10 minutes, 5% to 25% B; 10 to 25 minutes, 25% to 30% B; 25 to 85 minutes, 30% to 40% B; 85 to 130 minutes, 40% B (v/v, binary gradient). (b), (c), and (d) Elution profiles of fractions B, C, and D, respectively, on a prep-HPLC RPC column obtained with a UV detector at 280 nm (online). Solid line: UV signal at 280 nm, dashed line: gradient curve of EtOH, chromatography conditions for fractions B and C: a YMC-Pack ODS-AQ C₁₈ column (250 mm × I.D. 20 mm, S-5 μm, 12 nm), flow rate: 10 mL/min, solvent system: ultra-pure water (A) and methanol (B), elution mode: 0 to 5 minutes, 5% to 25% B; 5 to 25 minutes, 25% to 30% B; 25 to 45 minutes, 30% to 50% B (v/v, binary gradient); chromatography conditions for fraction D: a Grace adsorb sphere XL C₁₈ column (250 mm × I.D. 22 mm, S-5 μm, 12 nm), flow rate: 12 mL/min, solvent system: ultra-pure water (A) and methanol (B), elution mode: 0 to 15 minutes, 10% to 38% B; 15 to 75 minutes, 38% to 43% B; 75 to 115 minutes, 43% to 50% B (v/v, binary gradient).

Purification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

Fractions B, C, and D were separated individually by high-pressure preparative chromatography and detected by an online UV detector. The retention time of zizyphusine, spinosin, and 6′′′-feruloylspinosin can be determined according to the HPLC chromatogram. As shown in Figure 2(b) to (d), zizyphusine, spinosin, and 6′′′-feruloylspinosin were separated from impurities. The 3 compounds were obtained individually after separation, concentration, and drying; all the recoveries of this step were above 98%. The purities of zizyphusine, spinosin, and 6′′′-feruloylspinosin were all higher than 98%, as detected by HPLC-DAD.

Identification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

Under positive ionization mode, there was an intensive molecular ion peak of zizyphusine which had got a H⁺ at m/z 342.4. Under negative ionization mode, there were intensive molecular ion peaks of spinosin and 6′′′-feruloylspinosin which had lost a H⁺ at m/z 607.2 and m/z 783.0.

The MS and NMR spectra of the isolated compounds were consistent with those published for zizyphusine, spinosin, and 6′′′-feruloylspinosin.^{7,16,17,19

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Concluding Remarks

The purities of zizyphusine, spinosin, and 6′′′-feruloylspinosin were 98.6%, 98.2%, and 99.0%, respectively; 47.7 mg zizyphusine, 57.8 mg spinosin, and 80.5 mg 6′′′-feruloylspinosin were obtained from 31.20 g defatted ZSS through 4 steps: ultrasonic extraction, purification with macroporous resin, separation with flash chromatography, and purification with high-pressure preparative chromatography. The yields of the 3 compounds were 85.0%, 82.8%, and 83.0%, respectively. The extraction rate and purity of the 3 compounds were stable after 3 repetitive experiments. The described procedure is an efficient method that can be implemented to obtain gram scale samples of zizyphusine, spinosin, and 6′′′-feruloylspinosin from ZSS in the laboratory and to provide high-quality and stable analytical grade samples for further study on physiological functions and research applications.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the Beijing Natural Science Fund (KZ201710020014), the National Natural Science Foundation of China (31601658).

ORCID iDs

Jiong Ran Wang

Ke Ding

References

Cao

J-X.

Zhang

Q-Y.

Cui

S-Y

et al. Hypnotic effect of jujubosides from Semen Ziziphi Spinosae . J Ethnopharmacol. 2010;130(1):163-166.doi:10.1016/j.jep.2010.03.023

Han

Eun

et al. Anxiolytic-like effects of sanjoinine a isolated from Zizyphi Spinosi Semen: possible involvement of GABAergic transmission. Pharmacol Biochem Behav. 2009;92(2):206-213.doi:10.1016/j.pbb.2008.11.012

Sun

Y-F.

Liang

Z-S.

Shan

C-J.

Viernstein

Unger

et al. Comprehensive evaluation of natural antioxidants and antioxidant potentials in Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou fruits based on geographical origin by TOPSIS method. Food Chem. 2010;124(4):1612-1619.doi:10.1016/j.foodchem.2010.08.026

Zhao

Liu

Wen

et al. The pharmacokinetics and tissue distribution of coumaroylspinosin in rat: A novel flavone C-glycoside derived from Zizyphi Spinosi Semen . J Chromatogr B Analyt Technol Biomed Life Sci. 2017;1046:18-25.doi:10.1016/j.jchromb.2017.01.030

Cozzi

Ricordy

Aglitti

et al. Ascorbic acid and beta-carotene as modulators of oxidative damage. J Carcinog. 1997;18(1):223-228.doi:10.1093/carcin/18.1.223

Tan

BK-H.

Zhu

YC.

Linz

Zhu

. Comparison of cardioprotective effects using ramipril and Danshen for the treatment of acute myocardial infarction in rats. Life Sci. 2003;73(11):1413-1426.doi:10.1016/S0024-3205(03)00432-6

Niu

C-Y.

C-S.

Sheng

Y-X.

Zhang

J-L

. Identification and characterization of flavonoids from Semen Zizyphi Spinosae by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry. J Asian Nat Prod Res. 2010;12(4):300-312.doi:10.1080/10286021003752284

Jiang

Xiong

. Natural antioxidants as food and feed additives to promote health benefits and quality of meat products: a review. Meat Sci. 2016;120:107-117.doi:10.1016/j.meatsci.2016.04.005

Naveena

BM.

Sen

AR.

Vaithiyanathan

Babji

Kondaiah

. Comparative efficacy of pomegranate juice, pomegranate rind powder extract and BHT as antioxidants in cooked chicken patties. Meat Sci. 2008;80(4):1304-1308.doi:10.1016/j.meatsci.2008.06.005

10.

XF.

Xiao

et al. Neuroprotective effects of spinosin on recovery of learning and memory in a mouse model of Alzheimer's disease. Biomol Ther. 2019;27(1):71-77.

11.

Qiao

Liu

Chen

et al. A HPLC-MS/MS method for determination of 6'''-feruloylspinosin in rat plasma and tissues: pharmacokinetics and tissue distribution study. J Pharm Biomed Anal. 2016;121:77-83.doi:10.1016/j.jpba.2016.01.005

12.

Alañón

ME.

Alarcón

Marchante

Díaz-Maroto

MC.

Pérez-Coello

et al. Extraction of natural flavorings with antioxidant capacity from cooperage by-products by green extraction procedure with subcritical fluids. Ind Crops Prod. 2017;103:222-232.doi:10.1016/j.indcrop.2017.03.050

13.

Ding

JJ.

WJ.

Yuan

et al. Preparative chromatographic purification and separation of individual jujubasaponins from Zizyphi Spinosi Semen . Sep Sci Technol. 2011;46(16):2526-2530.doi:10.1080/01496395.2011.597041

14.

Cao

Song

et al. Preparative separation of nine flavonoids from Pericarpium Citri Reticulatae by preparative-HPLC and HSCCC. Sep Sci Technol. 2016;51(5):807-815.

15.

Abid

Azabou

Jridi

et al. Storage stability of traditional Tunisian butter enriched with antioxidant extract from tomato processing by-products. Food Chem. 2017;233:476-482.doi:10.1016/j.foodchem.2017.04.125

16.

Cheng

Bai

Zhao

et al. Flavonoids from Ziziphus jujuba Mill var. spinosa . Tetrahedron. 2000;56(45):8915-8920.doi:10.1016/S0040-4020(00)00842-5

17.

Wang

Luo

Kong

. HPLC-ESI-MS(n) analysis of chemical constituents in Semen Ziziphi Spinosae . Zhongguo Zhong Yao Za Zhi. 2009;34(21):2768-2773.

18.

Ding

Han

et al. Separation and interaction of the ingredients of antioxidant activities in Zizyphi Spinosi Semen . J Chem Eng Chin Univ.. 2013;27(5):842-853.

19.

Han

BH.

Park

MH.

Han

. Cyclic peptide and peptide alkaloids from seeds of Zizyphus vulgaris . Phytochemistry. 1990;29(10):3315-3319.doi:10.1016/0031-9422(90)80207-W

20.

Han

BH.

Park

MH.

Park

. Chemical and pharmacological studies on sedative cyclopeptide alkaloids in some Rhamnaceae plants. Pure Appl Chem. 1989;61(3):443-448.doi:10.1351/pac198961030443

21.

Han

BH.

Park

MH.

Han

. Aporphine and tetrahydrobenzylisoquinoline alkaloids from the seeds of Zizyphus vulgaris var. spinosus . Arch Pharm Res. 1989;12(4):263-268.doi:10.1007/BF02911058

22.

Han

BH.

Park

. Alkaloids are the sedative principles of the seeds of Zizyphus vulgaris var. spinosus . Arch Pharm Res. 1987;10(4):203-207.doi:10.1007/BF02857740

23.

Han

BH.

Park

. Sedative activity and its active components of Zizyphi fructus. Arch Pharm Res. 1989;12(4):263-268.

24.

Woo

WS.

Kang

SS.

Shim

. The structure of spinosin (2″-O-beta-glucosyl-swertisin) from Zizyphus vulgaris var. spinosus . Phytochemistry. 1979;18(2):353-355.

25.

Woo

WS.

Kang

SS.

Wanger

. Acylated flavone-C-glycosides from the seeds of Zizyphus jujuba . Phytochemistry. 1980;19:2791-2793.

26.

Zeng

Zhang

RY.

Wang

. Studies on the constituents of Zizyphus spinosus Hu. Yao Xue Xue Bao. 1987;22(2):114-120.

27.

Shin

KH.

Kim

HY.

Woo

. Determination of flavonoids in seeds of Zizyphus vulgaris var. spinosus by high performance liquid chromatography. Planta Med. 1982;44(2):94-96.doi:10.1055/s-2007-971410

Separation and Purification of Zizyphusine,Spinosin,and 6′′′-Feruloylspinosin From Zizyphi Spinosi Semen

Abstract

Keywords

Materials and Methods

Materials and Reagents

Extraction of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin From ZSS

Purification of Extract With Macroporous Resin

Separation by Flash Chromatography

Purification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

Chromatographic Analysis of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

MS and Nuclear Magnetic Resonance (NMR)

Statistical Analysis

Results and Discussion

Purification With SP825 Resin

Preliminary Fractionation by Flash Chromatography

Purification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

Identification of Zizyphusine, Spinosin, and 6′′′-Feruloylspinosin

Concluding Remarks

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iDs

References