Quality Evaluation And Pharmacological Activities Of Triterpes From Sour Jujube Fruits

Sour jujube [Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou] is a thorny, rhamnaceous deciduous plant widely distributed throughout the tropical and subtropical regions of the world. In China, sour jujube is a hardy fruit tree that grows well in wild areas. It adapts to dry and hot climates even in the arid regions of loess plateaus where other fruits cannot be grown. Its dried seeds named suanzaoren (Zizyphi Spinosi Semen) in Chinese, have a thousand-year history of use in traditional Asian medicine as sedative for treating mild anxiety, nervousness and sleep-related problems. Its fruits have long been used in folk medicine and are widely utilized in many herbal preparations for over one thousand years that claim to purify the blood and aid digestion. The fruits are also believed to promote anti-aging effects for women. Moreover, the fruits which are rich in nutrients such as sugar, organic acids, mineral, carotenoids and vitamins, have important pharmacological and nutritional functions. To strengthen comprehensively utilization of sour jujube fruit as natural antioxidants and other functional product, the systematical study on the quality control, chemical components and pharmacology functions is necessary. The major accomplishments and innovations in the paper as follows:1. Due to the complexity of ingredients in herbals, established a quality standard system is an important way to distinguish different Chinese medical herbs resources. In this study, a chromatographic fingerprint analysis was developed using high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) to gain more chemical information to create a representative fingerprint to quality assessment of sour jujube. The HPLC system (Waters, Milford, MA, USA) and Waters SunFire column (250×4.6 mm, 5μm), equipped with a binary pump and an ELSD-UM 3000. Data were acquired and processed by Trisep-2003 software. HPLC analysis was carried out by gradient elution with solvent A (acetonitrile) and solvent B (water containing 0.3% acetic acid, pH 2.8) at a flow rate of 0.8 mL/min. The percentage of acetonitrile (A) in the mobile phase was programmed as follows: 20% (0 min)-30% (10min)-60% (20min)-100% (50min). The optimized parameters of ELSD are 60℃for the evaporator tube temperature and 2.5 min/L for the nebulizing gas flow rate to obtain the best signal to noise (S/N) ratios. In order to compare and analysis the differernce of HPLC fingerprint, the chemometric methods including similarity evaluation (SA) and hierarchical cluster analysis (HCA) were applied to classify ten batches of sour jujube according to their cultivated origins. In addition, sour jujube was investigated referring to the qualitive TLC examination; total ash, acid-insoluble ash as well as water soluble extraction, alcohol extraction according to Chinese Pharmacopoeia. So the quality requirement of sour jujube was suggested to add that the contents of total ash and acid-insoluble ash were 1.0263-2.6409%, 0.0200-0.1874%, and water soluble extraction and alcohol extraction were 12.8790-14.5603%, 5.5652-8.0278%, respectively. All the results indicated that this reliable method was suitable for comprehensively quality control of sour jujube, and this methods established would lay a foundation for the quality evaluation of sour jujube.2. Technique for Order Performance by Similarity to Ideal Solution (TOPSIS) method was used to comprehensively evaluate total natural antioxidants and antioxidant activities in Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou fruits based on geographical origin. Correlations between natural antioxidants, antioxidant activities and geographical conditions were investigated. sour jujube fruits from Ningxia, Gansu, Shaanbei and Shaanxi, all of which are found the fruits from the semi-arid regions of loess plateaus, demonstrated significantly higher antioxidant effects. TOPSIS analysis revealed the following order of natural antioxidants and antioxidative properties of the fruits from different regions, according to the highest amount: Ningxia > Gansu > Shaanbei > Shanxi > Sichuan > Shaanxi > Henan > Shandong > Hebei > Liaoning. The results demonstrated that TOPSIS method is an efficient ranking method that can be efficiently utilized in the assessment of total natural antioxidant content and quality of sour jujube fruits. Consequently, manufacturing companies can obtain sour jujube fruits from locations known to enhance the antioxidant activities of these fruits. Growing conditions appear to have a direct impact on natural antioxidant content and activities. Correlation coefficients analyses showed that the altitude and annual precipitation exert significant influences on the fruit. Thus, fruits grown in arid harsh, and high-altitude areas can produce a larger amount of natural antioxidants and therefore exhibit higher antioxidant activities compared to fruits grown in other areas. While studies have yet to clearly confirm this, other herbs and fruits are underway.3. The power of sour jujube fruits was extracted with Pe, then extracted with 96% EtOH and concentrated. The residue was partitioned with saturated BuOH, eluted by microporous resin D101 adsorption technology and get four fractions (Fr-A, Fr-B, Fr-C, Fr-D). The Fr-C was further isolated and purified with silica gel and get five fractions (Fr-E, Fr-F, Fr-G, Fr-H, Fr-I). The Fr-H was precipitated with acetone and hydrolysed by 2M trifluoroacetic acid, purified with semi-HPLC to yield compound K (9.8 mg). The structure of compound K was established on the basis of extensive spectral evidence (TLC, UV,ESI-MS,1H-NMR,13C-NMR,DEPT), and identified was ursolic acid (3-β-hydroxy-urs-12- en-28-oic acid).4. The pharmacological potentials of TS from sour jujube fruits were evaluated by antimicrobial, antioxidant and antiproliferative activities: In the case of antimicrobial activity, the results possessed broad and remarkable antimicrobial activity against the microorganisms tested containing 8 bacteria by microplate bioassay. The minimal inhibitory concentration (MIC) value for bacteria was 0.078 mg/mL. Antioxidant activity was employed by in vitro test systems namely DPPH radical scavenging activity, ferric reducing antioxidant power (FRAP), reducing power, inhibition of lipid peroxidation, and bleaching ability ofβ-carotene in linoleic acid system. The results were found to be similar activity pattern with synthetic antioxidant BHT. In antiproliferative activity, we first examined the growth-inhibitory effect of TS on human breast cancer MCF-7 cells by the MTT assay. In order to explore the underlying mechanism of cell growth inhibition, cell cycle and apoptosis were studied by flow cytometry. The expression of Bax and Bcl-2 gene were analysis by RT-PCR and Bax and Bcl-2 protein were further assayed by Western blotting. TS inhibited the growth of MCF-7 cell in a dose-dependent manner. The IC50 value was 23.119μg/mL after 72h treatment, TS arrested the cell cycle in the G1 phase and induced apoptosis. RT-PCR and Western blotting analysis showed that TS inhibited Bcl-2 gene/protein expression and induced Bax gene/protein expression. To the best of our knowledge, the cytotoxic activity of sour jujube fruit towards MCF-7 was studied for the first time in the present study. Our studies encourage the evelopment of novel, efficient and less toxic natural product from sour jujube fruits for cancer chemotherapy.5. Although TS expressed excellent pharmacological potentials according to our test, it is very poorly water-soluble. In order to improve the utilization of TS in clinical practication, the inclusion complex between TS-β-CD was prepared by blending method. The solubilization of TS in aqueous solution ofβ-CD was investigated by the solubility method, and the solid inclusion complex was further identified by ultraviolet and scanning electron microscopy (SEM). The solubility of TS in water increased as the concentrations ofβ-CD, showing an AL type phase solubility diagram. The curvature closely corresponded to the simulation curve which was calculated on the basis of the 1:1 (guest:host) complexation model. The results indicated that the formation of a TS-β-CD inclusion complex is more water soluble than TS alone. New cosmetic formulation with TS-β-CD inclusion complex as cosmeceuticals was prepared and possessed good rheological properties, anti-aging and skin whitening effect.