PCR-RFLP for Distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method for rapid distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex， so as to avoid the influence of genetic confusion on drug safety. MethodThe DSS-tagged sequences of Periplocae Cortex were obtained from the Chloroplast Genome Information Resource （CGIR） and analyzed to find the enzymatic cleavage sites that were different from those of Acanthopanacis Cortex and Lycii Cortex. The specific enzymatic cleavage site， Cla I， of Periplocae Cortex was selected， on the basis of which the primers for PCR-RFLP were designed. Furthermore， the factors such as annealing temperature， number of cycles， Taq enzyme， PCR instruments， and enzymatic treatment time that may influence PCR-RFLP were studied. The established PCR-RFLP method was applied to the identification of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex samples produced in different regions. ResultThe PCR-RFLP at the annealing temperature of 59 ℃ and with 40 cycles showed clear bands of the samples. When the enzyme digestion time was 30 min. The reaction produced the target bands at about 140 bp and 290 bp for both Periplocae Cortex and its original plant and only a band at about 430 bp for Acanthopanacis Cortex， Lycii Cortex， and their original plants. The method can accurately distinguish Periplocae Cortex from its confounders Acanthopanacis Cortex and Lycii Cortex. ConclusionThe PCR-RFLP method for distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex was established. It has high stability， sensitivity， and applicability， providing a reference for the quality control of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex.

Identification of Original Plants of Lycii Cortex by Multiplex Allele-specific PCR / 中国实验方剂学杂志

ObjectiveTo identify Lycium chinense and L. barbarum as the original plants of Lycii Cortex simply and efficiently by multiple allele-specific polymerase chain reaction （PCR）. MethodThe chloroplast genome sequences of L. chinense and L. barbarum were downloaded from the Chloroplast Genome Information Resource （CGIR）， and then IdenDSS was employed to screen out the specific single nucleotide polymorphism （SNP） sites between the two plants. Primer 5.0 was used to design the specific primers， including primers GQ-F/R for identifying L. chinense and primers NX-F/R for identifying L. barbarum. Furthermore， the primer concentration ratio， annealing temperature， cycles， and Taq enzyme were optimized to establish the optimal PCR system and conditions for plant identification. Finally， the applicability of the established method was examined with the plant samples collected from different regions. ResultThe PCR with GQ-F/R∶NX-F/R concentration ratio of 2∶1 at the annealing temperature at 59 ℃ and for 30 cycles showed specific bands at 183 bp and 295 bp， respectively， for L. chinense and L. barbarum samples from different regions. ConclusionThe established PCR approach can simply， rapidly， and efficiently identify the original plants of Lycii Cortex， serving as a new method for the discrimination between L. chinense and L. barbarum. Moreover， the method provides technical support for the research and development of classic famous prescriptions containing Lycii Cortex.

Research progress on chemical constituents and pharmacological studies on root bark of Lycium barbarum / 中国中药杂志

Lycii Cortex, the dry root bark of Lycium barbarum(Solanaceae), is rich in chemical compositions with unique structures, such as organic acids, lipids, alkaloids, cyclopeptides and other components, and plays an important role in traditional Chinese medicine. It has the effect of cooling blood and removing steam, clearing lung and reducing fire. It is mainly used in the treatment of hot flashes due to Yin deficiency, hectic fever with night sweat, cough, hemoptysis and internal heat and diabetes. Modern pharmacological studies have shown that the crude extract or monomer of Lycii Cortex has a variety of pharmacological activities, such as hypoglycemic, hypotensive, hypolipidemic, antibacterial, and antiviral effects. In this paper, the chemical constituents and pharmacological effects of Cortex Lycii were reviewed in order to further clarify its effective substances, promote the development of medical undertakings, and ensure the "Healthy China" plan.

Chemical constituents from n-butanol parts of Lycii Cortex / 中草药

Objective: To study the chemical constituents from n-butanol-souluble part of Lycii Cortex (the root bark of Lycium chinense). Methods: The air-dried Lycii Cortex were powdered and extracted with 70% ethanol under reflux. After the removal of solvent under reduced pressure, the crude extract was extracted with petroleum ether, ethyl acetate and n-butanol successively. The compounds were isolated and purified by silica gel, Sephadex LH-20, ODS and semi-prepared high performance liquid chromatography from the n-butanol part of Lycii Cortex. The structures were identified by nuclear magnetic spectrometry, mass spectrometry and other spectral analyses. Results: Ten compounds were isolated from n-butanol parts of Lycii Cortex and characterized as (1'S,2R,5S,10R)-2-(1',2'-dihydroxy-1'-methylethyl)-6,10-dimethylspiro [4,5] dec-6-en-8-one 2'-O-β-D- glucopyranoside (1), (1'R,2R,5S,10R)-2-(1',2'-dihydroxy-1'-methylethyl)-6,10-dimethylspiro [4,5] dec-6-en-8-one 2'-O-β-D- glucopyranoside (2), (1R,6R,9S)-6,9,11-trihydroxy-4,7-megastigmadien-3-one 11-O-β-D-glucopyranoside (3), vanillic acid-4-O-β- D-glucopyranoside (4), 3,4-dihydroxyphenylpropionic acid (5), 3,4-dihydroxybenzenepropionic acid methyl ester (6), glucosyringic acid (7), dihydrophaseic acid 3'-O-β-D-glucopyranoside (8), isoscoploletin-β-D-glucoside (9) and fabiatrin (10). Conclusion: Compound 3 is isolated from Solanaceae family for the first time and compounds 1, 2 and 4 are isolated from Lycium genus for the first time. The NMR data of compound 2 is first reported as well.

Investigation of Xiebaisan Based on Ancient Literature / 中国实验方剂学杂志

Xiebaisan, which is one of the famous classical formulas in the Catalogue of Ancient Famous Classical Formulas (The First Batch) published by the National Administration of Traditional Chinese Medicine (TCM) in 2018, is derived from the Xiao' er Yaozheng Zhijue with definite clinical effect. By means of bibliometrics, 118 relevant ancient book data were collected, including 75 ancient books of TCM. It was found that the main symptoms of Xiebaisan had been expanded in the past dynasties. For example, the adaptive population of Xiebaisan had been extended from children to women and adults, and the scope of symptoms and signs had also been extended from pediatrics to internal medicine and gynecology. Meanwhile, the names, pathogenesis and syndromes of diseases are also described extensively, although the dosage ratio is different, the compatibility ratio of Mori Cortex, Lycii Cortex and Glycyrrhizae Radix et Rhizoma is relatively constant, and the decoction method is mostly followed by decocted power. At the same time, doctors in Ming and Qing dynasties have made a lot of general comments on explanation of this formula, but there is little controversy.

Textual Research on Lycii Cortex / 中国实验方剂学杂志

The research and development of classical prescriptions is an important way to promote the inheritance and innovation of traditional Chinese medicine(TCM) and promote the modernization and internationalization of TCM. It is particularly important to summarize the historical development of the composition of classical prescriptions, so as to define their origin, taste efficacy and syndrome taboos. It is also the source to ensure the safety and effectiveness of the whole prescription. As a commonly used traditional Chinese medicinal material, Lycii Cortex is widely used and has a good prospect for research and development. There are five prescriptions containing wolfberry bark in the Catalogue of Ancient Classical Prescriptions(the First Batch). There are only a few modern literatures on the development of Lycii Cortex. Therefore, this paper will summarize the development of skeleton skin alias, origin, nature, taste, meridian, efficacy, syndromes and taboos in ancient books of the previous dynasties. Textual analysis found that Lycii Cortex was first recorded in the Fifty-two Pathogenic Prescriptions. Ancient books before the Han dynasty did not mention the Chinese medicinal material named " Digupi" . There are little differences in the contents about Digupi between modern literatures and ancient books of the previous dynasties. With the changes of the times, Lycii Cortex has been rarely used, with a fault inheritance, suggesting that there are still many potential efficacy indications for clinical application. In addition to the traditional Chinese medicinal specifications, ancient books also recorded the processing methods with liquorice soup, fried, roasted, children's stool, wine soaking and steaming.

Effect and Mechanism of Amide Alkaloids of Lycii Cortex on Improving Lipid Metabolism in HepG2 Cells / 中国药学杂志

OBJECTIVE: To investigate the effect of two amide alkaloid derivatives (LCAA and LCAB) from Lycii Cortex on lipid metabolism in HepG2 cells and explore their possible mechanisms. METHODS: The lipid accumulation model of HepG2 cells induced by sodium oleate was established. The cells were pretreated with different concentrations of LCAA and LCAB, and the lipid accumulation was observed by oil red O staining. The content of TC in HepG2 cells was measured by oxidizing enzyme method, and the content of TG in HepG2 cells was measured by GPO-PAP method. The expression of lipid metabolism related proteins, such as SIRT1, FOXO1, AMPK and phospho-AMPK (p-AMPK), was detected by Western blot. The mRNA expression of AMPK, SIRT1, ACC2, COXII and COXIII were detected by RT-qPCR. RESULTS: Both LCAA and LCAB could significantly decrease the lipid accumulation in HepG2 cells induced by sodium oleate (200 μmol•L-1) (P<0.01) and the TC and TG levels (P<0.05 or P<0.01). At the same time, the sodium oleate-induced high expression of p-AMPK protein, the low expression of SIRT1, FOXO1 (P<0.01), and the low expression of AMPK, SIRT1, ACC2, COXII and COXIII mRNA (P<0 05 or P<0 01) were all improved by LCAA and LCAB. CONCLUSION: Both LCAA and LCAB can significantly improve lipid metabolism in HepG2 cells, and regulate lipid metabolism by regulating lipid metabolism-related proteins p-AMPK, SIRT1, FOXO1 and mitochondrial oxidation-related factors COXII and COXIII.

Identification of commercial Lycii Cortex and its adulterants using ITS2 sequence as DNA barcode / 中国药学杂志

OBJECTIVE: To identify the commercial medicinal materials and decoction pieces of Lycii Cortex and its adulterants using DNA barcoding technology. METHODS: A total of 137 samples, including 105 voucher samples belonging to nine species, seven GenBank sequences, and 25 test samples were involved in this study. Experiments were performed in accordance with the DNA barcoding standard operating procedures (DNA barcoding SOP) to get the ITS2 sequences. A DNA barcode database of Lycii Cortex and its adulterants were successfully constructed using 112 ITS2 sequences, which were amplified from the voucher samples and downloaded from the GenBank. This database was used to identify the commercial medicinal materials and decoction pieces of Lycii Cortex. RESULTS: The lengths of the ITS2 regions of the Lycii Cortex were 212-230 bp. The ITS2 sequences could clearly distinguish Lycii Cortex and its adulterants. Fifty percent of the commercial samples gained the ideal genomic DNA for the sequence amplification. Using the established database, the above-mentioned sequences were authenticated as Lycium chinense. CONCLUSION: ITS2 Sequence may be a suitable marker for the identification of Lycii Cortex and its adulterants. The DNA barcode databaseof Lycii Cortex and its adulterants constructed in this study are able to successfully identify the raw materials of the commercial medicinal materials and decoction pieces of Lycii Cortex that are currently available in the market.

Effects of the purified extracts from Lycii Cortex Radicis and ginger on lipid statusand serum cytokine levels in rats fed high fat diet

The present study was to investigate the effects of Lycii Cortex Radicis (LCR), the root bark of lycium (Lycium chenese Miller) and ginger (Gin) on body lipid status and serum levels of cytokines. Sprague-Dawley (SD) male rats weighing 193.6 +/- 16.8 g were divided into five groups, including one low fat (LF) and four high fat groups, i.e. HF-Control, HF-LCR, HF-Gin and HF-LCR + Gin groups. Diets for HF-LCR, HF-Gin and HF-LCR + Gin groups contained purified extracts having 0.2 g LCR tyramine, ginerol and 0.1 g tyramine plus 0.02 g gingerol per kg, respectively. Compared with those of the HF-Control total serum cholesterol level decreased, and HDL-cholesterol level increased in the HF-LCR group and serum triglyceride levels decreased in the three experimental groups fed the purified extracts. Liver cholesterol level was lower in the HF-LCR group than the HF-Control group, but triglyceride levels, which were increased by high fat diets were not changed by significantly by LCR or ginger extracts. Fecal lipid excretion was higher in the HF-LCR and HF-Gin groups, but cholesterol excretion was lower in the HF-Gin group than in the HF-Control group. The activities of liver cytosolic glucose-6-phosphate dehydrogenase and malic enzyme were lower in the HF-LCR + Gin group than in the HF-Control group. Serum adiponectin levels did not differ among the five groups, while leptin level was lower in the HF-Gin group and C-reactive protein levels were lower in the HF-Gin and the HF-LCR + Gin groups than in the HF-Control group. It is concluded that LCR can be utilized as an ingredient for lipid-lowering functional foods in the form of purified extract and addition of small amount of ginger extract would be useful for reducing one of the inflammatory cytokines to help prevent atherosclerosis.

Study on the hypochlolesterolemic and antioxidative effects of tyramine derivatives from the root bark of Lycium chenese Miller

The aim of the present study was to investigate the hypocholesterolemic effect and potential of tyramine derivatives from Lycii Cortex Radicis (LCR), the root bark of lycium (Lycium chenese Miller) in reducing lipid peroxidation. The activities of enzymes, hepatic 3-hydroxy 3-methylglutaryl (HMG) CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) and LDL oxidation were measured in vitro and animal experiments were also performed by feeding LCR extracts to rats. The test compounds employed for in vitro study were trans-N-p-coumaroyltyramine (CT) and trans-N-feruloyltyramine (FT), LCR components, N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS) from safflower seeds, ferulic acid (FA) and 10-gingerol. It was observed that FT and FS at the concentration of 1.2 mg/mL inhibited liver microsomal HMG CoA reductase activity by ~40%, but no inhibition of activity was seen in the cases of CT, CS, FA and 10-gingerol. Whereas, ACAT activity was inhibited ~50% by FT and CT, 34-43% by FS and CS and ~80% by 10-gingerol at the concentration of 1 mg/mL. A significant delay in LDL oxidation was induced by CT, FT, and 10-gingerol. For the animal experiment, five groups of Sprague-Dawley male rats were fed high fat diets containing no test material (HF-control), 1 and 2% of LCR ethanol extract (LCR1 and LCR2), and 1% of extracts from safflower seed (Saf) and ginger (Gin). The results indicated that total cholesterol level was significantly lower in Saf, LCR2 and Gin groups, and HDL cholesterol level was lower only in Gin group when compared with HF-control group; while there was no difference in the serum triglyceride levels among the five experimental groups. The level of liver cholesterol was significantly lower in LCR1 and LCR2 groups than HF-control. Serum levels of TBARS were significantly lower only in LCR2 group when compared with HF-control group. From the observed results, we concluded that LCR can be utilized as a hypocholesterolemic ingredient in combination with ginger, especially for functional foods.