Many triterpenoid compounds have been successfully heterologously synthesized in Saccharomyces cerevisiae. To increase the yield of triterpenoids, various metabolic engineering strategies have been developed. One commonly applied strategy is to enhance the supply of precursors, which has been widely used by researchers. Squalene, as a precursor to triterpenoid biosynthesis, plays a crucial role in the synthesis of these compounds. This study primarily investigates the effect of different squalene levels in chassis strains on the synthesis of triterpenoids(oleanolic acid and ursolic acid), and the underlying mechanisms are further explored using real-time quantitative PCR(qPCR) analysis. The results demonstrate that the chassis strain CB-9-5, which produces high levels of squalene, inhibits the synthesis of oleanolic acid and ursolic acid. In contrast, chassis strains with moderate to low squalene production, such as Y8-1 and CNPK, are more conducive to the synthesis of oleanolic acid and ursolic acid. The qPCR analysis reveals that the expression levels of ERG1, βAS, and CrCYP716A154 in the oleanolic acid-producing strain CB-OA are significantly lower than those in the control strains C-OA and Y-OA, suggesting that high squalene production in the chassis strains suppresses the transcription of certain genes, leading to a reduced yield of triterpenoids. Our findings indicate that when constructing S. cerevisiae strains for triterpenoid production, chassis strains with high squalene content may suppress the expression of certain genes, ultimately lowering their production, whereas chassis strains with moderate squalene levels are more favorable for triterpenoid biosynthesis.
Squalene/analysis* ; Saccharomyces cerevisiae/genetics* ; Triterpenes/metabolism* ; Metabolic Engineering ; Oleanolic Acid/biosynthesis* ; Ursolic Acid

It is generally believed that high-quality Bupleurum scorzonerifolium roots possess specific morphological characteristics, being red, robust, and long with strong odor. However, the scientific connotation of these characteristics has not been elucidated. According to the theory of "quality evaluation through morphological identification", we studied the correlations between appearance traits(the RGB value of root surface, root length, root diameter, dry weight, and ratio of phloem to xylem) and content of main chemical components(volatile oils, total saponins, total flavonoids, total polysaccharides, and seven saikosaponins) of B. scorzonerifolium roots. Epson Scanner and ImageJ were used to scan the root samples and measure the appearance traits. Ultraviolet spectrophotometry and HPLC were employed to determine the content of chemical components. The correlation, regression, and cluster analyses were performed to study the correlations between the appearance traits and the content of chemical components. The results showed that the content of volatile oils and saikosaponins were significantly correlated with RGB value, root length, and root diameter, indicating that within a certain range, the roots being redder, longer, and thicker had higher content of volatile oils and saikosaponins. According to the appearance traits and chemical component content, the 14 samples from different producing areas were classified into four grades, and the differences in morphological traits and chemical component content were consistent among different grades. The findings in this study demonstrate that appearance traits(RGB value, root length, and root diameter) can be used to evaluate the quality of B. scorzonerifolium roots. Meanwhile, this study lays a foundation for establishing an objective quality evaluation method for B. scorzonerifolium roots.
Bupleurum/chemistry* ; Saponins/analysis* ; Oleanolic Acid/analysis* ; Oils, Volatile/analysis* ; Plant Roots/chemistry*

Clinopodium chinense, a traditional folk medicinal herb, has been used to treat abnormal uterine bleeding(AUB) for many years. Saponins and flavonoids are the main active components in C. chinense. To study the pharmacokine-tics of multiple components from the total extract of C. chinense(TEC), we established a sensitive and rapid method of ultra-perfor-mance liquid chromatography coupled with tandem mass spectrometry(UPLC-MS/MS) for simultaneous determination of five compounds in the plasma of AUB rats. After validation, the AUB model was established with SD female rats which got pregnant on the same day by gavage with mifepristone(12.4 mg·kg~(-1)) and misoprostol(130 μg·kg~(-1)). The established method was applied to the detection of hesperidin, naringenin, apigenin, saikosaponin a, and buddlejasaponin Ⅳb in AUB rats after the administration of TEC. The pharmacokinetic parameters were calculated by DAS 2.0. The five compounds showed good linear relationship within the detection range. The specificity, accuracy, precision, recovery, matrix effect, and stability of the method all matched the requirements of biolo-gical sample detection. The above 5 compounds were detected in the plasma of AUB rats after the administration of TEC. The C_(max) va-lues of hesperidin, naringenin, apigenin, saikosaponin a, and clinoposide A were 701.6, 429.5, 860.7, 75.1, and 304.1 ng·mL~(-1), respectively. All the compounds owned short half-life and quick elimination rate in vivo, and the large apparent volume of distribution indicated that they were widely distributed in tissues. Being rapid, accurate, and sensitive, this method is suitable for the pharmacokinetic study of extracts of Chinese herbal medicines and provides a reference for the study of pharmacodynamic material basis of C. chinense in treating AUB.
Administration, Oral ; Animals ; Apigenin/analysis* ; Chromatography, High Pressure Liquid/methods* ; Chromatography, Liquid ; Drugs, Chinese Herbal/chemistry* ; Female ; Flavonoids/analysis* ; Hesperidin ; Lamiaceae ; Mifepristone ; Misoprostol ; Oleanolic Acid/analogs & derivatives* ; Plant Extracts/chemistry* ; Rats ; Saponins ; Tandem Mass Spectrometry/methods* ; Uterine Hemorrhage

This study is to establish the methods for determination of iridoid glycosides and triterpenic acids in Corni Fructus and provide technical support for the quality control of Corni Fructus. Morroniside, loganin and sweroside were determined by HPLC-UV method with acetonitrile and 0.1% formic acid as the mobile phase, and the detective wavelength was set at 240 nm. Oleanolic acid and ursolic acid were determined by HPLC-ELSD method with methanol-0.5% ammonium acetate (87:13) as the mobile phase. The results showed that the linear ranges of morroniside, loganin and sweroside were 5.335-213.4 mg · L(-1) (r = 0.9999), 5.515-220.6 mg · L(-1) (r = 1.0000), 1.992-79.68 mg · L(-1) (r = 1.0000), respectively. The average recoveries of the above three iridoid glycosides were 98.49%-99.28% with RSDs of recoveries being less than 2%. The linear ranges of oleanolic acid and ursolic acid were 7.74-154.8 mg · L(-1) (r = 0.9964), 10.82-216.4 mg · L(-1) (r = 0.9996), respectively. The average recoveries of the above two triterpenic acids were 98.11%-99.27% with RSDs of recoveries being less than 3%. The method established in this research is simple, rapid and reliable, and can be used for quality control of Corni Fructus. Furthermore, the research provided experimental data for the improvement of present quality standard of Corni Fructus, which has important significance to guarantee its quality and clinical curative effect.
Cornus ; chemistry ; Drug Stability ; Oleanolic Acid ; analysis ; Quality Control ; Triterpenes ; analysis

Four kinds of ionic liquids [BMIM] Br, [BMIM] BF4, [BMIM] PF6, [HMIM] PF6 were used to analyze the content of oleanic acid and paeoniflorin in Paeonia lactiflora with ultrasonic-assisted extraction coupled with HPLC. The chromatographic column, Purospher star RP-C18 (4.6 mm x 250 mm, 5 μm), was used. Acetonitrile and water (90:10) as mobile phase was used to determine the content of oleanic acid with a gradient elution and flow rate at 1.00 mL · min(-1), detection wavelength at 210 nm, chromatographic column temperature at room temperature. Paeoniflorin content was determined using acetonitrile and water (18:82) as mobile phase with a gradient elution and flow rate at 1.00 mL · min(-1), detection wavelength at 250 nm, the chromatographic column temperature at room temperature. The result show that oleanic acid has the highest extraction yield when the conditions are solid-liquid ratio of 1:80 (g · mL(-1)), and the [BMIM] Br methanol solution concentration of 0.6 mol · L(-1). Under the optimal extraction conditions, the content of oleanic acid from 0.24 to 3.76 μg showed a good linearity (r = 0.9999), the average recovery was 97.20%. Paeoniflorin has the highest extraction yield when the conditions are solid-liquid ratio of 1:130 (g · mL(-1)), and the [C4 MIM] PF6 methanol solution concentration of 0.6 mol · L(-1). Under the optimal extraction conditions, paeoniflorin content from 0.42 to 4.20 μg showed a good lin- earity (r = 1.000), the average recovery was 98.84%. This method is simple and reliable, its repeatability is also very good. It has important significance in the study P. lactiflora of ionic liquid microextraction.
Chromatography, Reverse-Phase ; methods ; Glucosides ; analysis ; Ionic Liquids ; chemistry ; Monoterpenes ; analysis ; Oleanolic Acid ; analysis ; Paeonia ; chemistry ; Ultrasonics

OBJECTIVETo establish quality standard of Zhuang medicine Lonicera dasystyla, and provide scientific basis for the quality control of L. dasystyla.

METHODCharacteristics of materia medica, microscopic features, TLC indentification, inspection, extractum and determination of chlorogenic acid, macranthoidin B, dipsacoside B were carried out through the experience, microscopic, physical and chemical methods, respectively. The standard of quality control was formulated thereafter.

RESULTThe characteristics of materia medica, microscopic features, TLC indentification were specified, the average contents of water, total ash, acid-insoluble ash, alcohol-soluble extracts, chlorogenic acid were 11.6%, 6.6%, 0.2% , 24.4%, 1.16%, respectively, the total amount of macranthoidin B and dipsacoside B was 3.13%. Quality standard of L. dasystyla was proposed according to experimental results.

CONCLUSIONThe quality of L. dasystyla can be controlled effectively with the quality standard.

Chlorogenic Acid ; analysis ; isolation & purification ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; chemistry ; standards ; Lonicera ; chemistry ; Oleanolic Acid ; analogs & derivatives ; analysis ; isolation & purification ; Quality Control ; Saponins ; analysis ; isolation & purification ; Solubility

OBJECTIVETo establish a method of TLC identification for Dida commonly used in Tibetan medicine from different species.

METHODWith silica gel G as the stationary phase, and chloroform-methanol (40: 1) as mobile phase, oleanolic acid from different species of Dida was separated and identified.

RESULTOleanolic acid was detected in 70 kinds of Dida derived from the Gentianaceae Swertia, Halenia, Gentianopsis, Lomatogonium, and Saxifragaceae saxifrage, except for the saxifrage, there are some differences among different genera or subjection.

CONCLUSIONThis TLC method can be used for identification of oleanolic acid in Dida from different species except saxifrage.

Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; methods ; Drugs, Chinese Herbal ; chemistry ; Medicine, Tibetan Traditional ; methods ; Oleanolic Acid ; analysis ; chemistry ; Species Specificity

OBJECTIVETo investigate the influence of storage period and grading standards on the quality of Prunella vulgaris in order to provide a theoretical basis for standardized cultivation.

METHODThe spices of P. vulgaris in different storage period (including current year, first-year and second-year) were collected from Lujiang base in Anhui Province. The samples were randomized, with the methods of morphologic characteristics and commercial size on P. vulgaris, the samples were graded into three grades; according to the methods from the Pharmacopoeia of the People's Republic of China, water-soluble extracts, ethanol-soluble extracts, total ash and acid insoluble ash were measured, respectively, utilizing the UV spectrophotometry to detection the contents of total flavonoids and polysaccharides, the levels of ursolic acid, oleanolic acid and rosmarinic acid were adopted by HPLC.

RESULTThe total ash and acid insoluble ash of P. vulgaris showed significantly increase during prolonged storage; however, the content of water-soluble extracts, ethanol-soluble, total flavonoids, polysaccharides, ursolic acid, oleanolic acid,rosmarinic acid showed significantly decrease during prolonged storage. The spica of P. vulgaris exhibited no rules differences between the quality and grading standards, The second-degree belongs to high content of total flavonoids; the high content of polysaccharides was first-degree; the total ash and acid insoluble ash of spicas exhibited gradually decreased from first-degree to third-grade, and the content of water-soluble extracts, ethanol-soluble extracts,ursolic acid,oleanolic acid, rosmarinic acid showed gradually increased from first-degree to third-grade.

CONCLUSIONThe current year was a suitable storage time of spicas from P. vulgaris could be selected; and the main bioactive components highest level of P. vulgaris spicas was third-degree, second-grade next, first-degree minimum.

China ; Chromatography, High Pressure Liquid ; Cinnamates ; analysis ; Depsides ; analysis ; Drug Storage ; Flavonoids ; analysis ; Oleanolic Acid ; analysis ; Polysaccharides ; analysis ; Prunella ; chemistry ; Triterpenes ; analysis

Five compounds were separated from Sambucus chinensis and identified as maslinic acid(1), 12alpha, 13-dihydroxyolean-3-oxo-28-oic acid(2), 13-hydroxyolean-3-oxo-28-oic acid (3), 3-oxo oleanolic acid (4), corosolic acid (5). Of them,compound 3 was a new compound, and compounds 1, 2, 4, and 5 were seperated from this plant for the first time.
Oleanolic Acid ; analysis ; Sambucus ; chemistry ; Triterpenes ; analysis

OBJECTIVETo develop an HPLC method for the determination of acteoside, oleanolic acid and ursolic acid in flowers of Campsis grandiflora.

METHODThe analysis was carried out on an Agilent ZORBAX Eclipse XDB-18 column eluted with methanol and 0.1% phosphoric acid in gradient mode. The detection wavelength was set at 334 mn at 0-30 min and 210 nm at 30-60 min.

RESULTThe peak areas and concentrations have a good linear relationship at 0.025 9-0.258 g x L(-1) for acteoside, 0.100-1.00 g x L(-1) for oleanolic acid and 0.104-1.04 g x L(-1) for ursolic acid, respectively. The average recoveries were 98.9%, 99.3% and 99.40%, respectively.

CONCLUSIONThe method can determinate the concentration of acteoside, oleanolic acid and ursolic acid simultaneously. It can be used for the quality control of flower of C. grandiflora.

Bignoniaceae ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Flowers ; chemistry ; Glucosides ; analysis ; Oleanolic Acid ; analysis ; Phenols ; analysis ; Spectrophotometry, Ultraviolet ; methods ; Triterpenes ; analysis