[Qualitative and quantitative analysis on crocins in fruits of Gardenia species].

The aim of this study was to compare crocins in the fruit of Gardenia jasminoides and Gardenia jasminoides var. radicans. Acchrom XCharge C_(18) column(4.6 mm×250 mm, 5 µm) was used for separation, with mobile phase of acetonitrile and 0.1% formic acid for gradient elution. The detection wavelength was set at 440 nm with a flow rate of 1.0 mL·min~(-1), and the column temperature was 30 ℃. The high performance liquid chromatography(HPLC) fingerprint of crocin in Gardenia species was established by testing 20 batches of G. jasminoides and 8 batches of G. jasminoides var. radicans samples from different sources, and UHPLC-ESI-Orbitrap-MS/MS technology and reference substances were used to predict and identify the common peaks. The results showed that 20 common chromatographic peaks from the samples were selected and the structures of 16 common peaks were predicted by mass spectrum. Four common peaks(crocin Ⅰ, Ⅱ, Ⅲ, and Ⅳ) were identified by the comparison with reference substances. The content of crocin Ⅰ, Ⅱ, Ⅲ, and Ⅳ was determined simultaneously under the same chromatographic condition, and both the system suitability and the methodological investigation results met the requirements of content determination. The relative similarity of HPLC fingerprint of 28 samples to the reference fingerprint was above 0.98. The results of cluster analysis(CA) showed that G. jasminoides and G. jasminoides var. radicans were separately grouped into one group. In the 20 batches of G. jasminoides, the content of crocin Ⅰ, Ⅱ, Ⅳ, and Ⅲ was between 3.58-9.58, 0.230-1.452, 0.014 5-0.135, and 0.301-1.12 mg·g~(-1), respectively, and the total content was between 4.12-12.25 mg·g~(-1). In the 8 batches of G. jasminoides var. radicans, the content of crocin Ⅰ, Ⅱ, Ⅳ, and Ⅲ was between 5.84-11.48, 0.308-0.898, 0.010 6-0.025 5, and 0.675-1.34 mg·g~(-1), respectively, and the total content was between 6.97-13.72 mg·g~(-1). The existing results showed that there is a certain similarity between G. jasminoides and G. jasminoides var. radicans in the composition of crocin, which needs further proved by more batches of samples. The method established in this paper provides references for the quality control of G. jasminoides, G. jasminoides var. radicans, and related products.

[Quality control of substance benchmarks for Danggui Sini Decoction].

A comprehensive quality control method was established to provide references for quality control and evaluation of substance benchmarks of Danggui Sini Decoction(DSD). The HPLC separation was performed on a Kromasil 100 C-8 column(4.6 mm×250 mm, 5 µm) with acetonitrile(A)-0.05% phosphoric acid in water(B) as mobile phase in a gradient elution mode at the flow rate of 1 mL·min~(-1). The column temperature was 25 ℃ and the detection wavelength was set at 275 nm. Under these conditions, the content of seven components, including paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhetate, ligustilide, and asarinin was simultaneously determined. Under the same chromatographic conditions, the HPLC fingerprint method for analysis of 15 batches of DSD was established. The content determination of aristolochic acid I, using the same test solution as the content determination item, was performed on an ACQUITY UPLC BEH C_(18) column(2.1 mm×50 mm, 1.7 µm) with methanol(A)-water(including 0.1% formic acid and 5 mmol·L~(-1) ammonium formate)(B) as the mobile phase in a gradient elution mode at the flow rate of 0.4 mL·min~(-1) and the column temperature of 40 ℃ by LC-MS/MS. The MS conditions included electrospray ionization(ESI) as an ion source, positive ion ionization, selective reaction monitoring(SRM), the parent ion of 359.3, and the daughter ion of 297.8. The results of the methodological investigation all met the requirements of content determination/fingerprint determination. As a result, the content ranges of paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhetate, ligustilide, and asarinin were 5.419 8-11.267 3, 1.023-3.669 8, 0.145 6-0.444 1, 0.099 1-0.321 9, 3.159 1-7.731 9, 0.146 4-0.471 7, and 0.237 3-0.401 0 mg·g~(-1), respectively. Twenty-two common peaks were selected and 10 of them were identified by the comparison with the reference substances. The fingerprint similarity of 15 batches of DSD was in the range of 0.91-0.996 and the content of aristolochic acid I in DSD was 300.03-638.13 ng·g~(-1). The method established in this study is reliable and easy to operate and has great practical value, which can be used for overall quality control of substance benchmarks for DSD.

[Correlation of non-crocin components of Gardeniae Fructus with its external properties].

This study aimed to explore the correlation of the content of 15 non-crocin components of Gardeniae Fructus with its external properties(shape and color). The fruit shape was quantified according to the length/diameter measured by ruler and vernier calliper and the chromaticity values L~*, a~*, b~*, and ΔE~* of all samples were determined by chroma meter. Chromatographic separation was conducted on a Welch Ultimate XB C_(18) column(4.6 mm×250 mm, 5 µm) under gradient elution with acetonitrile solution(A) and 0.1% formic acid aqueous solution(B) as the mobile phase at a flow rate of 1.0 mL·min~(-1). The column temperature was 30 ℃ and the detection wavelength was 238 nm. The high-performance liquid chromatography(HPLC) method was established for simultaneous determination of the content of eight iridoid glycosides, six phenolic acids, and one flavonoid in 21 batches of Gardeniae Fructus samples. The correlation of the content of the 15 components with shapes and chromaticity values in each sample was analyzed by multivariate statistical analysis. According to the circulation situation and traditional experience, 21 batches of Gardeniae Fructus samples were divided into three categories, namely 14 batches of Jiangxi products(small and round, red and yellow), 4 batches of Fujian products(oval, red) and 3 batches of Shuizhizi(Gardenia jasminoides, longest, reddest). The Gardeniae Fructus samples were sequenced as Jiangxi products(1.71) < Fujian products(1.99) < Shuizhizi(2.55) in terms of the length/diameter average, Jiangxi products(17.7) < Fujian products(19.7) ≈ Shuizhizi(19.6) in terms of average value of a~*(red and green), Jiangxi products(24.4) > Fujian products(19.2) ≈ Shuizhizi(19.3) in terms of b~*(yellow and blue), and Jiangxi products(49.8) > Fujian products(48.0) ≈ Shuizhizi(47.8) in terms of L~*(brightness). The total content of the 15 components, 8 iridoid glycosides, 6 phenolic acids, and rutin in Jiangxi products was in the ranges of 65.53-99.64, 52.15-89.16, 6.10-11.83, and 0.145-1.81 mg·g~(-1), respectively. The total amount of the 15 components, 8 iridoid glycosides, 6 phenolic acids, and rutin in Fujian products was in the ranges of 69.33-94.35, 63.52-85.19, 5.39-8.41, and 0.333-0.757 mg·g~(-1), respectively. In Shuizhizi, the total content of the 15 components, 8 iridoid glycosides, 6 phenolic acids, and rutin was in the ranges of 77.35-85.98, 68.69-76.56, 7.30-9.05, and 0.368-0.697 mg·g~(-1), respectively. Pearson correlation analysis revealed that Gardeniae Fructus with leaner and longer fruit shape possessed lower content of total phenolic acids(the sum of the six phenolic acids) and rutin, but the correlation with iridoid glycosides was not high. Additionally, the higher content of total phenolic acids and rutin denoted the yellow coloration of Gardeniae Fructus, and the higher content of cryptochlorogenic acid, chlorogenic acid, and rutin meant the brighter color of Gardeniae Fructus. However, the higher content of geniposide and neochlorogenic acid and the lower content of deacetyl asperulosidic acid methyl ester led to the red coloration of Gardeniae Fructus. The results indicated that the morphological characters of Gardeniae Fructus were closely related to its chemical components. The more round shape and the yellower color reflected the higher content of phenolic acids and flavonoid, and Gardeniae Fructus with redder color had higher content of geniposide. OPLA-DA showed that the length/diameter and the content of six iridoid glycosides(gardoside, shanzhiside, gardenoside, genipin 1-gentiobioside, 6ß-hydroxy geniposide, and deacetyl asperulosidic acid methyl ester), two phenolic acids(neochlorogenic acid and cryptochlorogenic acid) and rutin could be used as markers to distinguish three types of samples. This study provided experimental data for the scientific connotation of "quality evaluation through morphological identification" of Gardeniae Fructus.

Comprehensive profiling of phytochemicals in the fruits of Gardenia jasminoides Ellis and its variety using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

The fruits of Gardenia jasminoides Ellis are an important herb medicine in Traditional Chinese Medicine (TCM) and have been used for thousands of years for clearing away heat and toxic materials. It mainly contains iridoids, pigments, organic acids, and flavonoids. Although belonging to one species, it has two kinds of cultivars and one variety widely distributed and sold. This study aims to develop an integrated and efficient analytical strategy for comprehensive profiling of phytochemicals and clarify the differences in all three populations. Based on reversed-phase ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS), an optimized analytical approach for comprehensive profiling of phytochemicals in the fruits of G. jasminoides was established in negative ionization mode. The holistic metabolites profiling was carried out on UHPLC/ESI-QTOFMS and data analysis program Progenesis QI, and a total of 80 metabolites were obtained and interpreted by chromatographic and tandem mass spectral data. The interpretation of metabolites comprises iridoids, pigments, organic acids, and flavonoids. Principal component analysis and partial least square-discriminant analysis were performed, and 19 main different components could be obtained to distinguish the three populations. Combined with non-targeted and targeted data analysis, the integrated strategy developed in this study was feasibly applied to discern differences in the profiles of the phytochemicals accumulating in the fruits of three populations of G. jasminoides.

[Research progress on Polygalae Radix].

Polygalae Radix has long been used in China for calming the mind, promoting intelligence, communicating the heart and kidney, eliminating phlegm, and reducing swelling. At present, it is used to treat amnesia, insomnia, and malaise. Modern research has revealed that Polygalae Radix mainly contains triterpenoid saponins, xanthone, oligosaccharide esters, etc., with the activities of improving memory, resisting dementia, protecting the brain, relieving cough, and removing phlegm, as well as sedation and hypnosis. The present study reviews the research progress on chemical composition, pharmacological action, quality control, and metabolism of Polygalae Radix in the past 30 years, to provide a theoretical basis for further research and development.

[Content determination and transferring rules of seven components in Angelicae Sinensis Radix and its processed products].

A HPLC method was established for simultaneous determination of two organic acids(chlorogenic acid and ferulic acid) and five phthalides(senkyunolide I, senkyunolide H, senkyunolide A, ligustilide, and butylidenephthalide) in Angelicae Sinensis Radix and its processed products to clarify the underlying material transferring rules. The analysis was performed on a Welch Ultimate C_8 column(4.6 mm×250 mm, 5 µm) with acetonitrile(A)-0.085% phosphoric acid water(B) as the mobile phase in a gradient elution mode at the flow rate of 1.1 mL·min~(-1), the column temperature of 25 ℃, the detection wavelength of 280 nm, and the injection volume of 10 µL. Under these conditions, the content of the above-mentioned seven components was analyzed in 15 batches of Angelicae Sinensis Radix and its processed products, and the transfer rate of each compound was calculated. As a result, in the processed products, the average content of chlorogenic acid was slightly decreased and that of ferulic acid was equivalent to the medicinal materials. The content of senkyunolide I, senkyunolide H, senkyunolide A, and butylidenephthalide showed an increasing trend in the processed products as compared with the medicinal materials. The mass fraction of ligustilide in the medicinal materials was above 0.7%(0.94% on average), meeting the requirement of 0.6% in the Hong Kong Chinese Materia Medica Standards, but was 0.47% on average in the processed products, which was decreased by 50% approximately. Further investigation showed that the content of ligustilide in freshly made processed products of Angelicae Sinensis Radix did not change significantly compared with that in the medicinal materials, indicating that the loss of ligustilide in the processed products mainly occurred in the storage. Therefore, Angelicae Sinensis Radix is suitable for storing in the form of medicinal materials and the freshly made processed products should be used except for special cases. Additionally, it is recommended to control the content of volatile oils or ligustilide in medicinal materials and processed products of Angelicae Sinensis Radix to ensure its effectiveness in clinical medication.

[HPLC fingerprint of Tibetan medicinal plant ″Bangga″ and chemical components analysis of its two original plants by LC-MS/MS].

To obtain the chemical profile of Tibetan medicinal plant ″Bangga″, the present study established the HPLC fingerprint of ″Bangga″ and inferred common chemical constituents of its two original plants, Aconitum tanguticum and A. naviculare by LC-MS. The HPLC analysis was performed on a Kromasil 100 C_8 column(4.6 mm×250 mm, 5 µm) with acetonitrile(A)-0.1% formic acid in water(B) as mobile phase in a gradient elution mode. Besides, the flow rate was set at 1 mL·min~(-1) and the column temperature was 35 ℃. The detection wavelength was set at 255 nm and the injection volume was 10 µL. Seventeen batches of ″Bangga″ samples were analyzed and the HPLC fingerprint was established under the above conditions. Similarity evaluation was performed using Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012). As a result, 16 common peaks were selec-ted and the similarity values of 17 batches of ″Bangga″ were in the range of 0.702-0.966. Furthermore, one batch of A. tanguticum and one batch of A. naviculare were analyzed by LC-MS/MS and 74 common compounds were inferred, including 10 phenolic acids, 26 flavonoids, and 38 alkaloids. The established method, with good separation and strong specificity, is simple and feasible, and can be used for the quality control of ″Bangga″ and identification of its two original plants. A. tanguticum and A. naviculare are similar in chemical composition and component content, but are quite different in the content of flavonoids.

[Qualitative and quantitative analysis on triterpenoids in Ligustri Lucidi Fructus].

Triterpenoids are one of the most active constituents in Ligustri Lucidi Fructus, but only oleanolic acid has been mostly studied. In recent years, a growing number of studies have shown that other triterpenes from Ligustri Lucidi Fructus also have various biological activities, so it is necessary to build up a detailed profile of the triterpenoids in Ligustri Lucidi Fructus. The chromatographic separation was performed on a C_(18) column(4.6 mm×250 mm, 5 µm) with mobile phase of methanol-acetonitrile-0.2% formic acid for gradient elution. The detection wavelength was set at 210 nm, with a flow rate of 0.5 mL·min~(-1), and the column temperature of 25 ℃. The HPLC fingerprint of triterpenoids in Ligustri Lucidi Fructus was built by testing 21 batches of samples from different sources. The structures of the total 15 common chromatographic peaks were elucidated with UHPLC-ESI-Orbitrap-MS/MS technique and six of them were identified as tormentic acid, pomolic acid, maslinic acid, botulin, oleanolic acid and ursolic acid by comparison to the reference substances. Under the same chromatographic condition, four main triterpenes(podocarboxylic acid, hawthorn acid, oleanolic acid and ursolic acid) were quantified and the results of system adaptability and methodology investigation all met the requirements of content determination. Meanwhile, with oleanolic acid(A) as the internal reference substance, quantitative analysis of multi-components by single marker(QAMS) method was used to analyze the above four components. The relative correction factor of oleanolic acid(B), hawthorn acid(C) and ursolic acid(D) to oleanolic acid was f_(B/A)=1.12, f_(C/A)=1.02 and f_(D/A)=0.88, respectively, and the relative retention values of these three to oleanolic acid was RRV_(B/A)=0.46, RRV_(C/A)=0.70 and RRV_(D/A)=1.03, respectively. The contents determined by two methods were similar. In conclusion, the method built in this paper is proved to be simple, reliable and specific for the simultaneous qualitative and quantitative analysis of the triterpenoids in Ligustri Lucidi Fructus, which can lay foundation for further assays of the triterpenoids in Ligustri Lucidi Fructus and the relative products.

[Optimization of determination of astragaloside â £ in Astragali Radix by continuous single-factor method].

This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside Ⅳ in the legal method for the determination of astragaloside Ⅳ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside Ⅳ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside Ⅳ was 99.65% with RSD 2.2%. Astragaloside Ⅳ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 µg(r=0.999 6). The contents of astragaloside Ⅳ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside Ⅳ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside Ⅳ in Astragali Radix in the new edition of Chinese Pharmacopeia.

[Research progress of tannins in traditional Chinese medicines in recent ten years].

In this paper, the newly isolated tannins were sorted after a review of the literature concerning tannins in recent 10 years, and their research progress was summarized in terms of extraction, isolation, pharmacological activity and metabolism. Hydrolysable tannins and condensed tannins are the main structural types. Modern research shows that tannins have many pharmacological effects, such as bacteriostasis, antioxidation, antitumor, antivirus and blood glucose reduction, and have broad development prospects. They are usually extracted by water, ethanol and acetone and isolated and purified by macroporous resin and gel column chromatography. The packings commonly adopted for the column chromatography mainly included Sephadex LH-20, Diaion HP-20, MCI-gel CHP-20 and Toyopearl HW-40. Modern analytical techniques such as nuclear magnetic resonance spectroscopy(NMR), fast atom bombardment mass spectrometry(FAB-MS) and circular dichroism(CD) are generally used for the structural identification of tannins. Howe-ver, their isolation, purification and structural identification are still challenging. It is necessary to use a variety of high-throughput screening methods to explore their pharmacological activities and to explore the material basis responsible for their functions through experiments in vivo.

[Study on antipyretic effect of rhubarb on rats and its antipyretic ingredients].

A combination of LC-MS technology and activity evaluation was used to identify the antipyretic ingredients in rhubarb. The rat model of fever was established with dried yeast and then was administered ethanol extract and different polar fractions of rhubarb. Next, the anal temperature of these rats was measured and recorded at 0.5, 1, 2, 3, 4 and 5 h after administration, and the inhibition rate of each part on the rise of body temperature was calculated. The inhibition rate is higher and the antipyretic effect is better. The chemical composition of the effective fraction was analyzed with UPLC-ESI-Orbitrap-MS/MS technology. Compared with the model group, the increase of body temperature of ethanol extract group all reduced at each measurement time especially after 3 h, and the inhibition rate were 38.7%(P<0.05), 78.2%(P<0.01) and 72.4%(P<0.01) at 3 h, 4 h, and 5 h after administration, respectively. Both n-butanol and water fraction showed some antipyretic activity in the early stage, with the inhibition rate of 28.1%(P<0.01) and 24.9%(P<0.05) at 1 h after administration, respectively, while other fractions were not active. Thirty-three and twelve compounds were identified from n-butanol and water fraction by LC-MS/MS analysis, respectively, including ten tannins, fifteen anthraquinone glycosides, four anthrone glycosides, one phenolic glycoside, one naphthaline derivative, one anthraquinone and one sucrose. These results revealed that rhubarb had antipyretic activity on rats, and tannin and anthraquinone glycosides were the main active ingredients inside.

[Effect of different sulfur fumigation dosages on activity of browning enzymes and chemical constituents of Chrysanthemum morifolium cv. Boju].

This study aims to investigate the influence of the different dosages of sulfur on the quality and the browning enzyme activity of Chrysanthemum morifolium cv. Boju. In this experiment,UV-spectrophotometry was used to determine the activities of browning enzymes,including polyphenol oxidase( PPO) and peroxidase( POD),in 7 different dosages of 0,4,8,16,50,150,200 g·kg~(-1)( weight ratio of sulfur/fresh chrysanthemum). A comprehensively comparison of the 7 chemical constituents of C. morifolium cv. Boju fumigated with 7 different dosage of sulfur was conducted by HPLC analysis. In this paper,the results showed that the activities of PPO and POD enzymes decreased significantly in chrysanthemum processed by sulfur fumigation. The activities of PPO and POD enzymes decreased gradually with the increase of sulfur dosage. When the sulfur dosage was higher than 4 g·kg~(-1),the PPO enzyme was significantly reduced. When the sulfur dosage was higher than 8 g·kg~(-1),the PPO enzyme was completely inactivated. The effect of different sulfur dosage s on the chemical composition was investigated. In comparison,it was found that when the sulfur dosage was 8 g·kg~(-1),the content of chlorogenic acid was higher than the 4 g·kg~(-1) and that of the sample without sulfur fumigation. Thereafter,with the increase of the sulfur dosage,the content of chlorogenic acid was unchanged. It was speculated that when harvesting,the tissue of fresh flower was destroyed,which caused the activation of browning enzymes. Afterwards,the sulfur fumigation could significantly reduce the activity of browning enzymes,which prevented the conversion of phenols in the reaction substrates( chlorogenic acid and 3,5-dicaffeoylquninic acid) into terpenoids,and better retained quinic acid components. However,when the sulfur dosage reached 8 g·kg~(-1) or16 g·kg~(-1),the content of quinic acid components were no longer changed,which indicated that the sulfur dosage had reached the saturated dosage. Similarly,when the sulfur dosage was increased,the contents of flavonoid aglycones showed a downward trend except for luteolin-7-O-glucoside. It was speculated that the sulfur fumigation inhibited the activity of hydrolase,which reduced the hydrolysis of flavonoid glycosides to aglycones. However,the reaction mechanism needed further verification. In conclusion,although sulfur fumigation could significantly inhibit browning,different dosages of sulfur had a significant effect on the chemical composition of C. morifolium cv. Boju,which could affect the consistency of quality and the stability of the therapeutic effect. Excessive use of sulfur was likely to cause a large amount of SO2 residues in C. morifolium cv. Boju,Therefore,different Sulphur dosages had a significant effect on the quality of chrysanthemum,which therefore was not recommended in production. A small dose of sulfur could be used to prevent enzymatic browning. When the dosage of sulfur increased to a certain extent or reached a saturation state,a small dose of sulfur is recommended in necessary. In this paper,the correlation between the sulfur dosage,the enzyme activity,and the main chemical constituents of chrysanthemum was clarified. The experimental research provided the guidance for regulating the harvesting processing of chrysanthemum and the harvesting processing,and improving the quality of chrysanthemum.