In vivo Pharmacokinetic Analysis of Shenbai Nanosuspension in Rats Based on Overall Composition of Chinese Materia Medica / 中国实验方剂学杂志

ObjectiveTo clarify the scientific validity of in vivo pharmacokinetic determination of the whole drug composition in Shenbai nanosuspension in rats， and to provide methodological guidance and theoretical basis for the in vivo study of multi-component complex system of traditional Chinese medicine（TCM） preparations. MethodThe concentration of the overall components， mainly total saponins and total polysaccharides in Shenbai decoction and Shenbai nanosuspension， was determined in rat plasma at different times by area under the absorbance-wavelength curve method（AUAWC）， and the concentration of individual ginsenoside Rg1 was determined by high performance liquid chromatography（HPLC）， and the methodology was verified. The pharmacokinetic parameters of the whole component were compared with those of ginsenoside Rg1 to evaluate the in vivo operational characteristics of the two preparations. ResultThe methodological investigations of AUAWC and HPLC were in accordance with the requirements. AUAWC analysis showed that the overall components in both the decoction group and the nanosuspension group showed a one-compartment model， with half-life（t1/2） of 2.43 h and 2.04 h， respectively. The relative bioavailability of Shenbai nanosuspension was 138.99%. HPLC assay showed that ginsenoside Rg1 in the decoction group and the nanosuspension group showed a two-compartment model， with distribution half-life（t1/2α） of 0.13 h and 2.55 h， and elimination half-life（t1/2β） were 14.28 h and 3.85 h， respectively. The relative bioavailability of Shenbai nanosuspension was 127.49%. Compared with Shenbai decoction， the time to peak（tmax）， peak concentration（Cmax） and area under the drug-time curve（AUC） of the overall components and ginsenoside Rg1 in Shenbai nanosuspension were increased. ConclusionThe established AUAWC can be used for the pharmacokinetic study of the overall components of TCM preparations， which is complementary to the results of individual components measured by HPLC， and can provide useful reference for the in vivo study of new dosage forms of TCM.

Development and verification of a high performance liquid chromatography method for determination of aluminium adjuvant content in vaccine / 中国生物制品学杂志

@#Objective To develop a high performance liquid chromatography(HPLC)method for determination of aluminium adjuvant content in vaccine,and verify and preliminarily apply the method.Methods The 8-hydroxyquinoline derivatization method was used for determination. The chromatographic column was phenyl-hexyl column[Luna 5u PhenylHexyl(250 mm × 4. 6 mm)],and the mobile phase was composed of ammonium acetate solution-acetonitrile(with 8-hydroxyquinoline)(60 ∶ 40)containing 20 mg/L ascorbic acid,while eluted at a flow rate of 1. 0 mL/min with the isocratic eluent. The excitation wavelength and the emission wavelength of the fluorescence detector were 380 nm and 520 nm respectively. The column temperature was 40 ℃,and the sample injection was 50 μL. The developed method was verified for the specificity,linear range,accuracy,repeatability,stability and durability,and used to determine the aluminum content in 12 batches of vaccines. The results were compared with those determined by titration in general principle 3106of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).Results No interference peaks appeared in the sample chromatogram,and the non-aluminum adjuvant vaccine components and phosphate buffer had no interference with the determination. The linearity of aluminum standard was good in the concentration range of 6. 25 ～ 100 μg/mL,r = 0. 999 6. The average results of spike recoveries of aluminum content in inactivated hepatitis A vaccine,recombinant hepatitis B vaccine,adsorbed acellular DTP vaccine and inactivated enterovirus 71 vaccine were 98. 32%,100. 85%,101. 09% and 99. 31%,respectively in the verification for accuracy. The relative standard deviations(RSDs) of the determination results of aluminum content in the solution of six samples of the four vaccines in the same batch were 1. 09%,1. 42%,0. 97% and1. 30%,respectively. The RSDs of aluminum content of four vaccine samples stored at room tempe-rature for 0,2,4,6 and8 h were 0. 82%,0. 73%,0. 40% and 0. 48%,respectively. When the ratio of ammonium acetate solution to 8-hydroxyquinoline acetonitrile solution in mobile phase changed within 5%,the fluctuation range of aluminum content of four vaccines was less than 2%. There was no significant difference between the developed HPLC method and the titration method of Chinese Pharmacopoeia(VolumeⅢ,2020 edition)for determination of aluminum content in the 12 batches of vaccine samples.Conclusion A HPLC method for determination of aluminum adjuvant content in vaccines has been successfully established with good specificity,linearity,accuracy,repeatability,stability and durability,simple operation,high degree of automation and less interference of manual factors. It can realize the determination of aluminium content in single dose,which provides an effective means for the rapid and large-scale determination of aluminum content in vaccine products and monitoring the dispensing of semi-final products in the production process.

Comparison of Quality Change During Processing Process of Achyranthis Bidentatae Radix from Different Origins Based on Color-component Correlation Analysis / 中国实验方剂学杂志

ObjectiveTaking Achyranthis Bidentatae Radix（ABR） from different origins as samples， to quantitatively analyze the chemical composition and chromaticity of ABR with different processing degrees， and clarify the correlation and change law between color and composition in the processing process of ABR， so as to provide reference for the quality evaluation of processed products of ABR. MethodThe colorimeter is used to measure the chromaticity values of three kinds of processing degrees of ABR in different origins to show the color value change trend during the processing process， and the color parameters of wine-processed and salt-processed products of ABR with different processing degrees were analyzed by principal component analysis（PCA）， orthogonal partial least squares-discriminant analysis（OPLS-DA） and other analysis methods. The contents of eight representative components of ABR were measured by high performance liquid chromatography（HPLC）， the correlation between chromaticity and each representative component was analyzed by Pearson correlation analysis， and the applicability of the selected eight representative components was further verified by Fisher linear discriminant analysis， and the wine-processed and salt-processed products of ABR with different processing degrees were grouped according to the degree of processing， and 48 samples of wine-processed and salt-processed products with different processing degrees were used as training samples. Taking the contents of 5-hydroxymethylfurfural， polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone， ginsenoside Ro， chikusetsusaponin Ⅳa and polysaccharides as variables， the discriminant function was established respectively， and 12 samples of wine-processed and salt-processed products of ABR with different processing degrees were back-tested to verify the discriminant function and test the reliability of the function. ResultPCA and OPLS-DA results showed that ABR samples with different processing degrees were classified into clusters， and the results could significantly distinguish different processed products. During the process of wine and salt processing， the contents of 5-hydroxymethylfurfural， ginsenoside Ro， and chikusetsusaponin Ⅳa gradually increased with the deepening of the processing degree， while the contents of polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone and polysaccharides showed a gradual decreasing trend， indicating these 8 components increased and decreased to different degrees in the process of wine and salt processing. The results of Pearson correlation analysis showed that the 5-hydroxymethylfurfural content of the samples with different processing degrees of wine-processed and salt-processed products were negatively correlated with the brightness value（L*） and the total color difference value（E*ab）（P<0.01）， and positively correlated with the red-green value（a*） and the yellow-blue value（b*）（P<0.01）， and that the content of polypodine B and polysaccharides were positively correlated with L* and E*ab（P<0.01）. The discriminant functions of wine-processed and salt-processed products of ABR were established by Fisher linear discriminant analysis， and their accuracy rates in the training samples were 93.75% and 95.83%， respectively. Twelve test samples of wine-processed and salt-processed products with different processing degree were back substitution， and the correct rate was 100%. ConclusionThe trend of composition and color changes of ABR with different processing degrees in different production areas is relatively consistent， and the color value can better distinguish ABR with different processing degrees， and the color of ABR is related to some representative components in the processing process， indicating that the color can provide reference for the identification of the processing degree of ABR and the prediction of component content.

[Determination of three plant growth regulators in Dendrobium officinale and Anoectochilus roxburghii by three-phase hollow fiber liquid phase microextraction- high performance liquid chromatography].

Dendrobium officinale (D. officinale) and Anoectochilus roxburghii (A. roxburghii) are precious raw materials for traditional Chinese medicine. The growing demand for D. officinale and A. roxburghii cannot be met by current production techniques. Hence, the widespread artificial cultivation of D. officinale and A. roxburghii using substantial amounts of plant growth regulators (PGRs) has emerged. The excessive use of PGRs not only affects the quality and efficacy of medicinal materials but also causes a series of safety issues. Therefore, expanding research on residual PGRs in valuable Chinese medicinal materials is important to avoid the health hazards caused by these substances. Unfortunately, the identification of PGRs is challenging because of their trace and complex matrices. High performance liquid chromatography (HPLC) has become one of the mainstream analytical methods for PGR determination. An important consideration in the application of this technique to the detection of trace acidic PGRs is how to improve its accuracy and sensitivity. Three-phase hollow fiber liquid phase microextraction (3P-HF-LPME) has the advantages of a high enrichment factor, complex sample purification ability, low reagent consumption, low cost, and easy integration with chromatographic systems. Thus, the 3P-HF-LPME method overcomes the many shortcomings of traditional sample pretreatment methods. In this study, a novel, simple, and effective analytical method based on 3P-HF-LPME combined with HPLC was developed to extract, purify, enrich, and detect three trace acidic PGRs (indole-3-acetic acid, naphthyl acetic acid and indolebutyric acid) in D. officinale and A. roxburghii. The chromatographic separation conditions and 3P-HF-LPME model parameters were systematically optimized for this purpose. First, the sample solution was prepared by ultrasonication and low-temperature standing, and then adjusted to pH 3.0 using dilute hydrochloric acid. The sample solution (10 mL) and NaCl (1.50 g) were stored in a 15 mL brown extraction bottle with a built-in magnetic stirrer. Next, 30 µL of NaOH solution (pH 11.0) as the inner phase solution was injected into the inner cavity of a hollow fiber tube, which was subsequently sealed at both ends. The hollow fiber tube was soaked in n-octanol for 5 min and dried naturally to remove excess extraction solvent from its surface. Finally, the fiber tube was placed in a brown extraction bottle and stirred using a thermostatic magnetic stirrer at 40 ℃ and 1600 r/min for 2 h. After extraction, the three target analytes were separated on a Welch Ultimate XB-C18 column (250 mm×4.6 mm, 5 µm) under isocratic elution conditions using acetic acid aqueous solution and methanol (45∶55, v/v) as the eluent. The results indicated that the three PGRs showed good linearity in the range of 0.5-100.0 µg/L (coefficients of determination (r2)=0.9999), with limits of detection (LODs) of 0.02-0.15 µg/L. The method recoveries were 88.5-102.2%, with relative standard deviations (RSDs) of less than 3.7% (n=3). The extraction efficiencies and enrichment factors of the three PGRs in 15 batches of fresh D. officinale and A. roxburghii products were found to be 42.0%-86.8% and 140-289. Full-scan mass spectrometry was used to further identify positive samples to avoid false-positive results and enhance the reliability of the experimental method. In summary, the proposed method is sensitive, accurate, reliable, environment friendly, and capable of high enrichment. It could be used to determine the residues of three acidic PGRs in D. officinale and A. roxburghii. Moreover, it can provide technical support for the residue detection of PGRs in other Chinese medicinal materials.

[Rapid determination of aesculin and aesculetin in Fraxini Cortex by high performance liquid chromatography-ultraviolet at equal absorption wavelength].

Fraxini Cortex is a traditional Chinese herbal medicine that has been used for thousands of years to treat dampness-heat diarrhea, dysentery, red or white vaginal discharge, painful swelling or redness of the eyes, and nebula. It contains various chemical components, including coumarins, iridoids, phenolic acids, and flavonoids. Coumarins are important active ingredients in Fraxini Cortex and have antibacterial, anti-inflammatory, antioxidant, antitumor, and antiviral activities. Aesculin and aesculetin are two major coumarin components of Fraxini Cortex that are widely used in its quality evaluation. Previous HPLC methods for determination of aesculin and aesculetin present several limitations, such as long analysis times and high solvent and reference compound consumption. In this study, a rapid, eco-friendly and cost saving HPLC method for the determination of aesculin and aesculetin in Fraxini Cortex was established by using the core-shell column and equal absorption wavelength (EAW). Different factors influencing the extraction process, such as the extraction solvent, temperature, and time, were assessed to obtain the optimal extraction conditions. The results showed that Fraxini Cortex samples could be well extracted by ultrasonic extraction for 5 min with a 25% ethanol aqueous solution. A core-shell column was used, and different mobile phases and flow rates were investigated to obtain the best rapid-HPLC separation conditions. The optimized HPLC conditions were as follows: a Poroshell 120 EC-C18 column (50 mm×4.6 mm, 2.7 µm), acetonitrile-0.1% formic acid aqueous solution (6∶94, v/v) as the eluent, a flow rate of 1.5 mL/min, and a column temperature of 25 ℃. The EAW of aesculin and aesculetin was a key factor in their determination using a single reference compound. EAW selection was performed in two steps. First, the UV spectra of two equimolar concentrations of the reference compounds (aesculin and aesculetin) were compared to determine the EAW of the two analytes. The EAW results were then verified by the HPLC analysis of the reference compound solutions. The final EAW of aesculin and aesculetin was 341 nm. The determination of aesculin and aesculetin using only one reference compound (i. e., aesculin) was achieved by HPLC-UV at this EAW. The newly developed HPLC method revealed a good linear relationship between the two target analytes (r=1.0000). The limits of detection (LODs) and limits of quantification (LOQs) were 1.5 µmol/L and 3.0 µmol/L, respectively, and the average recoveries of aesculin and aesculetin were 99.0% and 97.5%. The stabilities of the sample solutions were examined, and the two analytes demonstrated good stability for 24 h. The contents of the target analytes in 10 batches of Fraxini Cortex were determined using the proposed EAW method and the classic external standard method (ESM), and comparable concentrations were obtained. The contents of aesculin and aesculetin in the 10 batches of Fraxini Cortex were 0.26%-2.80% and 0.11%-1.47%, respectively. A t-test was conducted to compare the results of the proposed EAW technique with those obtained via the method reported in the Chinese Pharmacopoeia, and no significant difference between the two assay methods was noted (P>0.05). Comparison of the newly established EAW method with those reported in the literature revealed that our method required only 10 min to complete and used as little as 0.5 mL of the solvent and only one standard. Therefore, the developed EAW method is a rapid, simple, eco-friendly, and cost-effective analytical method that is suitable for the determination of aesculin and aesculetin in Fraxini Cortex and its related products. The proposed technique is an improved method for determining aesculin and aesculetin and contributes to the enhancement of the quality evaluation of Fraxini Cortex.

[Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species].

In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 µL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.

[Simultaneous determination of five lignans from Schisandra chinensis by matrix solid-phase dispersion extraction-high performance liquid chromatography].

The kidney-shaped, red-colord fruit from the plant, Schisandra chinensis (Turcz.) Baill, which belongs to the Schisandraceae family, is among the most popular remedies used in traditional Chinese medicine. The English name of the plant is "Chinese magnolia vine". It has been used in Asia since ancient times to treat a variety of ailments, including chronic cough and dyspnea, frequent urination, diarrhea, and diabetes. This is because of the wide range of bioactive constituents, such as lignans, essential oils, triterpenoids, organic acids, polysaccharides, and sterols. In some cases, these constituents affects the pharmacological efficacy of the plant. Lignans with a dibenzocyclooctadiene-type skeleton are considered to be the major constituents and main bioactive ingredients of Schisandra chinensis. However, because of the complex composition of Schisandra chinensis, the extraction yields of lignans are low. Thus, it is particularly important to study pretreatment methods used during sample preparation for the quality control of traditional Chinese medicine. Matrix solid-phase dispersion extraction (MSPD) is a comprehensive process involving destruction, extraction, fractionation, and purification. The MSPD method is simple, it requires only a small number of samples and solvents, it does not require any special experimental equipments or instruments, and it can be used to prepare liquid, viscous, semi-solid, solid samples. In this study, a method combining matrix solid-phase dispersion extraction with high performance liquid chromatography (MSPD-HPLC) was established for the simultaneous determination of five lignans (schisandrol A, schisandrol B, deoxyschizandrin, schizandrin B, and schizandrin C) in Schisandra chinensis. The target compounds were separated on a C18 column with a gradient elution of 0.1% (v/v) formic acid aqueous solution and acetonitrile as the mobile phases, and detection was performed at a wavelength of 250 nm. First, the effects of 12 adsorbents, including silica gel, acidic alumina, neutral alumina, alkaline alumina, Florisil, Diol, XAmide, Xion, and the inverse adsorbents, C18, C18-ME, C18-G1, and C18-HC, on the extraction yields of lignans were investigated. Second, effects of the mass of the adsorbent, the type of eluent, and volume of eluent on the extraction yields of lignans were investigated. Xion was chosen as an adsorbent for MSPD-HPLC analysis of lignans from Schisandra chinensis. Optimization of the extraction parameters showed that the MSPD method had a high lignan extraction yield with Schisandra chinensis powder (0.25 g) as a fixed value, Xion as the adsorbent (0.75 g), and methanol as the elution solvent (15 mL). Analytical methods were developed for five lignans from Schisandra chinensis and these methods showed good linearity (correlation coefficients (R2)≥ 0.9999) for each target analyte. The limits of detection and quantification ranged from 0.0089 to 0.0294 µg/mL and 0.0267 to 0.0882 µg/mL, respectively. Lignans were tested at low, medium, and high levels. The average recovery rates were 92.2% to 111.2%, and the relative standard deviations were 0.23% to 3.54%. Both intra-day and inter-day precisions were less than 3.6%. Compared with hot reflux extraction and ultrasonic extraction methods, MSPD has the advantages of combined extraction and purification, being less time-consuming, and requiring lower solvent volumes. Finally, the optimized method was successfully applied to analyze five lignans from Schisandra chinensis samples from 17 cultivation areas.

[Quality evaluation methods of Poria based on yield and content of alkali extracted polysaccharide].

To improve the quality control methods of Poria and develop and utilize its resources fully, alkaline extraction was used in this study to determine the yield and content of alkali-soluble polysaccharides of Poria. The alkali-soluble extracts of Poria were obtained according to the optimum extraction conditions on the basis of single-factor test, and 30 batches of samples were determined. The structure and chemical composition of the alkali-soluble extracts was characterized by high-performance gel permeation chromatography(HPGPC), Fourier transform infrared spectrometry(FT-IR), nuclear magnetic resonance(NMR) spectroscopy and high-performance liquid chromatography(HPLC) with 1-phenyl-3-methyl-5-pyrazolone(PMP-HPLC). The results showed that the content of the alkali-soluble extracts was in the range of 46.98%-73.86%. The main component was ß-(1→3)-glucan, and its molecular mass was about 1.093×10~5. Further, the content of alkali-soluble polysaccharides of Poria was measured by UV-Vis spectrophotometry and HPLC coupled with the evaporative light scattering detector(HPLC-ELSD), and 30 batches of samples were measured. The results indicated that the content of alkali-soluble polysaccharides determined by UV-Vis spectrophotometry was in the range of 73.70%-92.57%, and the content of samples from Hubei province was slightly higher than that from Yunnan province, Anhui province and Hunan province. The content of alkali-soluble polysaccharides determined by HPLC-ELSD was in the range of 51.42%-76.69%, and the samples from Hunan province had slightly higher content than that from the other three provinces. The content determined by UV-Vis spectrophotometry was higher than that by HPLC-ELSD. However, the content determined by HPLC-ELSD was close to that of alkali-soluble extract, which could accurately characterize the content of alkali-soluble polysaccharides in Poria, and the method was simple and repeatable. Therefore, it is recommended that the quantitative analysis method for alkali-soluble extract and alkali-soluble polysaccharides by HPLC-ELSD be used in the quality standards of Poria in Chinese Pharmacopeia.

HPLC Fingerprint Analysis of Benchmark Sample of Yanghetang / 中国实验方剂学杂志

ObjectiveTo establish a high performance liquid chromatography（HPLC） fingerprint of Yanghetang benchmark sample， and evaluate its quality with chemometric methods， so as to provide a reference for the quality control of this benchmark sample. MethodHPLC was used to establish the fingerprint of Yanghetang benchmark sample with ZORBAX SB-C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was consisted of acetonitrile（A） -0.05% phosphoric acid aqueous solution （containing 0.05% triethylamine solution）（B） for gradient elution（0-5 min， 2%-3%A； 5-15 min， 3%-5%A； 15-65 min， 5%-30%A； 65-90 min， 30%-70%A）， the flow rate was 1.0 mL·min-1， the column temperature was 35 ℃， and the detection wavelength was 210， 260 nm. Traditional Chinese Medicine（TCM） Chromatographic Fingerprint Similarity Evaluation System （2012 edition） combined with cluster analysis， principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to evaluate the quality differences between different batches of Yanghetang benchmark samples， and to find the main chemical components responsible for the quality differences. ResultHPLC fingerprint of Yanghetang benchmark sample was established， 13 common peaks were identified and attributed to each common peak， including peaks 2 and 8 from Rehmanniae Radix Praeparata， peaks 10 and 11 from Cinnamomi Cortex， peaks 1， 3-6 from fried Sinapis Semen， peak 13 from Ephedrae Herba， and peaks 7， 9， 12 from Glycyrrhizae Radix et Rhizoma. Eight of them were identified by comparing with control substance， which were 5-hydroxymethylfurfural（peak 2）， sinapine thiocyanate（peak 4）， glycyrrhizin（peak 7）， verbascoside（peak 8）， cinnamic acid（peak 10）， cinnamaldehyde（peak 11）， glycyrrhizic acid（peak 12） and ephedrine hydrochloride（peak 13）. The similarities of the HPLC fingerprints of 15 batches of Yanghetang benchmark samples with the control fingerprint were all greater than 0.80. The three chemometric methods could classify the samples into two categories. Eight differential components were screened out， among which 5-hydroxymethylfurfural， sinapine thiocyanate， verbascoside and ephedrine hydrochloride were identified. ConclusionThe established fingerprint analysis method is accurate， stable and reproducible， which basically reflects the overall chemical composition of Yanghetang benchmark sample， and can provide a basis for establishment of quality standards for compound preparations of this famous classical formula.

Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species / 中国中药杂志

In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 μL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.

In Vitro and In Vivo Anti-Arthritic Potential of Caralluma tuberculata N. E. Brown. and Its Chemical Characterization.

Present research was planned to assess the in vitro and in vivo anti-arthritic potential of Caralluma tuberculata N. E. Brown. methanolic (CTME) and aqueous (CTAQ) extracts. Chemical characterization was done by high-performance liquid chromatography and gas chromatography−mass spectrometry analysis. The Complete Freund's Adjuvant (CFA) was injected in left hind paw of rat at day 1 and dosing at 150, 300 and 600 mg/kg was started on the 8th day via oral gavage in all groups except normal and disease control rats (which were given distilled water), whereas methotrexate (intraperitoneal; 1 mg/kg/mL) was administered to standard control. The CTME and CTAQ exerted significant (p < 0.01−0.0001) in vitro anti-arthritic action. Both extracts notably reduced paw edema, and restored weight loss, immune organs weight, arthritic score, RBCs, ESR, platelet count, rheumatoid factor (RF), C-reactive protein, and WBCs in treated rats. The plant extracts showed significant (p < 0.05−0.0001) downregulation of tumor necrosis factor-α, Interleukin-6, -1ß, NF-κB, and cyclooxygenase-2, while notably upregulated IL-4, IL-10, I-κBα in contrast to disease control rats. The plant extracts noticeably (p < 0.001−0.0001) restored the superoxide dismutase and catalase activities and MDA levels in treated rats. Both extracts exhibited significant anti-arthritic potential. The promising potential was exhibited by both extracts probably due to phenolic, and flavonoids compounds.

Genus Rauvolfia: A review of its ethnopharmacology, phytochemistry, quality control/quality assurance, pharmacological activities and clinical evidence.

ETHNOPHARMACOLOGICAL RELEVANCE: The plants are from the genus Rauvolfia Plum. ex L. (Apocynaceae), which is represented by 74 species with many synonyms, and distributed worldwide, especially in the Asian, and African continents. Traditionally, some of them are used for the treatment of various disorders related to the central nervous system (CNS), cardiovascular diseases (CVD), and as an antidote due to the presence of monoterpene indole alkaloids (MIAs) such as ajmaline (144), ajmalicine (164) serpentine (182), yohimbine (190) and reserpine (214). AIM: The present review provides comprehensive summarization and critical analysis of the traditional to modern applications of Rauvolfia species, and the major focus was to include traditional uses, phytochemistry, quality control, pharmacological properties, as well as clinical evidence that may be useful in the drug discovery process. MATERIALS AND METHODS: Information related to traditional uses, chemical constituents, separation techniques/analytical methods, and pharmacological properties of the genus Rauvolfia were obtained using electronic databases such as Web of Science, Scopus, SciFinder, PubMed, PubChem, ChemSpider, and Google Scholar between the years 1949-2021. The scientific name of the species and its synonyms were checked with the information of The Plant List. RESULTS: A total of seventeen Rauvolfia species have been traditionally explored for various therapeutic applications, out of which the roots of R. serpentina and R. vomitoria are used most commonly for the treatment of many diseases. About 287 alkaloids, seven terpenoids, nine flavonoids, and four phenolic acids have been reported in different parts of the forty-three species. Quality control (QC)/quality assurance (QA) of extracts/herbal formulations of Rauvolfia species was analyzed by qualitative and quantitative methods based on the major MIAs such as compounds 144, 164, 182, 190, and 214 using HPTLC, HPLC, and HPLC-MS. The various extracts of different plant parts of thirteen Rauvolfia species are explored for their pharmacological properties such as antimicrobial, antioxidant, antiprotozoal, antitrypanosomal, antipsychotic, cardioprotective, cholinesterase inhibitory, and hepatoprotective. Of which, clinical trials of herbal formulations/extracts of R. serpentina and MIAs have been reported for CVD, CNS, antihypertensive therapy, antidiabetic effects, and psoriasis therapy, while the extracts and phytoconstituents of remaining Rauvolfia species are predominantly significant, owning them to be additional attention for further investigation under clinical trials and QC/QA. CONCLUSION: The present communication has provided a comprehensive, systematic, and critically analyzed vision into the traditional uses, phytochemistry, and modern therapeutic applications of the genus Rauvolfia are validated by scientific evidence. In addition, different plant parts from this genus, especially raw and finished herbal products of the roots of R. serpentina have been demonstrated for the QC/QA.

Detection of 94 PDE-5is and Their Analogs Including N-Desmethylthiosildenafil in Various Formulations of Dietary Supplements and Food Samples Using HPLC and LC-Q-TOF/MS.

Consumption of foods and dietary supplements (DS) adulterated with unprescribed or non-permitted phosphodiesterase-5 inhibitors (PDE-5i) and their analogs can cause serious risk to human health. This study aims to analyze 93 PDE-5i and their analogs present in adulterated foods and DS using an established and validated method involving high-performance liquid chromatography (HPLC). The method was validated in solid and liquid samples, resulting in a limit of detection and quantitation of 0.03-0.5 and 0.08-1.6 µg/mL, respectively. Using the validated method, a total of 404 samples were screened. It was found that 32% of 404 samples were illegally adulterated with PDE-5i and their analogs; moreover, 16.9% of the adulterated samples were found to contain more than three compounds. HPLC-quadrupole-time-of-flight (TOF)/mass spectrometry (MS) analysis was conducted on all the samples to confirm the detected compounds accurately based on fragmentation ion patterns. In addition, sildenafil and tadalafil were detected from the capsule shells of DS unusually. Subsequently, the detected compounds were identified and quantified using HPLC at concentrations ranging from 0.007 to 370.0 mg/g. NMR analysis was carried out to confirm the accurate chemical structure of a compound found during the TOF/MS analysis, which did not match with the 93 reference standards.; it was identified to be N-desmethylthiosildenafil. In this study, various PDE-5i compounds and their analogs were detected from low to high concentrations in a sample. Therefore, the study sheds light on the misuse of PDE-5i and their analogs in consumable products, which pose a severe threat to public health.

[Preparation of cucurbitacin compounds in Siraitia grosvenorii roots by high speed countercurrent chromatography].

Siraitia grosvenorii (Swingle) C. Jeffrey, belonging to the family Cucurbitaceae, is a natural sweetener. The roots of this plant are used in folk medicine for the treatment of rheumatoid arthritis. Cucurbitacins play an important role in the resistance of this plant to insects and adversity, and have anti-inflammatory, anti-tumor, and other biological activities. They usually exist as a variety of similar structures in Cucurbitaceae plants. Separation of a large amount of high-purity monomer compounds by the conventional separation method based on column chromatography is difficult, which limits the research and application of their activities. Therefore, we chose a new method for this separation. High-speed countercurrent chromatography (HSCCC) is a liquid-liquid chromatographic technique characterized by high recovery and reproducibility, and is considered a very effective method for the separation of natural compounds present in various plant extracts. An appropriate solvent system is the key for efficient separation, but its selection is tedious, which hampers the wider implementation of HSCCC in chemical research involving preparative separations. In this study, based on the general estimation strategy by using the TLC solvent system (GUESS), the corresponding relationship between the partition distribution coefficient (K value) and the TLC retention factor (Rf value) of the compounds was established by the partition experiment. The Rf value and separation coefficient α were calculated using the water-saturated organic phase as the expansion agent, which could minimize the number of countercurrent separation experiments required in solvent system selection. In this study, HSCCC was used to establish an efficient method for the extraction of cucurbitacins from the root extract of Siraitia grosvenorii. A fraction rich in cucurbitacins was obtained from the ethanol extract of Siraitia grosvenorii roots after separation by column chromatography on HPD-100, MCI, and C18 columns. Six types of solvent systems with different compositions were investigated using the GUESS method. The results showed that employing the solvent system of n-hexane-ethyl acetate-methanol-water (3∶7∶3∶7, v/v/v/v) to partition the cucurbitacin fraction could remove a large number of impurities. The components retained in the upper phase in the partition experiment were subsequently purified by HSCCC. The favorable solvent system for HSCCC was n-hexane-ethyl acetate-methanol-water (4∶6∶5∶5, v/v/v/v), while the upper and lower phases were selected as the stationary and mobile phases, respectively, with a flow rate of 2.0 mL/min, a rotation speed of 860 r/min, and an injected sample weight of 280 mg. Five cucurbitacin compounds were obtained by one-time separation. The weights of the five compounds were 14.73, 8.82, 30.74, 5.03, and 3.81 mg. The purities of these compounds were 97.0%, 95.4%, 96.3%, 91.6%, and 95.3%, respectively. Their structures were identified as cucurbitacin Q1, 23,24-dihydrocucurbitacin F-25-acetate, cucurbitacin B, 23,24-dihydrocucurbitacin B, and dihydroisocucurbitacin B-25-acetate by1H-NMR and 13C-NMR spectroscopies, along with comparison with the literature. This study demonstrated how GUESS guidance accelerates the selection of HSCCC solvent systems, simplifies the workflow, and it provides an efficient preparative method for the separation of chemical constituents from the Siraitia grosvenorii roots, which can also be used as a new method for the large-scale preparation of cucurbitacin compounds.

Quantitative analysis of methoxyflavones discriminates between the two types of Kaempferia parviflora.

INTRODUCTION: Kaempferia parviflora or black ginger is abundantly cultivated because its rhizomes contain methoxyflavones that have many pharmacological properties. K. parviflora can be divided into two types, based on morphological characteristics, but differences in their chemical compositions have never been explored. OBJECTIVES: This research aims to find chemical markers that can be used to differentiate between the two types of K. parviflora, the red-leaf and green-leaf types, by quantifying the amounts of methoxyflavones. MATERIAL AND METHODS: K. parviflora samples were collected from 39 locations in Thailand. Their genetic diversity was assessed by a genotyping-by-sequencing (GBS) technique to construct the population structure. Their chemical compositions were analyzed by high performance liquid chromatography-photodiode array detection to determine the methoxyflavone contents. RESULTS: The population structure based on >3,000 single nucleotide polymorphism (SNP) markers showed that the samples can be divided into two groups, which were consistent with the classification by leaf margin color (red-leaf and green-leaf types). HPLC analysis revealed 3,5,7,3',4'-pentamethoxyflavone (PMF), 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), 3,5,7-trimethoxyflavone and 3,5,7,4'-tetramethoxyflavone as major methoxyflavones that can be used as chemical markers. The red-leaf type showed higher amounts of PMF, TMF and 3,5,7,4'-tetramethoxyflavone than the green-leaf type, while the green-leaf type showed higher amounts of DMF and 3,5,7-trimethoxyflavone than the red-leaf type. CONCLUSION: These results provide another approach to discriminate the two types of K. parviflora using chemical profiles alongside genetic and morphological analyses. Therefore, a specific type of K. parviflora can be selected over the other based on preferences for a certain methoxyflavone.

Evaluation of quality change in Salviae miltiorrhizae radix et rhizoma during drying by LF-NMR and HPLC.

Salviae miltiorrhizae radix et rhizoma (Danshen, in Chinese) is one of the traditional Chinese medicines commonly used in clinical practice. In this study, low field nuclear magnetic resonance (LF-NMR) was used to detect changes in the moisture content during the drying of Danshen. Three water states (bound, immobilized, and free) in Danshen were investigated by multi-exponential fitting of the NMR data. Mass changes during drying were analyzed using high-performance liquid chromatography and partial least squares discriminant analysis. The results revealed that two components, salvianolic acid B and tanshinone, were the main chemical substances that produced the differences. Correlations were found among chemical substances, color, and moisture. LF-NMR can quickly assess the moisture content during drying. It also provides a practical tool for the production and processing of medicines or slices.

Behaviour of Extractives in Norway Spruce (Picea abies) Bark during Pile Storage.

The current practices regarding the procurement chain of forest industry sidestreams, such as conifer bark, do not always lead to optimal conditions for preserving individual chemical compounds. This study investigates the standard way of storing bark in large piles in an open area. We mainly focus on the degradation of the most essential hydrophilic and hydrophobic extractives and carbohydrates. First, two large 450 m3 piles of bark from Norway spruce (Picea abies) were formed, one of which was covered with snow. The degradation of the bark extractives was monitored for 24 weeks. Samples were taken from the middle, side and top of the pile. Each sample was extracted at 120 °C with both n-hexane and water, and the extracts produced were then analysed chromatographically using gas chromatography with flame ionisation or mass selective detection and high-performance liquid chromatography. The carbohydrates were next analysed using acidic hydrolysis and acidic methanolysis, followed by chromatographic separation of the monosaccharides formed and their derivatives. The results showed that the most intensive degradation occurred during the first 4 weeks of storage. The levels of hydrophilic extractives were also found to decrease drastically (69% in normal pile and 73% in snow-covered pile) during storage, whereas the decrease in hydrophobic extractives was relatively stable (15% in normal pile and 8% in snow-covered pile). The top of the piles exhibited the most significant decrease in the total level of extractives (73% in normal and snow-covered pile), whereas the bark in the middle of the pile retained the highest amount of extractives (decreased by 51% in normal pile and 47% in snow-covered pile) after 24-week storage.

Absolute Quantification of Isoflavones in the Flowers of Pueraria lobata by qHNMR.

Pueraria lobata (Willd.) Ohwi. is a widely used medicinal plant in Korea, China, and Japan. The flower of P. lobata (Puerariae Flos) contains various bioactive substances such as triterpenoidal saponins and isoflavonoids. In this study, we developed a quantitative analysis of the isoflavones of Puerariae Flos by quantitative proton nuclear magnetic resonance (qHNMR) spectroscopy using the internal calibrant (IC). From the qHNMR results, the isoflavone content was found to be 7.99% and 10.57% for the MeOH sonication extract (PLs) and the MeOH reflux extract (PLr) of Puerariae Flos, respectively. The quantified isoflavone content was validated using the conventional analytical method, high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The present study shows that validated qHNMR spectroscopy is a reliable method for quantifying and standardizing the isoflavone content in Puerariae Flos.

Scientific Characterization of Traditional Softening Method of Corydalis Rhizoma / 中国实验方剂学杂志

ObjectiveModern scientific methods and techniques were used to scientifically characterize the traditional softening process of Corydalis Rhizoma， so as to clarify the scientificity and rationality of the traditional process， and provide reference for inheriting the processing methods and experience of traditional Chinese medicine. MethodLow-field nuclear magnetic resonance imaging （LF-NMR/MRI） was used to characterize the water types and distribution in the softening process of Corydalis Rhizoma. Samples during the softening process was cut into thick slices and its section was observed by stereoscopic microscope. High performance liquid chromatography （HPLC） was employed to determine the content change of tetrahydropalmatine during the softening process with the mobile phase of methanol-0.1% phosphoric acid solution （60∶40， triethylamine regulated to pH 6.5） and detection wavelength at 280 nm. The determination method of softening endpoint of Corydalis Rhizoma was simulated by texture analyzer （hand pinch method）， and the softening degree of the finished products was determined after optimizing the relevant parameters. ResultLF-NMR/MRI showed that the water could penetrate through the core and distribute evenly in Corydalis Rhizoma softened by Zhangbang method. The water first entered into the medicinal material from the epidermis and stem marks in the soaking stage as the form of free water， and then penetrated into the inner core to achieve redistribution in the moistening stage. Under stereoscopic microscope， it was observed that Corydalis Rhizoma softened by the Zhangbang method could be sliced well， but the core bursting slices were easy to appear if the softening time was not enough， and the softening of samples was caused by the keratine-like powder after absorbing water. HPLC measurement showed that the loss of tetrahydropalmatine in the softening method was small， its content decreased about 5% in the soaking process， and its content was almost unchanged during the moistening process. The softening degree of Corydalis Rhizoma could be quantified by the texture analyzer， and the optimum parameters were 2 mm·s-1 of speed before test， test speed and speed after test， 20 g of the trigger force， 20% of compression degree. The compressive force of the qualified softened Corydalis Rhizoma was 12.75-15.69 N with the relative standard deviation （RSD） of 6.8%. ConclusionModern scientific methods and techniques can characterize the scientificity and rationality of the traditional processing methods， and confirm that the Zhangbang softening method has the advantages of high efficiency， convenience and small loss of index components. The texture analyzer can simulate the softening endpoint judgment method （hand pinch method）， and realize the goal from subjective experience judgment to objective technology quantification， which has a good demonstration role for the modern inheritance of traditional processing technology.

Scientific Characterization of Traditional Softening Method of Corydalis Rhizoma / 中国实验方剂学杂志

ObjectiveModern scientific methods and techniques were used to scientifically characterize the traditional softening process of Corydalis Rhizoma， so as to clarify the scientificity and rationality of the traditional process， and provide reference for inheriting the processing methods and experience of traditional Chinese medicine. MethodLow-field nuclear magnetic resonance imaging （LF-NMR/MRI） was used to characterize the water types and distribution in the softening process of Corydalis Rhizoma. Samples during the softening process was cut into thick slices and its section was observed by stereoscopic microscope. High performance liquid chromatography （HPLC） was employed to determine the content change of tetrahydropalmatine during the softening process with the mobile phase of methanol-0.1% phosphoric acid solution （60∶40， triethylamine regulated to pH 6.5） and detection wavelength at 280 nm. The determination method of softening endpoint of Corydalis Rhizoma was simulated by texture analyzer （hand pinch method）， and the softening degree of the finished products was determined after optimizing the relevant parameters. ResultLF-NMR/MRI showed that the water could penetrate through the core and distribute evenly in Corydalis Rhizoma softened by Zhangbang method. The water first entered into the medicinal material from the epidermis and stem marks in the soaking stage as the form of free water， and then penetrated into the inner core to achieve redistribution in the moistening stage. Under stereoscopic microscope， it was observed that Corydalis Rhizoma softened by the Zhangbang method could be sliced well， but the core bursting slices were easy to appear if the softening time was not enough， and the softening of samples was caused by the keratine-like powder after absorbing water. HPLC measurement showed that the loss of tetrahydropalmatine in the softening method was small， its content decreased about 5% in the soaking process， and its content was almost unchanged during the moistening process. The softening degree of Corydalis Rhizoma could be quantified by the texture analyzer， and the optimum parameters were 2 mm·s-1 of speed before test， test speed and speed after test， 20 g of the trigger force， 20% of compression degree. The compressive force of the qualified softened Corydalis Rhizoma was 12.75-15.69 N with the relative standard deviation （RSD） of 6.8%. ConclusionModern scientific methods and techniques can characterize the scientificity and rationality of the traditional processing methods， and confirm that the Zhangbang softening method has the advantages of high efficiency， convenience and small loss of index components. The texture analyzer can simulate the softening endpoint judgment method （hand pinch method）， and realize the goal from subjective experience judgment to objective technology quantification， which has a good demonstration role for the modern inheritance of traditional processing technology.