Preparation of Rehmanniae Radix Juice for Baihe Dihuangtang Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo compare the four preparation methods of Rehmanniae Radix juice described in ancient literature and find the method that is most suitable for the preparation of Rehmanniae Radix juice used in Baihe Dihuangtang. MethodThe ancient medical books record four methods for preparing Rehmanniae Radix juice： crushing fresh Rehmanniae Radix for juice， steaming fresh Rehmanniae Radix for juice， boiling fresh Rehmanniae Radix for juice， and boiling dry Rehmanniae Radix for juice. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS） was employed to detect the compounds in the four juice samples， followed by principal component analysis （PCA）. Result① Totally 27 compounds were identified in the juice samples， including 10 iridoid glycosides， 14 phenylethanoid glycosides， 2 phenolic acids， and 1 irisone. Among them， 15 common compounds were shared by the four juice samples， including 7 iridoid glycosides， 7 phenylethanoid glycosides， and 1 phenolic acid. ② Five common compounds in the four juice samples can be matched with the reference standards， which were catalpol， aucubin， rehmannioside D， ajugol， and purpureaside C. ③ Verbascoside and isoacteoside were not detected in the juice prepared by crushing fresh Rehmanniae Radix， while it was detected in the other three juice samples， which indicated that the two components were produced after heating rather than being the original components in fresh Rehmanniae Radix. ④ The comparison of the ion fragments demonstrated that verbascoside was produced from purpureaside C after the cleavage of the glycosidic bond and removal of a molecule of mannose. ⑤ Isoacteoside could be isomerized from verbascoside， and its relative content increased with the extension of heating time. However， the relative content of verbascoside and purpureaside C did not decrease significantly. Therefore， it was hypothesized that purpureaside C was produced from its upstream component. ConclusionThe juice prepared by crushing fresh Rehmanniae Radix has the chemical composition significantly different from the juice samples prepared with the other 3 methods， while the latter 3 juice samples had similar chemical composition. Although all the four methods can be used， it is more suitable to prepare Rehmanniae Radix juice by steaming fresh Rehmanniae Radix， boiling fresh Rehmanniae Radix， and boiling dry Rehmanniae Radix.

Study on antipyretic effect of rhubarb on rats and its antipyretic ingredients / 中国中药杂志

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.

Study on quality status of mineral medicine Calamina / 中国中药杂志

In this paper, some quality problems of mineral medicine Calamina and calcined Calamina have been discussed after determination and analysis of the quality parameters of a large number of market samples, and the countermeasures are put forward. According to the XRD results, as well as the results of tests included in Chinese Pharmacopoeia(2015 edition), the authenticity of Calamina and calcined Calamina samples were identified. The content of zinc oxide in samples were determined by the method of determination in Chinese Pharmacopoeia. Individually, inductively coupled plasma mass spectrometry(ICP-MS), inductively coupled plasma atomic emission spectrometry(ICP-AES) and atomic fluorescence spectrometry(AFS) methods were used for the determination of impurity elements and harmful elements in Calamina and calcined Calamina samples. Four kinds of impurity elements of magnesium(Mg), iron(Fe), aluminum(Al), calcium(Ca) and five harmful elements such as lead(Pb), cadmium(Cd), arsenic(As), copper(Cu), mercury(Hg) were measured. The study showed that: ① Fake Calamina products on the market were overflowing; ② The mineral origin of the mainstream Calamina in the market is inconsistent with that stipulated in Chinese Pharmacopoeia(2015 edition); ③ The contents of harmful elements Pb and Cd in Calamina and calcined Calamina are generally higher, while the contents of harmful elements As and Cu in some inferior Calaminae are higher; ④ Parts of calcined Calamina were improperly or inadequately processed. In view of these quality problems, the countermeasures are put forward as follows: ① It is suggested that hydrozincite should be approved as the mineral source of Calamina, and be included by Chinese Pharmacopoeia; ② Strengthen the research on the specificity of Calamina identification methods to improve the quality control level; ③ Strengthen the research on the processing of Calamina, and formulate the limit standards for the content of Pb and Cd in Calamina; ④ Carry out research on the artificial synthesis of Calamina and calcined Calamina, in order to cope with the current shortage of Calamina resources and ensure the sustainable development of Calamina medicinal materials.

Morphological and microscopical identification of Genkwa Ramulus and its adulterants / 中国中药杂志

The purpose of this article is to identify Daphne genkwa and its adulterants, Wikstroemia chamaedaphne, according to the morphological and microstructure characteristics of their stem and foliage. The root of D.genkwa was studied simultaneously. The results indicated that the crude drug and processed pieces of Genkwa Ramulus were mainly composed of stems and branches where obvious opposite petiole scars and branch marks were able to be seen on their nodes. Otherwise, foliage or peduncles generally couldn't be found. Moreover, the fine silver flocculent fibers could be observed in the bark of fracture surface. The adulterants were the plant segments which were composed of stems, foliage and peduncles with spikelet-pedicel scars. There existed microstructures differences between Genkwa Ramulus and its adulterants. In the former, single thick lignified phloem fibers were interspersed in the stem phloem of the transverse section with very thick wall and unicellular non-glandular hairs could be observed on the lower epidermis of foliage. Nevertheless, in the latter, there was no thick lignified phloem fibers in cross section of stem phloem, the outer wall of epidermal cells of foliage hadthick cuticles and no non-glandular hairs in lower epidermis of foliage. The results can be used for the identification and the quality standard of the crude drug and processed pieces of D.genkwa.The characteristics of the microstructures and the transverse section can be used to identify the radix D.genkwa.

Exploration research on hepatotoxic constituents from Polygonum multiflorum root / 中国中药杂志

By observing the cytotoxic effects of anthraquinones on HepG2 cell and using the precision-cut liver slices technique to authenticate the cytotoxic constituents, the paper aims to explore the material basis of Polygonum multiflorum root to cause liver toxicity. Firstly, MTT method was used to detect the effect of 11 anthraquinone derivatives on HepG2 cell. Then, the clear cytotoxic ingredients were co-cultured with rat liver slices for 6h respectively, and the liver tissue homogenate was prepared. BCA method was used to determine the content of protein in the homogenate and continuous monitoring method was used to monitor the leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamine amino transpeptidase (GGT) and lactate dehydrogenase (LDH). The toxic effect of these ingredients on liver tissue was tested by calculating the leakage rate of the monitored enzymes. As a result, rhein, emodin, physcion-8-O-β-D-glucopyranoside and physcion-8-O-(6'-O-acetyl)-β-D-glucopyranoside showed cytotoxic effects on HepG2 cell and their IC₅₀ values were 71.07, 125.62, 242.27, 402.32 μmol•L⁻¹ respectively, but the other 7 compounds are less toxic and their IC₅₀ values can not be calculated. The precision-cut liver slices tests showed that rhein group of 400 μmol•L⁻¹ concentration significantly increased the leakage rate of ALT, AST and LDH (P<0.01), and the rhein group of 100 μmol•L⁻¹ concentration only increased the leakage rate of LDH (P<0.05). With the increase of rhein concentration, the protein content in liver slices decreased significantly (P<0.05) with a certain range of does. Emodin group of 400 μmol•L⁻¹ concentration significantly increased the leakage rate of ALT, GGT and LDH (P<0.01). Physcion-8-O-β-D-glucopyranoside group of 800 μmol•L⁻¹ concentration also significantly increased the leakage rate of ALT, AST and LDH (P<0.01 or P<0.05), but the group of 200 μmol•L⁻¹ concentration only significantly increased the LDH leakage (P<0.05). Along with the increase of the concentration of physcion-8-O-β-D-glucopyranoside, the leakage rate of ALT, AST and LDH showed a trend of increase, but the protein content in liver slices was in decline. Furthermore, MTT reduction ability of liver slices significantly decreased (P<0.01) in the physcion-8-O-β-D-glucopyranoside group of 800 μmol•L⁻¹ concentration. The results suggested that rhein, emodin and physcion-8-O-β-D-glucopyranoside at high concentrations (≥400 μmol•L⁻¹) can produce some damage to the liver tissue. However, the exposure levels of these constituents are very low, so to reach the toxic concentration (400 μmol•L⁻¹ or 800 μmol•L⁻¹) an adult of 65 kg body weight will need at least a single oral 4 898 g, 339 g and 5 581 g of P.multiflorum root respectively, which is far from the statutory dose of crude P. multiflorum root (3-6 g) or its processed product (6-12 g). Therefore, the conclusion that anthraquinones are the prime constituents of the hepatotoxicity of P. multiflorum root are still not be proved.

Simultaneous determination of 12 mycotoxins in Polygoni Multiflori Radix by UPLC-ESI-MS/MS combined with modified QuEChERS / 中国中药杂志

To investigate the cause of liver toxicity induced by Polygoni Multiflori Radix through determining various mycotoxins in it. An UPLC-ESI-MS/MS method was developed and established to simultaneously determine 12 mycotoxins, Aflatoxins B₁, B₂, G₁, G₂, Ochratoxins A and B, Fumonisins B₁ and B₂,T-2 toxin, HT-2 toxin, Deoxynivalenol and Zearalenone, in rawand processed Polygon iMultiflori Radix. The sample was extracted with modified QuEChERS method, and then was separatedon a WelchUltimate XBC₁₈ column by gradient elution using methanol and 2 mmol•L⁻¹ ammonium acetate aqueous solution containing 0.1% acetic acidas mobile phase. The analytes were detected in MRM mode by mass spectrometry and determined by external standard method. This method made a good linearity in the 0.1-200 μg•kg⁻¹ with correlation coefficients of 0.996 3-0.999 9. The average recoveries of 12 mycotoxins at three spiked concentration levels were ranged from 71.19% to 98.68% with relative standard deviations of 1.7%-13%. This method is simple, sensitive, accurate and suitable for the quantification of 12 mycotoxins in Polygoni Multiflori Radix.As a result, 15 batches were found fungus contamination and total 8 kinds of mycotoxins including AFB₁, AFG₂, FB₁, OTB, T-2, HT-2, FB2 and OTA were detected, and their contentswere between 0.51-1 643.2 μg•kg⁻¹. Among these contaminated samples, AFB1 was detected in one batch of processed Polygoni Multiflori Radix with the content of 6.8 μg•kg⁻¹ beyond its limit standard 5 μg•kg⁻¹. Since AFB₁ has clear liver toxicity, we deduced that the mouldy samples may be one of the important causes of Polygoni Multiflori Radix causing liver toxicity.

Rapid extraction of DNA from Chinese medicinal products by alkaline lysis / 中国中药杂志

The study is aimed to explore a rapid method to extract DNA from fried Chinese medicinal products. The alkaline lysis buffer was made of sodium hydroxide, 1% PVP and 1% TritonX-100 and Tris-HCl solution was neutralized, through heat cracking and neutralization two step to extract DNA from processed and prepared products of traditional Chinese medicine. Then universal primes were used to amplify PCR products for fired Chinese medicinal materials. The results indicated the optimized alkaline lysis method for extracting DNA is quick and easy. Extracting of the different processed Sophora japonica of DNA concentration was (420.61 ± 123.91) g x L(-1). Using 5% Chelex-100 resin purification can improve the DNA concentration. Our results showed that the optimized alkaline lysis method is suitable for Chinese medicinal materials for quickly DNA extraction.

Studies on chemical constituents in Flos Sophorae Carbonisatus / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the chemical constituents in Flos Sophorae Carbonisatus.</p><p><b>METHOD</b>Silica gel column chromatography was used to separate and purify the chemical constituents. The structures were elucidated by spectral analysis.</p><p><b>RESULT</b>Six compounds were isolated from Flos Sophorae Carbionisatus, and their structures were elucidated as maltol (1), 3-hydroxypyridine (2), malto-3-O-[6'-O-(4"-hydroxy-tans-cinnamoyl)-beta-D-glucopyranoside (3), 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] sophoradiol ethyl ester (4), 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] sophoradiol methyl ester (5), rutin (6).</p><p><b>CONCLUSION</b>4 is a new compound, and 1,2,3,5 were first reported from Flos Sophorae Carbonisatus.</p>

Study on the history of the processing of flos sophorae / 中国中药杂志

By collecting and analyzing the information of the processing of Flos Sophorae in ancient and recent literatures, we discovered that such methods as steaming, boiling, stir-frying and baking had been used before Qing Dynasty. There were more than 10 kinds of different decoction pieces due to different subsidiary agents and distinction of processing degree. In modern times, besides stir-frying with vinegar used in Jilin, stir-flying with honey used in Henan and Shandong, being crude, yellowing by stir-frying and carbonizing by stir-frying are used in other places. This research has provided useful information for the modern processing study by summarizing the previous experiences earnestly.