This study aims to separate and characterize self-assembled nanoparticles(SAN) from Shaoyao Gancao Decoction(SGD) and determine the content of active compounds. Further, we aimed to observe the therapeutic effect of SGD-SAN on imiquimod-induced psoriasis in mice. The separation of SGD was performed by dialysis, and the separation process was optimized by single factor experiment. The SGD-SAN isolated under the optimal process was characterized, and the content of gallic acid, albiflorin, paeoniflorin, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid in each part of SGD was determined by HPLC. In the animal experiment, mice were assigned into a normal group, a model group, a methotrexate group(0.001 g·kg~(-1)), and SGD, SGD sediment, SGD dialysate, and SGD-SAN groups of different doses(1, 2, and 4 g·kg~(-1)) respectively. The psoriasis grade of mice was evaluated based on the pathological changes of skin lesions, the content of inflammatory cytokines, organ index and other indicators. The results showed that SAN obtained by centrifugation at 13 000 r·min~(-1) for 30 min was stable after dialysis for 4 times, which were uniform spherical nanoparticles with the particle size of(164.43±1.34) nm, the polydispersity index of(0.28±0.05), and the Zeta potential of(-12.35±0.80) mV. The active compound content accounted for more than 70% of SGD. Compared with the model group, SAN and SGD decreased the skin lesion score, spleen index, and inflammatory cytokine levels(P<0.05 or P<0.01) and alleviated the skin thickening and infiltration of inflammatory cells. However, the sediment group and the dialysate group had no obvious effect. SGD showed a good therapeutic effect on imiquimod-induced psoriasis in mice, and SAN demonstrated the effect equivalent to SGD in a dose-dependent manner. Therefore, we conclude that the SAN formed during decocting is the main active form of SGD, which can lower the levels of inflammatory cytokines, promote the normal differentiation of keratinocytes, and reduce the infiltration of inflammatory cells in the treatment of psoriasis lesions in mice.
Mice ; Animals ; Imiquimod ; Drugs, Chinese Herbal/pharmacology* ; Glycyrrhizic Acid ; Chromatography, High Pressure Liquid/methods*

OBJECTIVE: To assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer. METHODS: TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-β production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed. RESULTS: The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P < 0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P < 0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti-tumor immunity (P < 0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P < 0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P < 0.05). CONCLUSION Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DCbased immunotherapy for LLC in mice.
Animals ; Humans ; Mice ; Apoptosis ; Dendritic Cells/immunology* ; Glycyrrhizic Acid/pharmacology* ; HMGB1 Protein ; Lung Neoplasms/immunology*

This study focused on the separation, characterization, content determination, and antiviral efficacy research on colloidal particles with different sizes in Maxing Shigan Decoction(MXSG). The mixed colloidal phase of MXSG was initially separated into small colloidal particle segment(S), medium colloidal particle segment(M), and big colloidal particle segment(B) using ultrafiltration. Further fine separation was performed using size-exclusion chromatography. Dynamic light scattering(DLS) and transmission electron microscopy(TEM) were employed to characterize the size and morphology of the separated colloidal particles. UPLC-MS/MS was used to determine the content of ephedrine, amygdalin, glycyrrhizic acid, and the EDTA complexometric titration was used to measure the calcium(Ca~(2+)) content in different colloidal phases. Finally, a respiratory syncytial virus(RSV) infection mouse model was established using intranasal administration. The experimental groups included a blank group, a model group, a ribavirin group, an MXSG group, an S group, an M group, and a B group. Oral administration was given for treatment, and pathological changes in mouse lung tissue and organ indices were evaluated. The results of the study showed that the distribution of ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) content was not uniform among different colloidal segments. Among them, the B segment had the highest proportions of the three components, except for Ca~(2+), accounting for 46.35%, 53.72%, and 92.36%, respectively. Size-exclusion chromatography separated colloidal particles with uniform morphology in the size range of 100-500 nm. Compared to the S and M segments, the B segment showed an increased lung index inhibition rate(38.31%), spleen index, and thymus index in RSV-infected mice, and it improved the infiltration of inflammatory cells and lung injury in the lung tissue of mice. The complex components in MXSG form colloidal particles of various sizes and morphologies through heating, and small-molecule active components such as ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) participate in the assembly to varying degrees. The main material basis for the antiviral effect of MXSG is the colloidal particles with certain particle sizes formed by the assembly of active components during the heating process.
Mice ; Animals ; Amygdalin/chemistry* ; Drugs, Chinese Herbal/chemistry* ; Glycyrrhizic Acid/analysis* ; Ephedrine/analysis* ; Chromatography, Liquid ; Tandem Mass Spectrometry ; Antiviral Agents/pharmacology*

Objective: To investigate the effects and mechanism of diammonium glycyrrhizinate (DG) on liver injury in severely scalded rats. Methods: The experimental research method was used. Fifty-four female Sprague-Dawley rats aged 7-9 weeks were divided into sham injury group with simulated injury on the back, and simple scald group and scald+DG group with scald of 30% total body surface area on the back, with 18 rats in each group. Rats in sham injury group were not specially treated after injury, and rats in simple scald group and scald+DG group were rehydrated for antishock. Besides, rats in scald+DG group were injected intraperitoneally with 50 mg/kg DG at post injury hour (PIH) 1, 25, and 49. Rats in the three groups were collected, the serum content of liver function injury related indexes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total protein, and albumin was measured by automatic biochemical assay analyzer, and serum content of ornithine carbamoyl transferase (OCT) was measured by enzyme-linked immunosorbent assay method at PIH 24, 48, and 72; hepatic histopathological changes at PIH 72 were observed by hematoxylin-eosin staining; the mRNA expressions of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), glucose regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), and protein kinase R-like endoplasmic reticulum kinase (PERK) in liver tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction at PIH 24, 48, and 72. The protein expressions of Bcl-2, Bax, GRP78, PERK, and ATF4 in liver tissue were detected by Western blotting at PIH 72 in sham injury group and PIH 24, 48, and 72 in simple scald group and scald+DG group. The number of samples was 6 in each group at each time point. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni test. Results: Compared with that in sham injury group, the serum content of AST, ALT, and LDH was significantly increased (P<0.01), and the serum content of total protein and albumin was significantly decreased (P<0.05 or P<0.01) of rats in simple scald group at all post-injury time points. Compared with those in simple scald group, the serum AST content of rats in scald+DG group at PIH 24 was decreased significantly (P<0.05); the serum AST, ALT, and LDH content of rats in scald+DG group at PIH 48 was decreased significantly (P<0.01), and the serum total protein content was increased significantly (P<0.01); the serum AST, ALT, and LDH content of rats in scald+DG group at PIH 72 was decreased significantly (P<0.01), and the serum total protein and albumin content was increased significantly (P<0.01). At PIH 24, 48, and 72, the serum OCT content of rats in simple scald group was (48.5±3.9), (40.8±2.4), and (38.7±2.0) U/L, which was significantly higher than (15.1±2.5), (15.7±2.6), and (16.4±3.7) U/L in sham injury group (P<0.01), and (39.0±4.5), (31.8±2.0), and (22.1±2.6) U/L in scald+DG group (P<0.05 or P<0.01). At PIH 72, the cells in liver tissue of rats in sham injury group had normal morphology and regular arrangement, with no obvious inflammatory cell infiltration; the cells in liver tissue of rats in simple scald group had disordered arrangement, diffuse steatosis, and moderate inflammatory cell infiltration; the cells in liver tissue of rats in scald+DG group arranged regularly, with scattered steatosis and a small amount of inflammatory cell infiltration. Compared with those in sham injury group, the Bcl-2 mRNA (P<0.05 or P<0.01) and protein expressions of liver tissue were significantly decreased, and the mRNA (P<0.01) and protein expressions of Bax were significantly increased in rats in simple scald group at PIH 24, 48, and 72. Compared with those in simple scald group, the mRNA (P<0.05) and protein expressions of Bax in liver tissue of rats in scald+DG group were decreased significantly at PIH 48; the mRNA (P<0.01) and protein expressions of Bax in liver tissue of rats in scald+DG group were significantly decreased, and the mRNA (P<0.01) and protein expressions of Bcl-2 were significantly increased at PIH 72. Compared with those in sham injury group, the mRNA (P<0.05 or P<0.01) and protein expressions of ATF4, GRP78, and PERK in liver tissue were significantly increased in rats in simple scald group at all post-injury time points. Compared with those in simple scald group, the mRNA (P<0.01) and protein expressions of ATF4 in liver tissue of rats in scald+DG group at PIH 48 were significantly decreased, and the mRNA (P<0.05 or P<0.01) and protein expressions of ATF4, GRP78, and PERK were significantly decreased in liver tissue of rats in scald+DG group at PIH 72. Conclusions: DG can effectively reduce the degree of liver injury in rats after severe scald, and the mechanism may involve alleviating endoplasmic reticulum stress and mitigating mitochondrial damage.
Albumins/pharmacology* ; Animals ; Burns/pathology* ; Female ; Glycyrrhizic Acid/pharmacology* ; Liver ; RNA, Messenger/genetics* ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein/pharmacology*

This paper aims to study the difference in the intestinal absorption kinetics of main active components of Sini decoction and its separated recipes and explain the scientificity and rationality of the compatibility of Sini Decoction. A in situ intestinal perfusion rat model was established to evaluate the differences in the absorption of benzoylmesaconine, benzoylaconine, benzoylhypacoitine, mesaconitine, hypaconitine, glycyrrhizic acid, liquiritin and 6-gingerol from Sini Decoction and its separated recipes in the duodenum, jejunum and ileum by high performance liquid chromatography(HPLC). The results indicated that the Sini Decoction group was superior to the Aconiti Lateralis Radix Praeparata group in terms of absorption degree and rate for aconitum alkaloids. The absorption of benzoylmesaconine and hypaconitine in the duodenum, jejunum and ileum was faster and stronger in the Sini Decoction group(P<0.05). The absorption degree of glycyrrhizic acid in the duodenum was significantly higher in the Sini Decoction group than in the Glycyrrhizae Radix et Rhizoma group and the Glycyrrhizae Radix et Rhizoma-Zingiberis Rhizoma group(P<0.05). The absorption rate and degree of 6-gingerol in the ileum in the Sini Decoction group were significantly higher than those in the Zingiberis Rhizoma group(P<0.05). In short, Zingiberis Rhizoma and Glycyrrhizae Radix et Rhizoma can promote the absorption of aconitum alkaloids in different intestinal segments, which reflects the scientific composition of Sini Decoction.
Aconitine/analogs & derivatives* ; Aconitum ; Alkaloids ; Animals ; Catechols ; Drugs, Chinese Herbal ; Fatty Alcohols ; Glycyrrhizic Acid ; Intestinal Absorption ; Kinetics ; Rats

Glycyrrhizic Acid/adverse effects* ; Humans ; Hypertension

With reference to the production process documented in Chinese Pharmacopoeia, this paper prepared the calibrator samples of Xiaochaihu Granules from multiple batches and established a method for fingerprint analysis and content determination that could be used to evaluate Xiaochaihu Granules available in market. Multiple batches of Chinese herbal pieces contained in Xiaochaihu Granules were collected for preparing the calibrator samples according to the process in Chinese Pharmacopoeia. Following the establishment of fingerprints for calibrator samples by UHPLC, the method for determining the contents of saikosaponin B2, saikosaponin B1, baicalin, wogonoside, baicalein, liquiritin, glycyrrhizin G2 and glycyrrhizic acid in Xiaochaihu Granules was established. The experimental results showed that the fingerprints of calibrator samples had 26 common peaks, covering the chemical compounds of main herbs Bupleuri Radix, Scutellariae Radix, Changii Radix, Glycyrrhizae Radix et Rhizoma, and Rhizoma Zingiberis Recens. The similarity of fingerprints for 47 batches of Xiaochaihu Granules from 31 companies with the calibrator sample fingerprint ranged from 0.74 to 0.99, indicating good applicability of the established fingerprint. The contents of main components baicalin, saikosaponin B2, and glycyrrhizic acid in Xiaochaihu Granules were within the ranges of 22.917-49.108 mg per bag(RSD 19%), 0.28-2.19 mg per bag(RSD 62%), and 0.897-6.541 mg per bag(RSD 41%), respectively. The quality difference in saikosaponin B2, and glycyrrhizic acid among different manufacturers was significant. The fingerprint analysis and content determination method for calibrator samples of Xiaochaihu Granules prepared according to the production process in Chinese Pharmacopoeia has been proved suitable for evaluating the quality of Xiaochaihu Granules from different manufacturers. Saikosaponin B2, glycyrrhizic acid, and liquiritin should be added as content control indicators for Xiaochaihu Granules, aiming to further improve the product quality.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Glycyrrhizic Acid/analysis* ; Rhizome/chemistry* ; Scutellaria baicalensis

A total of 18 batches of Zhuru Decoction samples were prepared. Chromatographic fingerprints were established for Zhuru Decoction and single decoction pieces, the content of which was then determined. The extraction rate ranges, content, and transfer rate ranges of puerarin, liquiritin, and glycyrrhizic acid, together with the common peaks and the similarity range of the fingerprints, were determined to clarify key quality attributes of Zhuru Decoction. The 18 batches of Zhuru Decoction samples had 25 common peaks and the fingerprint similarity higher than 0.95. Puerariae Lobatae Radix, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens had 21, 3, and 1 characteristic peaks, respectively. The 18 batches of samples showed the extraction rates within the range of 18.45%-25.29%. Puerarin had the content of 2.20%-3.07% and the transfer rate of 38.5%-45.9%; liquiritin had the content of 0.24%-0.85% and the transfer rate of 15.9%-37.5%; glycyrrhizic acid had the content of 0.39%-1.87% and the transfer rate of 16.2%-32.8%. In this paper, the quality value transmitting of substance benchmarks of Zhuru Decoction was analyzed based on chromatographic fingerprints, extraction rate, and the content of index components. A scientific and stable method was preliminarily established, which provided a scientific basis for the quality control and formulation development of Zhuru Decoction.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/standards* ; Glycyrrhizic Acid/analysis* ; Quality Control ; Rhizome/chemistry*

The methods for determining the characteristic chromatogram and index components content of Xuanfu Daizhe Decoction were established to provide a scientific basis for the quality evaluation of substance benchmarks and preparations. Eighteen batches of Xuanfu Daizhe Decoction were prepared with the decoction pieces of different batches and of the same batch were prepared respectively, and the HPLC characteristic chromatograms of these samples were established. The similarities of the chromatographic fingerprints were analyzed. With liquiritin, glycyrrhizic acid, 6-gingerol, ginsenoside Rg_1, and ginsenoside Re as index components, the high performance liquid chromatography was established for content determination with no more than 70%-130% of the mass average as the limit. The results showed that there were 19 characteristic peaks corresponding to the characteristic chromatograms of 18 batches of Xuanfu Daizhe Decoction, including 8 peaks representing liquiritin, 1,5-O-dicaffeoylqunic acid, ginsenoside Rg_1, ginsenoside Re, 1-O-acetyl britannilactone, ginsenoside Rb_1, glycyrrhizic acid, and 6-gingerol, and the fingerprint similarity was greater than 0.97. The contents of liquiritin, glycyrrhizic acid, 6-gingerol, and ginsenosides Rg_1 + Re in the prepared Xuanfu Daizhe Decoction samples were 0.53%-0.86%, 0.61%-1.2%, 0.023%-0.068%, and 0.33%-0.66%, respectively. Except for several batches, most batches of Xuanfu Daizhe Decoction showed stable contents of index components, with no discrete values. The characteristic chromatograms and index components content characterized the information of Inulae Flos, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens in Xuanfu Daizhe Decoction. This study provides a scientific basis for the further research on the key chemical properties of substance benchmark and preparations of Xuanfu Daizhe Decoction.
Benchmarking ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/chemistry* ; Ginsenosides/analysis* ; Glycyrrhizic Acid/analysis* ; Quality Control

According to the polarity of different components in Sanpian Decoction, two fingerprints were established. Then the substance benchmark freeze-dried powder of 15 batches of Sanpian Decoction was prepared, followed by the determination of the fingerprints, index component content, and dry extract rates, the identification of attribution of characteristic peaks, and the calculation of similarities between these fingerprints and the reference(R), the content and transfer rate ranges of ferulic acid, sinapine thiocyanate, liquiritin, and glycyrrhizic acid, and the dry extract rate range. The results showed that the similarities of 15 batches of the substance benchmark fingerprints with R were all greater than 0.900.Further summarization of the characteristic peaks revealed that there were a total of 20 characteristic peaks in fingerprint 1, among which, eight were from Sinapis Semen, four from Paeoniae Radix Alba, six from Chuanxiong Rhizoma, and two from Glycyrrhizae Radix et Rhizoma. A total of 16 characteristic peaks were observed in fingerprint 2, including one from Sinapis Semen, three from Paeoniae Radix Alba, eight from Chuanxiong Rhizoma, and four from Glycyrrhizae Radix et Rhizoma. The average dry extract rate of 15 batches of substance benchmarks was 18.25%, with a dry extract rate range of 16.28%-20.76%. The index component content and transfer rate ranges were listed as follows: 0.15%-0.18% and 38.81%-58.05% for ferulic acid; 0.26%-0.42% and 36.51%-51.02% for sinapine thiocyanate; 0.09%-0.15% and 48.80%-76.61% for liquiritin; 0.13%-0.24% and 23.45%-35.61% for glycyrrhizic acid. The fingerprint, dry extract rate, and index component content determination was combined for analyzing the quality value transfer of substance benchmarks in the classic prescription Sanpian Decoction.The established quality evaluation method for the substance benchmarks was stable and feasible, which has provided a basis for the quality control of Sanpian Decoction and the follow-up development of related preparations.
Benchmarking ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Glycyrrhizic Acid/analysis* ; Paeonia ; Quality Control ; Thiocyanates