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 observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro. METHODS: Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl₄)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed. RESULTS: High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01). CONCLUSIONS Amygdalin can dramatically alleviate liver fibrosis induced by CCl₄ in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.
Rats ; Male ; Mice ; Animals ; Transforming Growth Factor beta/metabolism* ; Amygdalin/therapeutic use* ; Endothelial Cells/metabolism* ; Olive Oil/therapeutic use* ; Rats, Wistar ; Smad Proteins/metabolism* ; Liver Cirrhosis/metabolism* ; Liver ; Transforming Growth Factor beta1/metabolism* ; Signal Transduction ; Collagen Type I/metabolism* ; Carbon Tetrachloride ; Hepatic Stellate Cells

To discuss the effect of deterioration on the quality of Armeniacae Semen Amarum by observing the changes of macroscopic characteristics, active components and rancidness degrees of Armeniacae Semen Amarum in deterioration process. The traditional macroscopic identification was used to observe, identify and classify the morphologic and organleptic characteristics of Armeniacae Semen Amarum. The contents of amygdalin and fatty oil(two representatives of active components) were detected by HPLC and general rule 0713 in Chinese Pharmacopoeia, respectively. Acid value and peroxide value of the samples were selected as the representative indices of different rancidness degrees, and the general rule 2303 was adopted as the method for quantitative analysis. Then principal component analysis(PCA), partial least square analysis discrimination analysis(PLS-DA) were further utilized to establish the discriminative models of samples with different rancidness degrees, and also to screen out the largest contribution factors. In sensory evaluation, Armeniacae Semen Amarum samples were divided into three groups: non-rancid, slightly-rancid, and noticeably-rancid. The color of seed coat, cotyledon and surface of noticeably-rancid samples was deepened, and the odor differed much from non-rancid samples. Average content of amygdalin and fatty oil in non-rancid samples was 4.12% and 67.77%, respectively, both meeting the requirements of Chinese Pharmacopoeia; and decreased to some extent in slightly-rancid samples. However, the content of amygdalin sharply dropped to 0.074% in noticeably-rancid samples. The acid value and peroxide value were increased significantly with the intensifying of the rancidness degree, from only 1.363 and 0.016 74 in non-rancid samples to 1.865 and 0.023 70 in slightly-rancid samples, even doubled in noticeably-rancid samples(2.167 and 0.033 82). The discriminative models established by PCA and PLS-DA could complete the task of distinguishing the non-rancid samples from noticeably-rancid ones. The contribution degree of amygdalin content as one of the input attributes of discriminative model was higher than 1. Rancidness affected the quality of Armeniacae Semen Amarum, resulting in appearance changes, decrease in content of active components, and increase in acid value and peroxide value. Obviously, noticeably-rancid samples were non-conforming to Chinese Pharmacopoeia and no longer suitable for medicinal use. Rancidness can significantly reduce the quality of Armeniacae Semen Amarum, and even could possibly produce toxicity, which should attach more attention.
Amygdalin ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Semen

This study aimed to establish characteristic chromatogram and content determination method for Chan Taoren formula granules,evaluate the production processes of Chan Taoren formula granules based on the correlation of characteristic chromatogram and the transfer rate of D-amygdalin,and clarify the key control points. The optimized analytical method was carried out on a Waters CORTECS C18 column(4. 6 mm×150 mm,2. 7 μm) with acetonitrile-0. 1% phosphoric acid aqueous solution as the mobile phase at a flow rate of 0. 6 m L·min-1. The detection wavelength was 207 nm,and the column temperature was 20 ℃ ． As compared with the standard decoction of Chan Taoren,there were five characteristic peaks in the decoction pieces,extracts,concentrates,spray-dried powders and formula granules,basically consistent in relative retention time and peak pattern; in addition,the transfer rate of D-amygdalin from Chan Taoren pieces to the formula granules was within the transfer rate range of standard decoction. The average transfer rate of D-amygdalin was 56.65%,72.85%,94.58% and 99.29% respectively in the extraction,concentration,spray drying and granulation processes. Therefore,the factors affecting D-amygdalin in the extraction process were further studied. The results showed that D-amygdalin was easily converted to L-amygdalin in the water extraction process,leading to a low transfer rate of D-amygdalin in this process.D-amygdalin was unstable under alkaline conditions and prone to isomerization. Both liquid to solid ratio and extraction time had significant effects on the extraction rate of D-amygdalin. In this study,the key links in the production process of Chan Taoren formula granules was clarified based on the characteristic chromatogram and the quantity transmission of D-amygdalin,which provided a theoretical basis for production and quality control.
Amygdalin ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Quality Control ; Water

Amarogentin is an efficacious Chinese herbal medicine and a component of the bitter apricot kernel. It is commonly used as an expectorant and supplementary anti-cancer drug. β-Glucosidase is an enzyme that hydrolyzes the glycosidic bond between aryl and saccharide groups to release glucose. Upon their interaction, β-glucosidase catalyzes amarogentin to produce considerable amounts of hydrocyanic acid, which inhibits cytochrome C oxidase, the terminal enzyme in the mitochondrial respiration chain, and suspends adenosine triphosphate synthesis, resulting in cell death. Hydrocyanic acid is a cell-cycle-stage-nonspecific agent that kills cancer cells. Thus, β-glucosidase can be coupled with a tumor-specific monoclonal antibody. β-Glucosidase can combine with cancer-cell-surface antigens and specifically convert amarogentin to an active drug that acts on cancer cells and the surrounding antibodies to achieve a killing effect. β-Glucosidase is injected intravenously and recognizes cancer-cell-surface antigens with the help of an antibody. The prodrug amarogentin is infused after β-glucosidase has reached the target position. Coupling of cell membrane peptides with β-glucosidase allows the enzyme to penetrate capillary endothelial cells and clear extracellular deep solid tumors to kill the cells therein. The Chinese medicine amarogentin and β-glucosidase will become an important treatment for various tumors when an appropriate monoclonal antibody is developed.
Amygdalin ; therapeutic use ; Antibodies, Monoclonal ; therapeutic use ; Antineoplastic Agents ; therapeutic use ; Cell-Penetrating Peptides ; therapeutic use ; Humans ; Iridoids ; therapeutic use ; Prodrugs ; therapeutic use ; beta-Glucosidase ; therapeutic use

Amygdalin, D-mandelonitrile-beta-D-glucoside-6-beta-glucoside, belongs to aromatic cyanogenic glycoside group derived from rosaceous plant seed. Mounting evidence has supported the anti-cancer effects of amygdalin. However, whether amygdalin indeed acts as an anti-tumor agent against breast cancer cells is not clear. The present study aimed to investigate the effect of amygdalin on the proliferation of human breast cancer cells. Here, we show that amygdalin exerted cytotoxic activities on estrogen receptors (ER)-positive MCF7 cells, and MDA-MB-231 and Hs578T triple-negative breast cancer (TNBC) cells. Amygdalin induced apoptosis of Hs578T TNBC cells. Amygdalin downregulated B-cell lymphoma 2 (Bcl-2), upregulated Bcl-2-associated X protein (Bax), activated of caspase-3 and cleaved poly ADP-ribose polymerase (PARP). Amygdalin activated a pro-apoptotic signaling molecule p38 mitogen-activated protein kinases (p38 MAPK) in Hs578T cells. Treatment of amygdalin significantly inhibited the adhesion of Hs578T cells, in which integrin alpha5 may be involved. Taken together, this study demonstrates that amygdalin induces apoptosis and inhibits adhesion of breast cancer cells. The results suggest a potential application of amygdalin as a chemopreventive agent to prevent or alleviate progression of breast cancer, especially TNBC.
Adenosine Diphosphate Ribose ; Amygdalin* ; Apoptosis* ; bcl-2-Associated X Protein ; Breast Neoplasms ; Caspase 3 ; Humans ; Integrin alpha5 ; Lymphoma, B-Cell ; MCF-7 Cells ; p38 Mitogen-Activated Protein Kinases ; Plants ; Receptors, Estrogen ; Triple Negative Breast Neoplasms*

The study aims to develop an LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in urine samples, and to investigate their urinary excretion characteristics in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection (HTLPI). The urine samples were extracted by methanol, and then separated on a Hedera ODS-2 column with a mobile phase of acetonitrile and 5 mmol · L(-1) ammonium acetate buffer solution containing 0.05% formic acid (20:80). Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited good linearity over the concentration range of 0.03 -40 µg · mL(-1). The values on both the occasions (intra- and inter-day) were all within 15% at three concentration levels. No matrix effect and carry-over effect were observed. Amygdalin and paeoniflorin were stable in human urine under different storage conditions. Approximately 79.6% of the administered amount of amygdalin was excreted unchanged in urine within 24 h and which was 48.4% for paeoniflorin. The developed LC-MS/MS method can be applied to evaluate the urinary excretion of amygdalin and paeoniflorin.
Amygdalin ; urine ; Chromatography, Liquid ; Drugs, Chinese Herbal ; Glucosides ; urine ; Humans ; Monoterpenes ; urine ; Tandem Mass Spectrometry

The effect of amygdalin joint hydroxysafflor yellow A (HSYA) on the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and the possible mechanism were studied and explored. Chondrocytes were obtained from endplate of one-month SD rat intervertebral discs and cultured primary endplate chondrocytes. After identification, they were divided into normal group, induced group, amygdalin group, HSYA group and combined group. CCK-8 kit was adopted to detect the proliferation of the endplate chondrocytes. FCM was measured to detect the apoptosis. Real-time PCR method was adopted to observe the mRNA expression of Aggrecan, Col 2 alpha1, Col 10 alpha1, MMP-13 and the inflammatory cytokines IL-1beta. The protein expression of Col II, Col X was tested through immunofluorescence. Compared with the normal group, the proliferation of the endplate chondrocytes decreased while the apoptosis increased (P < 0.05). With down regulation of the mRNA expressions of Aggrecan, Col 2 alpha1 and up regulation of the mRNA expressions of Col 10 alpha1, MMP-13, IL-1beta (P < 0.05), the protein expression of Col II decreased while the protein expression of Col X increased. Compared with the induced group, amygdalin group, HSYA group, the combined group could inhibit the apoptosis and promote the proliferation (P < 0.05). They could increase the mRNA expressions of Aggrecan and Col 2 alpha1 while decrease the mRNA expressions of Col 10 alpha1, MMP-13 and IL-1beta (P < 0.05). They could also enhance the protein expression of Col II while reduce the protein expression of Col X. The effect of the combined group was significantly better than that of amygdalin and HSYA. Amygdalin joint HSYA could inhibit the degeneration of the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and better than the single use of amygdalin or HSYA.
Amygdalin ; pharmacology ; Animals ; Apoptosis ; Cells, Cultured ; Chalcone ; analogs & derivatives ; pharmacology ; Chondrocytes ; drug effects ; Collagen ; metabolism ; Drug Synergism ; Interleukin-1beta ; Intervertebral Disc ; cytology ; Quinones ; pharmacology ; Rats

OBJECTIVETo establish an HPLC method for the determination of ephedrine hydrochloride, D-pseudo-ephedrine and amygdalin in Xiao'er Pingchuan Qutan granule.

METHODPheny ether chromatographic column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.1% phosphoric acid (containing 0.1% three ethylamine) (3:97) as the mobile phase. The UV detection wavelength was at 210 nm, with the flow rate of 1 mL x min(-1), and column temperature was at 35 degrees C.

RESULTThe linearity of ephedrine hydrochloride, D-pseudo-ephedrine and amygdalin ranged between 0.078 60-3.144 microg (r = 1.000 0), 0.103 4-2.068 microg (r = 0.999 7) and 0.430 5-3.157 microg (r = 0.999 8), respectively. Their average recoveries were 98.46% (RSD 1.1%), 103.0% (RSD 1.5%) and 97.15% (RSD 2.1%), respectively.

CONCLUSIONThe method is simple, stable and reliable that it can be used to determine the content of ephedrine hydrochloride, D-pseudo-ephedrine and amygdalin in Xiao'er Pingchuan Qutan granule.

Amygdalin ; analysis ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Ephedrine ; analysis ; chemistry ; Linear Models ; Pseudoephedrine ; analysis ; chemistry ; Reproducibility of Results ; Time Factors

Armeniacae semen (AS) has been considered a toxic herb in the Korean medicine as it contains hydrogen cyanide and amygdalin, especially in its endocarp. Therefore, prebrewed AS that is devoid of endocarp has been traditionally used. In the present study, amygdalin content of the prebrewed AS was significantly lower (2.73 +/- 0.32 microg/ml; p < 0.01) than the content in the extract that contained the endocarps (28.50 +/- 6.71 microg/ml); amygdalin content corresponded to 10% of the extract in the present study. Because of single oral dose toxicity of prebrewed AS according to the recommendation of Korea Food and Drug Administration Guidelines (2009-116, 2009), which was based on single oral dose toxicity study of prebrewed AS, mortality due to toxic principles was significantly reduced. In this study, 2,000 mg/kg of prebrewed AS led to death of 1 female rat and 1 male rat at the end of 2 hr of administration. Based on these results, the 50% lethal dose in both male and female rats was determined to be 9279.5 mg/kg. Seizure, loss of locomotion, and increases in respiration and heart rate were observed as prebrewed AS treatment-related toxicological signs; these signs were restrictedly manifested in the prebrewed AS (2,000 mg/kg)-treated rats. In addition, no changes were observed in body weight, organ weight, gross features, and histopathological parameters with 2,000 mg/kg of AS in both male and female rats. These findings serve as direct evidence that amygdalin in AS is the toxic principle, which can be reduced by the traditional prebrewing method involving the exclusion of endocarp.
Amygdalin ; Animals ; Body Weight ; Female ; Heart Rate ; Humans ; Hydrogen Cyanide ; Korea ; Locomotion ; Male ; Organ Size ; Rats ; Respiration ; Seizures ; Semen ; United States Food and Drug Administration