[Metabolic and excretory process of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium in rats' urine and feces analyzed by RRLC-QqQ-MSn].

This present study was to investigate the metabolism and excretion of characteristic polyphenols such as flavonoids and coumarins in urine and feces of rats after intragastric administration of ethanol extracts of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium. The urine and feces of rats were collected after intragastric administration of 70% ethanol extracts of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium. Rapid resolution liquid chromatography coupled with quadrupole tandem mass spectrometry (RRLC-QqQ-MSn) was applied to compare the contents of polyphenols in ethanol extract, urine and feces. By comparing with reference substance, 30 polyphenols were identified from the ethanol extracts of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium, including flavone glycosides, flavones, flavonone glycosides, flavonones, flavonol glycosides, polymethoxyflavones, coumarins, and limonoids and so on. The detection of various types of compounds showed differences in contents between the intestinal metabolism and excretion in the feces after systemic circulatory metabolism and renal excretion. The results showed that the polymethoxyflavones and flavonones were primarily excreted through urine, and the flavonone glycosides and limonoids were primarily excreted through feces. However, coumarins were hardly detected in feces and urine, indicating that coumarins may be metabolized in the body.

System Pharmacology-Based Strategy to Decode the Synergistic Mechanism of Zhi-zhu Wan for Functional Dyspepsia.

Functional dyspepsia (FD) is a widely prevalent gastrointestinal disorder throughout the world, whereas the efficacy of current treatment in the Western countries is limited. As the symptom is equivalent to the traditional Chinese medicine (TCM) term "stuffiness and fullness," FD can be treated with Zhi-zhu Wan (ZZW) which is a kind of Chinese patent medicine. However, the "multi-component" and "multi-target" feature of Chinese patent medicine makes it challenge to elucidate the potential therapeutic mechanisms of ZZW on FD. Presently, a novel system pharmacology model including pharmacokinetic parameters, pharmacological data, and component contribution score (CS) is constructed to decipher the potential therapeutic mechanism of ZZW on FD. Finally, 61 components with favorable pharmacokinetic profiles and biological activities were obtained through ADME (absorption, distribution, metabolism, and excretion) screening in silico. The related targets of these components are identified by component targeting process followed by GO analysis and pathway enrichment analysis. And systematic analysis found that through acting on the target related to inflammation, gastrointestinal peristalsis, and mental disorder, ZZW plays a synergistic and complementary effect on FD at the pathway level. Furthermore, the component CS showed that 29 components contributed 90.18% of the total CS values of ZZW for the FD treatment, which suggested that the effective therapeutic effects of ZZW for FD are derived from all active components, not a few components. This study proposes the system pharmacology method and discovers the potent combination therapeutic mechanisms of ZZW for FD. This strategy will provide a reference method for other TCM mechanism research.

Quality Control of the Fuzi Lizhong Pill Through Simultaneous Determination of 16 Major Bioactive Constituents by RRLC-MS-MS.

Fuzi Lizhong pill (FLP) is used to treat gastritis, and the monarch drug of it is Aconiti Lateralis Radix Praeparata (Fuzi, aconite roots) which is a toxic herbal medicine. To better control the safety and quality of FLP, an effective method to analyze the contents of 16 toxic and bioactive components using rapid resolution liquid chromatography-tandem triple-quadrupole mass spectrometer was established. The 16 constituents included aconine, mesaconine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, adenosine, liquiritin, liquiritigenin, glycyrrhizic acid, isoliquiritigenin, 6-gingerol, atractylenolide III, atractylenolide I, atractylenolide II and glycyrrhetic acid. Ideal separation was performed using gradient elution in 13 min by optimized conditions. All the isomerides were isolated to baseline. The improved method with a polarity switch in contiguous time segments could analyze the five types of components, including polar and nonpolar compounds, without decreasing sensitivity. The proposed method was fully validated. The results revealed that contents of six alkaloids from Fuzi were significantly different among the samples. Using the established method and multivariate statistical method, the quality consistency of two dosage forms of FLP from different companies were analyzed. The optimized method could be used for the quality control of FLP and investigate index compound variation between two dosage forms.

Chemotaxonomic Classification Applied to the Identification of Two Closely-Related Citrus TCMs Using UPLC-Q-TOF-MS-Based Metabolomics.

This manuscript elaborates on the establishment of a chemotaxonomic classification strategy for closely-related Citrus fruits in Traditional Chinese Medicines (TCMs). UPLC-Q-TOF-MS-based metabolomics was applied to depict the variable chemotaxonomic markers and elucidate the metabolic mechanism of Citrus TCMs from different species and at different ripening stages. Metabolomics can capture a comprehensive analysis of small molecule metabolites and can provide a powerful approach to establish metabolic profiling, creating a bridge between genotype and phenotype. To further investigate the different metabolites in four closely-related Citrus TCMs, non-targeted metabolite profiling analysis was employed as an efficient technique to profile the primary and secondary metabolites. The results presented in this manuscript indicate that primary metabolites enable the discrimination of species, whereas secondary metabolites are associated with species and the ripening process. In addition, analysis of the biosynthetic pathway highlighted that the syntheses of flavone and flavone glycosides are deeply affected in Citrus ripening stages. Ultimately, this work might provide a feasible strategy for the authentication of Citrus fruits from different species and ripening stages and facilitate a better understanding of their different medicinal uses.

[Study on HPLC fingerprint and chemical constituent difference of different species of Aurantii Fructus Immaturus].

This study is to establish an HPLC fingerprint by HPLC-DAD method and simultaneous quantitative analysis of 17 components of 18 batches of Citrus aurantium and 10 batches of C. sinensis. The separation was performed on an Agilent Poroshell 120 SB-C18 (4.6 mm×100 mm,2.7 µm) column with the gradient elution of methanol-0.1% formic acid water, the flow was 0.6 mL•min⁻¹. The detection wavelength was set at 318 nm. The column temperature was maintained at 30 ℃. The data calculation was performed with similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (Version 2004A) together with SIMCA-P 13.0 software to clarify the differential marker between these two different species of Aurantii Fructus Immaturus. This method has good precision stability and repeatability that could provide basis for quality control and evaluation of Aurantii Fructus Immaturus.

[Research on concentration of 5 different ginsenosides in Panax japonica collected from different cultivated geographic regions].

In this paper, an HPLC-QqQ-MS method for determination of 5 different ginsenosides of Panax japonica collected from different cultivated geographic regions was established. The separation was performed on a Zorbax XDB-C18 (4.6 mm×100 mm, 1.8 µm) column with the gradient elution of acetonitrile (contained 0.1% formic acid)-0.1% formic acid water. The flow rate was 0.5 mL•min⁻¹. The colunm temperature was maintained at 30 ℃. The analytes were detected using electrospray ionization (ESI) in multiple reaction monitoring (MRM) modes. Reaction selected ions were 203.2 for ginsenoside Re, 202.9 for ginsenoside Rg1, 365.0 for ginsenoside Rf, 789.1 for ginsenoside Rd, 360.9 for ginsenoside Ro. Ginsenosides Re, ginsenosides Rg1, ginsenosides Rf, ginsenosides Rd, ginsenosides Ro had good linearity in the ranges of 3.33-66.60 µg (r=0.999 1),2.83-56.54 µg (r=0.999 2), 0.32-6.51 µg (r=0.999 2), 12.55-251.00 µg (r=0.999 3), 0.85-16.90 µg (r=0.999 5), respectively. The results of recovery were among 100.8% to 104.6%, and the values of RSD were blow 3.0%. This method is simple, reliable and accurate, and can provide basis for P. japonica basic research.

Short-time QiBaoMeiRan Formula Treatment Exerts Estrogenic Activities without Side Effects on Reproductive Tissues in Immature Mice.

The Chinese herbal preparation QiBaoMeiRan formula (QBMR) displayed estrogenic effects in ovariectomized rats after long-term administration in a previous study. The uterus and vagina are negatively influenced by estrogens in hormone therapy. While QBMR is known to be a phytoestrogen, its estrogenic effects and safety on reproductive tissues after short-term administration and its mechanism via estrogen receptor (ER) pathway haven't been studied. Here, we characterized its estrogenic effects using immature mice together with in vitro studies for further molecular characterization. Immature mice were treated with QBMR at doses of 1.125, 2.25, and 4.5 g/kg for 7 days. 1.125 and 2.25 g/kg QBMR promoted the growth and development of uterus and vagina, and upregulated ERα and ERß expression in reproductive tissues. QBMR had a stimulatory effect on proliferating cell nuclear antigen in vagina but not in uterus, and was without any influence on ki-67 antigen in uterus and vagina. QBMR significantly induced luciferase expression from the ERα/ß-estrogen response element (ERE) luciferase reporter and upregulated ERα and ERß expressions in MCF-7 cells, which were significantly inhibited by estrogen antagonist ICI182,780. This study demonstrated QBMR exerts estrogenic effects on reproductive tissues without side effects and through ER-ERE-dependent pathway.

Sanmiao formula inhibits chondrocyte apoptosis and cartilage matrix degradation in a rat model of osteoarthritis.

Sanmiao formula (SM) is a basic prescription for the treatment of gouty and rheumatoid arthritis that has been used in China over a long period of history. However, there is no evidence associating SM with the treatment of osteoarthritis (OA). In this study, a characterization of the anti-OA effect of SM was conducted using an in vivo rat model induced by anterior cruciate ligament transection and medial meniscus resection (ACLT plus MMx), together with in vitro studies using chondrocytes for further molecular characterization. Rats subjected to ACLT plus MMx were treated with SM at doses of 0.63, 1.25 and 2.5 g/kg per day for three or six weeks. SM treatment significantly inhibited the histopathological changes of articular cartilage damage and synovial inflammation in the rats following ACLT plus MMx. SM (2.5 g/kg) clearly inhibited chondrocyte apoptosis and prevented cartilage matrix degradation, which was indicated by the increased proteoglycan and collagen content, particularly with regard to type II collagen expression in articular cartilage. Furthermore, SM (2.5 g/kg) markedly inhibited the release of interleukin (IL)-1ß, tumor necrosis factor-α and nitric oxide in serum, while simultaneously increasing the levels of bone morphogenetic protein-2 and transforming growth factor-ß in the circulation. Notably, SM (2.5 g/kg) clearly attenuated the OA-augmented expression of matrix metalloproteinase (MMP)-13 and augmented the OA-reduced expression of tissue inhibitor of metalloproteinase (TIMP)-1 in the knee joints. In addition, SM significantly reduced the proportion of early and late apoptotic and sub-G1 phase cells, and clearly decreased the expression of MMP-13 and increased that of TIMP-1 at the mRNA and protein levels in IL-1ß-induced chondrocytes. These findings provide the first evidence that SM effectively treats OA by inhibiting chondrocyte apoptosis, cartilage matrix degradation and the inflammatory response.

Treatment with qibaomeiran, a kidney-invigorating Chinese herbal formula, antagonizes estrogen decline in ovariectomized rats.

Traditional Chinese medicines (TCM) contain multi-interactive compounds that have been used for treatment of peri-menopausal syndrome and have become a new phytoestrogens resource. The QiBaoMeiRan formula (QBMR), including Polygoni multiflori radix, Angelicae sinensis radix, Achyranthis bidentatae radix, semen Cuscutae, fructus Lycii, Poria, and fructus Psoraleae, has been used clinically for treating osteoporosis in post-menopausal women by virtue of its kidney-invigorating function. However, no evidence base links QBMR to estrogen replacement therapy. In this study, we undertook a characterization of estrogenic activity of QBMR using ovariectomized (OVX) rats. OVX rats were treated with QBMR at doses of 0.875, 1.75, and 3.5 grams/kg per day for 8 weeks. QBMR treatments demonstrated significant estrogenic activity, as indicated by vaginal cornification, reversal of atrophy of uterus, vagina, and mammary gland, and up-regulation of estrogen receptor α (ERα) and estrogen receptor ß (ERß) expression in the reproductive target tissues, where ERß up-regulation was stronger than that of ERα. Meanwhile, treatment with QBMR significantly increased adrenal weight and serum estradiol levels and tended to decrease serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in a dose-dependent manner. Moreover, QBMR significantly decreased weight gain and rectal temperature increase caused by ovariectomy, and the largest changes in rectal temperature were found at the lowest dose. The data suggest that QBMR's estrogenic responses show tissue variation that reflects different affinities of ERs for QBMR components. This study demonstrates that QBMR activity is mediated through estrogenic components and provides an evidence base for QBMR treatment of post-menopausal symptoms.

[Exploring the association rules of clinical application of shenmai injection through text mining].

OBJECTIVE: To explore the rules of clinical application of Shenmai Injection (SI). METHODS: The data sets of SI were downloaded from CBM database by the method of literature retrieved from Jan. 1980 to May 2012. Rules of Chinese medical patterns, diseases, symptoms, Chinese patent medicines (CPM), and Western medicine (WM) were mined out by data slicing algorithm, and they were demonstrated in frequency tables and two-dimension based network. RESULTS: Totally 3 159 literature were recruited. Results showed that SI was most frequently correlated with stasis syndrome and deficiency syndrome. Heart failure, arrhythmia, myocarditis, myocardial infarction, and shock were core diseases treated by SI. Symptoms such as angina pectoris, fatigue, chest tightness/pain were mainly relieved by SI. For CPM, SI was most commonly used with Compound Danshen Injection, Astragalus Injection, and so on. As for WM, SI was most commonly used with nitroglycerin, fructose, captopril, and so on. CONCLUSIONS: The syndrome types and mining results of SI were the same with its instructions. Stasis syndrome was the potential Chinese medical pattern of SI. Heart failure, arrhythmia, and myocardial infarction were potential diseases treated by SI. For CPM, SI was most commonly used with Danshen Injection, Compound Danshen Injection, and so on. And for WM, SI was most commonly used with nitroglycerin, fructose, captopril, and so on.

Treatment with SiMiaoFang, an anti-arthritis chinese herbal formula, inhibits cartilage matrix degradation in osteoarthritis rat model.

A Chinese herbal preparation, SiMiaoFang (SMF), has been used clinically for treating arthralgia by virtue of its anti-inflammatory and pain-relieving activities. However, no evidence base links SMF to anti-osteoarthritis (OA), particularly its link to inhibiting cartilage matrix degradation. In this study, we undertook a characterization of anti-OA activity of SMF using an in vivo rat model induced by anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx) together with in vitro studies with chondrocytes for further molecular characterization. ACLT+MMx rats were treated with SMF at doses of 0.63, 1.25, and 2.5 grams/kg per day for 6 weeks. SMF treatments significantly inhibited cartilage matrix degradation, as indicated by increasing proteoglycan and collagen content, particularly type II collagen expression in articular cartilage, decreasing CTX-II (collagen type II degradation marker), and increasing CPII (collagen type II synthesis marker) in circulation. Moreover, SMF suppressed synovial inflammation and inhibited release of interleukin-1ß (IL-1ß) and tumor necrosis factor-α in serum. The levels of serum prostaglandin E2 and nitric oxide productions were decreased via suppression of the production of cyclooxygenase-2 and inducible nitric oxide synthase, respectively. Importantly, SMF interfered with OA-augmented expression of matrix metalloproteinases (MMPs) -3 and -13 and aggrecanases (ADAMTS) -4 and -5, which are considered to be key enzymes in cartilage matrix degradation, and simultaneously augmented OA-reduced tissue inhibitors of metalloproteinases (TIMPs) -1 and -3 expression in the joints. The largest changes in these parameters were found at the highest dose. Meanwhile, SMF significantly decreased MMP-3 and -13 and increased TIMP-1 and -3 at mRNA and protein levels in IL-1ß-induced chondrocytes. These findings provide the first evidence that SMF effectively treats OA by inhibiting cartilage matrix degradation.

Genotoxicity evaluation of Mequindox in different short-term tests.

Quinoxaline-1,4-dioxides (QdNOs) are the potent heterocyclic N-oxides with interesting biological properties such as antibacterial, anticandida, antitubercular, anticancer and antiprotozoal activities. Here, we tested and compared the mequindox (MEQ) for mutagenic abilities in a battery of different short term tests according to OECD guidelines. When compared with the controls, a strong mutagenicity of MEQ and carbadox (CBX) was observed with an approximate concentration-effect relationship in Salmonella reverse mutation test, chromosome aberration test, unscheduled DNA synthesis assay and HGPRT gene mutation test, in the absence and presence of S(9)-mix. In in vivo micronucleus test, CBX produced significant increase in the proportion of micronucleus formation than MEQ in mice bone marrow cells. From these results, we can conclude that MEQ had a strong genotoxic potential to mammalian cells in vitro as well as in vivo and its mutagenicity is slightly higher than CBX. Our results, for the 1st time, discuss the genotoxic potential of MEQ. These results not only confirm the earlier findings about CBX but also extend the knowledge and awareness about the genotoxic risk of QdNO derivatives.

Two generation reproduction and teratogenicity studies of feeding quinocetone fed to Wistar rats.

To investigate the reproductive toxicity and teratogenic potential of quinocetone, a growth promoting agent, Wistar rats were fed different diets containing 0, 50, 300 and 1800 mg/kg quinocetone or 300 mg/kg olaquindox. Groups of 15 males and 30 females (F(0)) were fed through a 10-week prebreed period as well as during mating, gestation, parturition and lactation. At weaning, 12 males and 24 females of F(1) generation weanlings per group were selected randomly as parents for F(2) generation. Selected F(1) weanlings were exposed to the same diet and treatment as their parents. At the highest quinocetone group, body weights in F(0) and F(1) rats, fetal body weight on day 21 after birth and number of viable fetuses in F(0) and F(1) generation significantly decreased. In teratogenicity study, groups of 12 males and 24 females were fed with the same diets through a 12-week prebreed period and matting periods. Pregnant rats were subjected to cesarean section on GD 20 for teratogenic examination. At the highest quinocetone group, body weights and feed efficiency, fetal body lengths, tail lengths, litter weights and number of viable fetuses significantly decreased. The NOAEL for reproduction/development of quinocetone for rats was estimated to be 300 mg/kg diet.

Two generation reproduction and teratogenicity studies of feeding cyadox in Wistar rats.

To investigate the teratogenic potential and reproductive toxicity of cyadox, a growth promoting agent, Wistar rats (F(0)) were fed with diets containing cyadox (0, 50, 150 and 2500 mg/kg) or olaquindox (150 mg/kg), approximately equivalent to cyadox 5, 15, 250 or olaquindox 15 mg/kg b.w./day across two generations. Half of the pregnant rats (F(0), F(1b)) were subjected to caesarean section on gestational day 20 for teratogenic examination and the other half produced pups F(1a) and F(2a), respectively. At the 250 mg/kg b.w./day cyadox group, body weights of F(1b) pregnant rats and F(2a) on day 21 after birth decreased; fetal body lengths and tail lengths decreased; the number of fetal resorptions increased significantly; litter weights, number of viable fetuses decreased; number of embryo resorptions increased significantly; number of liveborn F(1a), F(1b) and F(2a) decreased. No macroscopic or microscopic change of any significance was found in the reproductive organs. Significant increases in the incidence of cervical ribs or lumbar ribs in F(2a) pups and significant increases of relative organ weight of testis and epididymis in F(1b) were observed at the 250 mg/kg b.w./day cyadox group. The NOAEL for reproduction/development of cyadox for rats was estimated to be 150 mg/kg diet, which was equivalent to approximately 15 mg/kg b.w./day.

A chronic toxicity study of cyadox in Wistar rats.

To investigate the chronic toxicity of cyadox, a growth promoting agent, five groups of Wistar rats (30 rats/group/sex) were fed with the diets containing cyadox (0, 100, 400 and 2000 mg/kg) or olaquindox (400 mg/kg) for 78weeks. There were significant decreases in body weights in both genders during most of the study period in 2000 mg/kg cyadox and 400 mg/kg olaquindox rats. Significant decreases in serum alkaline aminotransferase in the 2000 mg/kg cyadox rats at weeks 26, 52 and 78 were observed. Relative weights of liver and kidney were significantly increased in 2000 mg/kg cyadox and 400 mg/kg olaquindox rats at weeks 26, 52 and 78. A significant increase in relative brain and heart weights in 2000 mg/kg cyadox males was observed. The histopathological examinations revealed that 2000 mg/kg cyadox diet or 400 mg/kg olaquindox diet could induce proliferation of bile canaliculi in the portal area of liver and swelling and fatty degeneration of the proximal renal tubular epithelial cells in kidneys. In conclusion, the target organs of cyadox for rats were liver and kidney. The no-observed-adverse-effect level of cyadox in this study was estimated to be 400 mg/kg diet.

Population pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in chicken based on retrospective data, incorporating first-pass metabolism.

A population pharmacokinetic (PPK) model for enrofloxacin and its metabolite ciprofloxacin in chicken based on retrospective data was developed. Plasma concentrations of enrofloxacin and its metabolite ciprofloxacin were determined in blood samples from chicken administered either enrofloxacin via oral and intravenous routes or ciprofloxacin via intravenous injection. The disposition of enrofloxacin and ciprofloxacin was described simultaneously by an integrated mathematic model. Two compartments were used to describe the enrofloxacin and ciprofloxacin disposition profiles. The formation of ciprofloxacin was through the central compartment of enrofloxacin. The integrated model was estimated with nonlinear mixed effects model (NONMEM). The total clearance of enrofloxacin (CLEN) and ciprofloxacin (CLCP) was 0.613 L/h and 1.15 L/h, respectively. Correlation between CLEN, the central compartment volume of distribution for enrofloxacin (V2) and CLCP was estimated. After intravenous administration of enrofloxacin, the transformation rate of enrofloxacin to ciprofloxacin was 0.429 L/h. The bioavailability factor after oral administration was 0.926, and 12.6% of enrofloxacin after oral administration was transformed to ciprofloxacin via first-pass effect. Pharmacodynamic (PD) evaluation was performed using area under concentration time curve of active moiety from 0 to 24 h and MIC collected from literature. This study is the first one to use PPK method to investigate parent drug and its metabolite disposition and PDs using an integrated model in veterinary medicine.

[HPLC determination of chemical constituents produced in Radix Polygoni Multiflori after processing].

OBJECTIVE: To analysis the changes of two chemical constituents, namely 2, 3-dihydro-3, 5- dihydroxy-6-methyl-4H-pyran-4-one (DDMP) and 5-hydryoxymethyl-furfural (5-HMF) produced in Radix Polygoni Multiflori after processing, with processing time, and to determine the contents of 5-HMF in samples of Radix Polygoni Multiflori and Radix Polygoni Multiflori preparata. METHOD: An HPLC method was applied with a Zobax SB-C18 (3.9 mm x 150 mm, 5 microm) column by a elution using methanol-water (10: 90) as the mobile phase. The detection was set at UV 280 nm. RESULT: The contents of DDMP were increasing with the processing time until 24 hour, followed by a decrease until 60 hour process. The contents of 5-HMF were increasing gradually throughout the 60 hour steaming process. The contents of 5-HMF in 11 samples of Radix Polygoni Multiflori preparata were from 0.013% to 0.101%, and only one in 4 samples of Radix Polygoni Multiflori containing trace amount of 5-HMF. CONCLUSION: The chemical components in Radix Polygoni Multiflori were changed during the processing procedures. Therefore, the processing of Radix Polygoni Multiflori should be controlled and standardized.

[Review about structure-function relationships of anthraquinone derivatives from Radix et Rhizoma Rhei].

It review the structure-function relationship of natural anthraquinone derivatives from Radix et Rhizoma Rhei. The anthraquinone derivatives had many identical activities because they have the identical mother nucleus; but the strength of their activities were different, because they have different substitution groups. The anthraquinone derivatives shown the obvious structure-function relationship in many respects, such as antioxygenation, antibiosis, anticancer, the influence of immunity and so on.

[New chemical constituents from Radix Polygoni Multiflori after processing].

OBJECTIVE: To study the new chemical constituents from Radix Polygoni Multiflori after processing. METHODS: Various kinds of chromatographic methods were used to deparate the chemical constituents from Radix Polygoni Multiflori after processing. Their structures were determined by NMR and MS spectral data. RESULTS: The two new compounds were 2,3-dihydro-3,5-dihydroxy-6-meth-yl-4 (H)-pyran-4-one(I) and 5-hydruoxymethyl-furfuran(II). CONCLUSION: It is the first time that compound I and 1I were isolated from Polygoni.