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The development of single Chinese materia medica is an important direction of technological innovation in the field of Chinese materia medica at present， and the study of its comprehensive intellectual property protection system is of great significance to the intellectual property protection of the whole chain of innovative enterprises of single Chinese materia medica. Based on this， this paper takes the comprehensive protection system of intellectual property of Callicarpa nudiflora constructed by Jiuzhitang Pharmaceutical as a model to conduct empirical research， analyzes the protection forms applicable to intellectual property of Chinese materia medica， such as patents， administrative protection， trademarks， designs and intangible cultural heritages， and discusses the valuable and insufficient aspects of the protection system currently constructed by Jiuzhitang Pharmaceutical and puts forward the following suggestions：①paying attention to patent applications for planting/processing methods of raw medicinal materials， ②emphasizing the protection of geographical indications， authentic medicinal herbs， and new plant varieties， ③actively promoting product and technology upgrades， ④applying for data protection during product iteration， ⑤emphasizing the layout timing of patent and administrative protection， ⑥focusing on improving goodwill， ⑦enhancing awareness of intellectual property protection and promoting deep integration of industry， academia， and research. We hope that innovative enterprises engaged in the development of single Chinese materia medica can learn from the experience of the case， and optimize the strategy to better protect related products.
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OBJECTIVE To establish the fingerprint of the ethanol extract from Callicarpa nudiflora， analyze its anti- respiratory syncytial virus （RSV） activity in vitro， and study the relationship between spectrum and effect. METHODS Using 10%， 30%， 50%， 70% and 90% ethanol as solvent， 20 batches of ethanol extracts from 4 batches of C. nudiflora were prepared. The fingerprints for 20 batches of ethanol extracts from C. nudiflora were mapped by ultra-high-performance liquid chromatography （UPLC）， and the similarity evaluation was conducted by using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints （2012 edition）. The cytopathic effect method and MTT method were used to investigate the in vitro inhibitory activity of the ethanol extracts from C. nudiflora on RSV. Pearson correlation analysis， grey correlation degree and orthogonal partial least squares （OPLS） analysis were used to study the spectrum-effect relationship. RESULTS There were 25 common peaks in 20 batches of ethanol extracts from C. nudiflora， and the similarities ranged from 0.912 to 0.998， and the RSDs of common peak areas were 33.54%-162.28%. The average values of IC50 for RSV of 20 batches of ethanol extracts from C. nudiflora were 9.55-272.23 μg/mL. The results of Pearson correlation analysis， grey correlation analysis and OPLS analysis showed that the Pearson correlation coefficients （P＜0.05） of the common peaks 8， 10， 12， 16， 18-19， 22-24 with pharmacodynamic indicators and regression coefficients were all negative， the correlation coefficients were all greater than 0.6， and the values of variable importance in projection were all greater than 1. CONCLUSIONS Twenty batches of ethanol extracts from C. nudiflora have similar components but significant differences in content， and exhibit different degrees of anti-RSV activity in vitro. The corresponding components of common peaks 8， 10， 12， 16， 18-19， 22-24 may be the characteristic components of anti-RSV of C. nudiflora.
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ObjectiveTo explore the effect of Wuzhishan Callicarpa nudiflora （LHZZ） on the sensitivity of nasopharyngeal carcinoma （NPC） cells to cisplatin （DDP） and the mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the survival rate of NPC HNE1 cells after treatment with different concentration of DDP （0，2，4，8，16，32 mg·L-1） and different concentration of LHZZ （0，25，50，75，100 mg·L-1）. The following groups were designed： control group （normal HNE1 cells），DDP group （8 mg·L-1 DDP，24 h），LHZZ group （50 mg·L-1 LHZZ，24 h），DDP + LHZZ group （8 mg·L-1 DDP + 50 mg·L-1 LHZZ，24 h），DDP + LHZZ + nuclear factor erythroid 2-related factor 2 （Nrf2） activator sulforaphane （SFN） group （8 mg·L-1 DDP + 50 mg·L-1 LHZZ，24 h， 10 μmol·L-1 SFN，24 h）. Then CCK-8 assay was employed to examine cell survival rate，colony formation test the colony-forming ability，flow cytometry and in situ terminal end-labeling（TUNEL） staining cell apoptosis，fluorescent probe 2'，7'-dichlorofluorescin diacetate （DCFH-DA） the level of reactive oxygen species （ROS） in cultured cells，Western blot the expression of apoptosis-related proteins in cells，such as B-cell lymphoma/leukemia-2 （Bcl-2），Bcl-2 associated X protein （Bax），and cysteinyl aspartate specific proteinase-3 （Caspase-3），and Real-time quantitative polymerase chain reaction （Real-time PCR） the expression of Nrf2 and antioxidant response element （ARE） mRNA in cells. ResultThe survival rates of cells treated with different concentration of DDP and different concentration of LHZZ decreased compared with that in the control group （P<0.05）. Compared with the DDP group and the LHZZ group，DDP + LHZZ group demonstrated decrease in cell survival rate，number of cell colonies，and Bcl-2 level，and increase in the apoptosis level and the expression of Bax and Caspase-3 （P<0.05）. However，after the addition of SFN，the Nrf2/ARE signaling pathway was activated and the above variation was inhibited （P<0.05）. In addition，the level of intracellular ROS in the LHZZ group was lower than that in the control group （P<0.05） and the level in the DDP + LHZZ group was lower than that in the DDP group （P<0.05）. Moreover，the ROS level in the DDP + LHZZ + SFN group was higher than that in the DDP+LHZZ group （P<0.05）. ConclusionLHZZ can enhance the sensitivity of DDP-induced NPC apoptosis，possibly by blocking the Nrf2/ARE signaling pathway and inhibiting the level of ROS.
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A phytochemical investigation was carried out on the extract of a medicinal plant Callicarpa nudiflora, resulting in the characterization of five new 3, 4-seco-isopimarane (1-5) and one new 3, 4-seco-pimarane diterpenoid (6), together with four known compounds. The structures of the new compounds were fully elucidated by extensive analysis of MS, 1D and 2D NMR spectroscopic data, and time-dependent density functional theory (TDDFT) calculation of electronic circular dichroism (ECD) spectra, and DFT calculations for NMR chemical shifts and optical rotations.
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Abietanes/isolation & purification , Callicarpa/chemistry , Diterpenes/isolation & purification , Molecular Structure , Phytochemicals/isolation & purification , Plant LeavesABSTRACT
The purpose of the research is to study the bioactive constituents of Callicarpa nudiflora. From the 65% ethanol extract of C. nudiflora leaves, ten compounds were isolated by macroporous adsorption resin, Sephadex LH-20, ODS, silica gel, and preparative HPLC. These compounds were identified as callicapene M6(1), sterebin A(2), isomartynoside(3), crenatoside(4), luteolin-7-O-neohesperidoside(5), apigenin-7-O-β-D-neohesperidoside(6), isoacteoside(7), acteoside(8),(7R)-campneoside I(9), and(7S)-campneoside I(10) on the basis of NMR, HR-ESI-MS, and optical rotation data. Compound 1 was obtained as a new compound. Compounds 2 and 4 were isolated from the genus Callicarpa for the first time. Compounds 9 and 10 were isolated from C. nudiflora for the first time.
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Callicarpa , Chromatography, High Pressure Liquid , Diterpenes , Molecular Structure , Plant LeavesABSTRACT
Objective: To provide the theoretical basis for determining the best harvesting plant organ and harvesting period, and investigate the content of chemical constituents of Callicarpa nudiflora in different plant organs and different growth periods. Methods: The contents of total flavonoids, total phenolic acid and total saponins were determined by ultraviolet spectrophotometry, and the seven components were determined by HPLC. The ANOVA and PCA methods were used to analyze the content of each constituent. Results: The dry extract rate, the contents of total flavonoids, total phenolic acid, total saponins, forsythiaside B, acteoside, isoacteoside, and apigenin-7-O-β-D-glucopyranoside in functional leaves were the highest, while the contents of caffeic acid, galuteolin and luteolin in tender leaves were the highest, and all of them were significantly different from the young shoots (P 0.05). The contents of total phenolic acid, total saponins, forsythiaside B, and acteoside were the highest in the FB period, and there was no significant difference with the EFS period (P > 0.05). The contents of galuteolin and apigenin-7-O-β-D-glucopyranoside were the highest in the earlier fruit maturation (EFM) period and the later fruit maturation (LFM) period, respectively, and there was no significant difference with the EFS period (P > 0.05). The contents of each chemical component were reduced to the minimum at the fruit-drop (FD) period, and it was significantly different from that at the EFS period and the FB period (P < 0.05). According to the comprehensive evaluation model constructed by PCA, the comprehensive score of the EFS period was the highest (F = 3.252), followed by the FB period (F = 3.011). Conclusion: Main chemical constituents of C. nudiflora were significantly different in harvesting parts and growth periods. The contents of main chemical constituents were higher in functional leaves and tender leaves, and the contents of main chemical constituents were higher from FB period to EFS period.
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This article is based on basic data such as field surveys and literature surveys, contrasting and analyzing the distribution of Callicarpa nudiflora by different zoning methods, different data sources, and different spatial scales. The results showed that there were certain differences in the distribution results obtained by using different methods, such as qualitative description, similar ecological environment, and niche model, to divide the distribution of the C. nudiflora, but all of them could reflect the distribution of C. nudiflora to different degrees. Among them, the qualitative description division method has certain advantages in macro guidance in a large scale. The distribution range obtained by the ecological environment similar division method is wider than that obtained by applying the qualitative description method and the niche model method. The results of the zoning of the distribution of the C. nudiflora obtained from different data sources were different. The number and representativeness of the survey data have an impact on the zoning results. Through the analysis of the distribution of different spatial scales, the ecological factors and contribution rates that affect the distribution of C. nudiflora are different in China and in the world. The comprehensive multi-source data analysis showed that C. nudiflora mainly distributed in southern coastal provinces such as Hainan, Guangdong, Guangxi and Fujian in China, and also in Jiangxi, Guizhou, Yunnan, Sichuan, Chongqing, Hunan, Gansu, Taiwan and other provinces. Globally, C. nudiflora are suitable for distribution in Southeast Asia, such as China, Vietnam, Laos, Myanmar, India, etc. There are also potential distribution areas in the southern United States and Mexico.
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Callicarpa , China , Data Collection , Information Storage and Retrieval , VietnamABSTRACT
Objective: To investigate the adsorption kinetics and thermodynamic characteristics of phenylethanoid glycosides (PGs) in the leaves of Callicarpa nudiflora on macroporous resins and provide a reference for the separation and purification of these compounds. Methods: With adsorption and desorption ratio as indexes, optimum types of macroporous resin for purification of PGs were selected from 8 kinds of macroporous resins by static adsorption and desorption tests, and then adsorption kinetics model and adsorption isotherm model of PGs were established to investigate their adsorption processes. Results: SP-825 and SP-207 resin were selected and they have similar adsorption process for PGs. Both of them showed a fast adsorption in 0-60 min, a slow adsorption in 60-360 min, and an equilibrium adsorption stage after 360 min. Adsorption dynamic behavior was well described by quasi-second-order equation of both SP-825 and SP-207 macroporous resins, and adsorption rate was mainly controlled by liquid film diffusion and intraparticle diffusion. Equilibrium adsorption data fitted Langmuir and Freundlich isotherm equations well. Both of the two kinds of resins showed good adsorption properties for PGs, and the adsorption process belongs to favorable adsorption. Conclusions: Both of the kinetic model and thermodynamic model can well describe the adsorption process of SP-825 and SP-207 macroporous resins and the two resins were regarded as excellent adsorption resins for the purification of PGs from the leaves of Callicarpa nudiflora.
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Objective: To evaluate the antidiabetic effect of Callicarpa nudiflora extract in streptozotocin-induced diabetic rats. Methods: The chemical constituents in Callicarpa nudiflora extract were identified by UPLC-Q-TOF-MS. Callicarpa nudiflora extract (0.15 and 0.3 g/kg) was orally administered to streptozotocin-induced diabetic rats for 42 d. The effects of Callicarpa nudiflora extract on body weight, blood glucose, serum insulin, total cholesterol, triglyceride, LDL-C and HDL-C were investigated, and its effect on liver and pancreatic pathology was assessed by histopathological analysis. Moreover, the expression levels of adenosine 5'-monophosphate-activated protein kinase (AMPK), glucose transporter type 4 (GLUT4), phospho-AMPK/AMPK, and p-acetyl-coA carboxylase (P-ACC)/ACC in the skeletal muscles and liver were determined by reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Results: A total of 34 compounds, including 8 iridoids, 14 phenylpropanoids, and 12 flavonoids, were identified from Callicarpa nudiflora extract. Callicarpa nudiflora extract significantly reduced blood glucose and significantly restored all other biochemical parameters to near normal levels, including serum insulin, total cholesterol, triglyceride, LDL-C, and HDL-C. Callicarpa nudiflora extract improved insulin resistance and reversed the damage in the liver and pancreas caused by diabetes. Furthermore, Callicarpa nudiflora extract increased the expression levels of phospho-AMPK, GLUT4, P-ACC, and insulin receptor substrate-1 and decreased the expression level of PPAR毭 in diabetic rats.Conclusions: Callicarpa nudiflora extract improved oral glucose tolerance, lipid metabolism, insulin resistance, and reversed the diabetes-related damage in the liver and pancreas by activating the AMPK-ACC pathway.
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Four new 3, 4-seco-labdane diterpenoids, nudiflopenes J-M, were isolated from the leaves of Callicarpa nudiflora along with six known compounds. The structures of these diterpenoids were determined by comprehensive spectroscopic analysis. All the isolated compounds were evaluated for their inhibitory effects on NO production in LPS-stimulated RPMs and RAW264.7 cells. The results suggest that nudiflopenes J-M and other four known compounds showed significant inhibitory effects against NO production comparable to the positive control dexamethasone.
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Objective To study the chemical constituents of the leaves of Callicarpa nudiflora. Methods The chemical constituents were isolated and purified by column chromatography on silica gel, MPLC, and PHPLC. Their structures were elucidated on the basis of physicochemical properties and spectroscopic analysis. Results Five compounds were isolated from the leaves of C. nudiflora and elucidated as 6’-O-caffeoyl-ajugol (1), luteolin (2), 5,4’-dihydroxy-3,7,3’-trimethoxyflavone (3), luteolin-4’-O-(6’’-E-caffeoyl)- β-D-glucopyranoside (4), and 2α,3α,19α,23-tetrahydroxyurs-12-en-28-ursolic acid (5). Conclusion Compound 1 is a new compound named nudifloside A1.
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OBJECTIVE: To study the chemical constituents of Callicarpa nudiflora. METHODS: The chemical constituents were isolated and purified by column chromatography on silica gel, ODS, Sephadex LH-20 and MPLC. Their structures were elucidated by spectroscopic evidence and compared with those in literature. RESULTS: Nine compounds were isolated and identified as 6-O-caffeoyl ajugol(1), leucosceptoslde A(2), 6-O-caffeoly-β-glucose(3), nudifloside(4), luteolin-7-O-glucoside(5), quercetin 3'-O-β-D-glucoside(6), cistaneside C(7), acteoside(8), and syringalide A 3'-α-L-rhamnopyranoside (9). CONCLUSION: Compounds 1, 2, 6, 7, and 9 are isolated from this plant for the first time.
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Callicarpa nudiflora is the genuine medicinal material in Hainan province, and is successfully approved as a new variety in Chinese Pharmacopoeia 2015. Based on a detailed investigation on the literature reports at home and abroad from 1996 to 2016, we have found that the principal chemical constituents were recognized as phenylpropanoids, flavonoids, phenolic acids, triterpenoids, diterpenoids, iridoids, sterols, etc. The biological activities of chemical constituents from C. nudiflora were mainly studied on the hemostasis, anti-inflammation, anti-bacterial, immune-strengthening, and cytotoxic activities. The present paper systematically summarized the previously phytochemical work of C. nudiflora, and the biological activities were also reviewed briefly. The current paper provides the references for the further quality control, pharmacological action study, and utilization of this medicinal plant.
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Objective: To investigate the synergistic antibacterial effect of Callicarpa nudiflora associated with Vancomycin Hydrochloride on the pneumonia model rats infected by methicillin-resistant staphylococcus aureus (MRSA), and to provide a safer and more effective treatment of clinical ideas for the treatment of infections caused by MRSA. Methods: Totally 80 NIH female rats were randomly divided into eight groups, ten rats as the control group, and 200 μL MRSA (1 × 108 CFU/ mL) bacteria was dropped in the left nasal cavity of the remaining rats to produce infected animal model. The administration with drug respectively by group was continued for 10 d. The common state including activity and intake of water and food were observed and noted. After administration for 10 d, the count of leucocyte, microbial load, and histopathological change of lung were observed. Results: Compared with the model group, the absolute value of leucocyte and microbial load of association of C. nudiflora and Vancomycin Hydrochloride were reduced signicantly (P < 0.05). The pathological damage alleviated significantly. Conclusion: Association of C. nudiflora and Vancomycin Hydrochloride has the synergistic antibacterial effect towards MRSA, which can improve the curative effect of antibiotics and shorten the period of treatment.
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Objective: An UHPLC-MS/MS method was developed for the simultaneous determination of acteoside, isoacteoside, and forsythoside B in plasma of rats and the pharmacokinetic parameters for three phenolic glycosides were calculated as well. Methods: Samples of plasma of rats were obtained at different time after rats were administrated with Callicarpa nudiflora extract (5 g/kg). After the addition of acidification (hydrochloric acid, 0.25 mol/L) and deproteinization by acetonitrile, plasma samples were separated on a Phenomenex® Kinetex C18 column (50 mm × 2.1 mm, 1.7 μm) with gradient elution using acetonitrile-0.005% formic acid as mobile phase. Mass spectrometric detection was carried out by multiple reaction monitoring (MRM) using electrospray ionization in negative ion mode. Results: A good linearity of acteoside, isoacteoside, and forsythoside B was shown in the ranges of 7.77 - 3 880.00 ng/mL (r2 = 0.995 5), 5.04 - 2 520.00 ng/mL (r2 = 0.994 9), and 1.78 - 890.00 ng/mL (r2 = 0.995 1), respectively. The mean extraction recoveries of analytes were in the range of 75.2% - 89.9%, and the intra- and inter-day RSD values were less than 8.8%. The tmax of acteoside, isoacteoside, and forsythoside B was about 30 min, AUC0~t were (93 881.65 ± 18 326.65), (29 204.97 ± 8 499.88), and (15 027.05 ± 3763.82) ng∙min/mL, Cmax were (2 179.00 ± 355.60), (737.57 ± 210.31), and (227.30 ± 48.38) ng/mL, t1/2z were (235.41 ± 117.90), (151.56 ± 49.23), and (161.68 ± 63.92) min, respectively. Conclusion: The method is proved to be simple, rapid, and specific, and to be suitable for the simultaneous determination of acteoside, isoacteoside, and forsythoside B in plasma of rats and the pharmacokinetic study.
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Objective To optimize purification technology of total flavonoids in Callicarpa nudiflora Hook. et Arm. by macroporous adsorption resin. Methods Macroporous resin models including AB-8, D-101, HP-20, HP2MG, were optimized by static adsorption and desorption experiments regarding to adsorption rate and desorption rate of total flavonoids. Purification technology parameters of total flavonoids were optimized by single factor test. Results HP-20 macroporous resin presented the best purification efficiency,the optimum purification conditions were that taking 4. 46 mg·mL-1 of total flavonoidsat pH 3. 0, loading at 3 BV·h-1, washed with 3BV of water at 3 BV·h-1,then eluted with 4 BV 75% ethanol at 2 BV·h-1, finally obtaining the total flavonoids from the dry extract of Callicarpa nudiflora Hook. et Arn. with the purity of 47. 4%. Conclusion HP-20 macroporous resin is suitable for preliminary purification of total flavonoids in Callicarpa nudiflora Hook. et Arn.
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Objective: To develop a UPLC method for the stimultaneous determination of five active ingredients in Callicarpa nudiflora. Methods Analysis was performed on an Agilent Eclipse XDB-C18 column (100 mm ×3.0 mm, 1.8 μm) eluted with acetonitrile (A) and 0.1% methanoic acid (B) in a gradient program. The flow rate was 0.7 mL/min, the detection wavelength was 350 nm, and the column temperature was 40°C. Results Luteoloside, acteoside, luteolin-4'-O-β-D-glucopyranoside, luteolin, and 5, 4'-dihydroxy-3, 7, 3'-trimethoxyflavone showed a good linearity in the ranges of 2.44-122.0 μg/mL (r = 0.999 8), 9.06-453.0 μg/mL (r = 0.999 9), 4.42-221.0 μg/mL (r = 0.999 9), 3.36-168.0 μg/mL (r = 0.999 8), and 2.52-126.0 μg/mL (r = 0.999 8). The average recoveries, measured at three concentration levels, varied from 98.5%-100.8%. Conclusion The method is simple, accurate, and can be used for the quality control of C. nudiflora.
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From the n-butanol extract of the leaves of Callicarpa nudiflora,thirteen compounds were isolated by repeated column chromatography with silica gel,C_(18) and Sephadex LH-20.Their structures were identified by spectroscopic (~1H,~(13)C NMR and so on) and chemical methods as luteolin (1),luteolin-7-O-β-D-glucopyran coside (2),apigenin (3),5,7,4'-trihydroxy-3'-methoxyflavone (4),luteolin-3'-O-β-D-glucopyranoside (5),api genin-7-O-β-D-glucopyranoside (6),luteolin-7-O-(6-trans-caffeoyl)-β-D-glucopyranoside (7),luteolin-7-O-(6-trans-feruloyl)-β-D-glucopyranoside (8),arjunglucoside I(9),luteolin-7-O-(6-p-coumaryl)-β-D-glucopyranoside (10),forsythoside B (11),isomartynoside (12),deacylisomartynoside B (13).Among them,compound 11 was isolated from this plant for the first time and compounds 4,7-10,12-13 were isolated from this genus for the first time.
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Objective To determine the content of luteolin in Callicarpa nudiflora Hook.et Arn..Methods HPLC was performed with chromatographic conditions as follows:Alltima C18 column(250 mm? 4.6 mm,5 ? m)with column temperature at 25 ℃,the mobile phase of methanol-water-phosphate(55:45:0.4),flow rate at 1 mL/min,and the detection wavelength at 348 nm.Results The linearity of luteolin was good in the range of 0.032 46～ 0.129 84 ? g(r=0.999 5).The average recovery was 99.52 % and RSD was 1.58 %.Conclusion The method is simple,feasible and reproducible,and can be used to control the quality of Callicarpa nudiflora Hook.et Arn..