Preparation of kakkatin derivatives and their anti-tumor activity

BACKGROUND Modern pharmacological studies have confirmed that plant-derived compounds from Puerariae flos(PF)has significant biological activities against liver damage,tumors and inflammation.Kakkatin is an isoflavone polyphenolic compound isolated from PF flower.However,the effect of kakkatin and its derivatives on anti-tumor has not been well explored.AIM To design and synthesize a kakkatin derivative[6-(hept-6-yn-1-yloxy)-3-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one(HK)]to explore its anti-tumor biological activity.METHODS Hept-6-yn-1-yl ethanesulfonate was introduced to replace hydrogen at the hydroxyl position of kakkatin phenol,and the derivative of kakkatin was prepared;the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect cell viability,a clone formation assay was adopted to detect cell proliferation,apoptosis,necrosis,and cell cycles were analyzed by Annexin V/propidium iodide staining and flow cytometry.Cell migration and invasion ability were evaluated by cell scratch assay and transwell assay.The potential mechanism of HK on hepatocellular carcinoma(HCC)SMMC-7721 cells was explored through network pharmacology and molecular docking,and finally real-time PCR assays was used to verify the potential targets and evaluate the biological activity of HK.RESULTS Compared with kakkatin,the modified HK did not significantly increase the inhibitory activity of gastric cancer MGC803 cells,but the inhibitory activity of HCC SMMC-7721 cells was increased by about 30 times,with an IC50 value of 2.5μM,and the tumor inhibition effect was better than cisplatin,which could significantly inhibit the cloning,invasion and metastasis of HCC SMMC-7721 cells,and induce apoptosis and G2/M cycle arrest.Its mechanism of action is mainly related to the upregulation of PDE3B and NFKB1 target proteins in the cAMP pathway.CONCLUSION HK have a significant inhibitory effect on HCC SMMC-7721 cells,and the targets of their action may be PDE3B and NFKB1 proteins in the cAMP pathway,making it a good lead drug for the treatment of HCC.

Screening for Anti-tumor Activity of Fractions from Buddleja officinalis Maxim

[Objectives]The anti-tumor activity of fractions from Buddleja officinalis Maxim.by petroleum ether,ethyl acetate,n-butanol and water solvent was studied.[Methods]The ethanol extract from B.officinalis Maxim.was extracted and then concentrated with petroleum ether,ethyl acetate,n-butanol and water,respectively,and the extracts were obtained.The inhibitory effects of the four different fractions on the growth of three tumor cell lines in vitro were detected by CCK-8 method,and the median inhibitory concentration(IC 50 value)was calculated.[Results]The four fractions inhibited the growth of the three tumor cell lines in vitro,among which the n-butanol fraction had the best anti-tumor activity.The IC 50 values of the n-butanol fraction on human gastric cancer(SGC-7901),human breast cancer(MCF-7)and human liver cancer(BEL-7404)cell lines were 0.08,1.58 and 0.12 mg/mL,respectively.[Conclusions]Petroleum ether,ethyl acetate,n-butanol and water fractions from the ethanol extract of B.officinalis Maxim.had certain anti-tumor effects,and the n-butanol fraction had the best anti-tumor activity.

3D-QSAR Studies on the Anti-tumor Activity of N-Aryl-salicylamide Derivatives

In the present work,comparative molecular field analysis(CoMFA)techniques were used to perform three-dimensional quantitative structure-activity relationship(3D-QSAR)studies on the anti-tumor activity(pHi,i=1,2,3,4)of N-aryl-salicylamide derivatives against four cancer cell lines,including A549,MCF-7,SGC-7901,and Bel-7402.12 compounds were randomly selected as the training set to establish the prediction models,which were verified by the test set of 5 compounds containing template molecule.The contributions of steric and electrostatic fields to pH1,pH2,pH3,and pH4 were 23.8% and 76.2%,20.1% and 79.9%,18.7% and 81.3%,and 14.3%and 85.7%,respectively.The cross-validation(Rcv 2)and non-cross-validation coefficients(R2)were 0.826 and 0.963 for pH1,0.867 and 0.974 for pH2,0.941 and 0.989 for pH3,and 0.797 and 0.961 for pH4,respectively.The CoMFA models were then used to predict the activities of the compounds,and it was found that the models had strong stability and good predictability.Based on the CoMFA contour maps,some key structural factors responsible for the anticancer activity of the series of compounds were revealed.The results provide some useful theoretical references for understanding the mechanism of action,designing new N-aryl-salicylamide derivatives with high anti-tumor activity,and predicting their activities.

Antiviral Activity of the Effective Monomers from Folium Isatidis Against Influenza Virus in Vivo

In order to evaluate the anti-influenza virus activity of the effective monomer from Folium Isatidis (FI) in vivo,we established mice model with viral pneumonia and divided them into 3 different dose groups,then observed their lung indexes,pulmonary pathological changes,pulmonary virus hemagglitination titers,living time and death rates.The results showed that the monomer could reduce the pulmonary index from 2.64 to 1.93,1.63 and 1.40 (P<0.01) and decrease the hemagglitination titer from 1.15 to 0.84,0.70 and 0.59 (P<0.01).In addition,different groups of FI could significantly lessen the mortality rate from 100% to 30%,25% and 15%,and prolong the living time from 5.1d to 6.5d,8.4d and 8.9d respectively(P<0.01).The high dose (75 mg/kg/d) has the similar effect with 100 mg/kg/d dose of virazole(P>0.05),and more effective than 200 mg/kg/d dose of antiviral liquor (P<0.05).

Synthesis and Anti-tumor Activity of Novel Amide Derivatives of Ursolic Acid

Ursolic acid was modified at C3 and C28 position to obtain fourteen derivatives including twelve novel compounds, and their chemical structures were characterized by IR, ^1H NMR and MS. Cell growth inhibitory effects of the derivatives against Hela cell were evaluated by MTT assay. All these derivatives were found to have stronger cell growth inhibitory than their parent compound, ursolic acid. The derivatives with a substituted acetyl group at C3 hydroxyl group show better activities than those with an unsubstituted hydroxyl group.

<i>In Vivo</i>Antioxidant Activity of Fucoxanthin on Obese/Diabetes KK-<i>A^y</i>Mice

Dietary intake of 0.1% fucoxanthin significantly reduced lipid hydroperoxide levels of liver and abdominal white adipose tissue (WAT) of obese/diabetes KK-Ay mice. The fucoxanthin supplementation also significantly reduced blood glucose level and hepatic lipid contents of the mice. Oxidative stress is known to be induced in hyperglycemia and high fat conditions. Therefore, in vivo antioxidant activity of fucoxanthin found in the present study could be attributed to its anti-diabetic effect and its decreasing effect on hepatic lipids. On the other hand, little effect of fucoxanthin on lipid hydroperoxide levels was found in normal ICR mice. Although the content of fucoxanthin metabolites in the abdominal WAT of KK-Ay mice was about 50 times higher that in the liver, there was little difference in its In Vivo antioxidant activity between in the liver and in the abdominal WAT. These results suggest that well-known ability of fucoxanthin to scavenge active oxygen species and/or free radicals would not be a main reason to explain its In Vivo antioxidant activity.

Characterization and Anti-tumor Activity of Glycopeptides from Ganoderma sinensis

The water-soluble part（GS） of Ganoderma sinense Zhao, Xu et Zhang was divided into high molecular（GS-H） and low molecular（GS-L） parts by Cellulose Super Filtration, and GS was also fractionated into four fractions, GS-1, 2, 3, and 4 by ethanol precipitation according to their molecular weights. Chemical analysis shows that GS and GS-1, 2, 3, 4 were complexes of polysaccharide and peptide. The fractions with molecular weights over 4000, GS-1, 2, 3, and GS-H show anti-tumor activities, however, the fractions with molecular weights lower than 4000, GS-4, and GS-L have no anti-tumor activity, indicating that the anti-tumor activity of Ganoderma Sinensis was caused by glucopeptides with molecular weight ranging from 4000 to 20000. Two purified glucopeptides, GS-6b and GS-7b were obtained from GS-H by ion-exchange and gel-permeation chromatography. Their molecular weights, glycosidic linkages, and configurations were detected by means of IR spectrum, sugar composition analysis, and methylation analysis. The polysaccharide parts of GS-6b and GS-7b had glucan backbone consisting of β-1→3 Glc, and side chain containing glucosyl, mannosyl, fucosyl, xylosyl, galactosyl, and glucuronic acid residues attached on 1-2, 1-4, 1-6 positions of the backbone of GS-6b, or 1-6, 1-4 positions of the backbone of GS-7b. The peptide parts in GS-6b and GS-7b were composed of 10 kinds of amino acids, including Asp, Ser, Arg, Gly, Thr, Pro, Ala, Val, Met, and Lys.

Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.

Anti-tumor activities of macromolecular fractions of fresh gecko vivo and their induction of Bel-7402 cell differentiation

Objective:To investigate the anti-tumor effect of macromolecular fractions of fresh gecko (M-AG) in vivo and their differentiation-inducing activity in Bel-7402 cells in vitro.Methods:An H22 hepatocarcinoma-bearing mouse model was used to evaluate the anti-tumor activity of M-AG samples.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was applied to analyze cell viability.Cell morphology was observed by phase contrast microscopy.The quantity of the alpha-fetoprotein was detected by a radioimmunoassay.Chromatometry was used to assay the albumin quantity.Activities of alkaline phosphatase and γ-glutamyl trans-peptidase were measured by biochemical methods.Finally,western blotting was applied to assess proteins in the mitogen-activated protein kinase (MAPK) signaling pathway.Results:Macromolecular fractions of fresh gecko exerted a significant anti-tumor effect in mice.The inhibition rate of tumor growth was 63% in the moderate M-AG dose group.Cells treated with M-AG displayed a differentiated state.The treatment lowered alphafetoprotein secretion and significantly decreased the activities of γ-glutamyl trans-peptidase and alkaline phosphatase in Bel-7402 cells.In contrast,M-AG increased the amount of albumin in the cell culture medium.All biochemical indices demonstrated that M-AG induced Bel7402 cell differentiation.Western blotting showed no changes in the quantities of extracellular signal-regulated kinase (ERK) 1/2,p38MAPK,or c-Jun N-terminal protein kinase 1/2.However,M-AG significantly activated the phosphorylation of ERK1/2 in a dose-dependent manner.In addition,M-AG had no significant influence on the expression of nuclear factor-kappa B.Conclusion:Macromolecular fractions of fresh gecko has an anti-tumor activity in H22 hepatocarcinoma-bearing mice in vivo and inhibits Bel-7402 cell proliferation in vitro by inducing cell differentiation related to activation of ERK1/2.

Synthesis and Anti-tumor Activity of Novel Glycyrrhetinic Acid Derivatives

Twenty-five derivatives of glycyrrhetinic acid（GA） modified on the A-ring,at C30 and C11 positions were synthesized.Their in vitro cytotoxicity against various cancer cell lines[henrietta lacks strain of cancer cells（HeLa）,human hepatocellular liver carcinoma cells（HepG2） and human gastric carcinoma cells（BGC-823）] was evaluated by standard MTT[3-（4,5-dimethyl-2-thiazol-yl）-2,5-diphenyl-2H-tetrazolium bromide] assay.All the tested derivatives were found to have stronger cell growth inhibitory than their parent compound GA.Among them,compounds 3a,5a,and 8d have similar activity on HeLa cell line,and compound 8a has similar activity on HeLa,HepG2 and BGC-823 cell lines as Gefitinib.

Anti-tumor Activity of Isovanillin and Its Molecular Mechanism

Taking isovanillin and the important products synthesized from raw materials of isovanillin as objects,this paper reviews its pharmacological effects and molecular mechanisms,including inhibiting tumor angiogenesis,blocking cancer cell self-repair,inducing cancer cell DNA fragmentation,cytotoxicity,inhibiting tumor growth,regulating brain nerves,inhibiting renin and other pharmacological effects,etc.,which provide new ideas for the in-depth research and development of isovanillin.

Establishment of Quality Standard for Freeze-dried Tablets of Polygonatum sibiricum and Study on Anti-tumor Activity of Diosgenin

[Objectives]The paper was to establish the quality standard for freeze-dried tablets of Polygonatum sibiricum and to explore the antitumor activity of its extract diosgenin.[Methods]Taking freeze-dried powder samples of P.sibiricum from 4 different producing areas as materials,and referring to the quality standard of P.sibiricum in the Chinese Pharmacopoeia(2020 edition),the contents of total ash,moisture,extract,total sugar and diosgenin were determined by total ash determination method,drying method,hot dipping method,0.2%anthrone-sulfuric acid method and HPLC,respectively.The antitumor activities of diosgenin against A431(human epidermal carcinoma cells),H1975(human lung adenocarcinoma cells)and Ramos(human B lymphoblastoma cells)were investigated by MTT assay.[Results]The moisture content of the samples was 2.8%-4.7%(not more than 18.0%);the total ash content was 1.9%-3.4%(not more than 4.0%);the ethanol-soluble extract content was 72.99%-78.99%(not less than 45.0%);and the total sugar content was 7.95%-9.94%(not less than 7.0%).The lowest content of diosgenin was 0.18%,and diosgenin was significantly resistant to A431.[Conclusions]The content determination method established in the study is simple,accurate and reproducible.

Synthesis,Crystal Structure and Anti-tumor Activity of Ethyl 3-(4-methoxyphenyl)-4-oxo-3,3a,4,6-tetrahydro-1H-furo[3,4-c]pyran-3a-carboxylate

The title compound（ethyl 3-（4-methoxyphenyl）-4-oxo-3,3a,4,6-tetrahydro-1H-furo[3,4-c]pyran-3a-carboxylate） has been synthesized,and its crystal structure was characterized by X-ray single-crystal diffraction.The crystal belongs to monoclinic,space group P21/n,with a = 10.124（4）,b = 11.754（4）,c = 13.792（5） ,β = 111.533（3）o,V = 1526.6（10） 3,Z = 4,C17H19O6,Mr = 319.32,Dc = 1.389 g/cm3,F（000） = 676,λ（MoKα） = 0.71073 ,μ = 0.105 mm-1,R = 0.0660 and wR = 0.2027 for 2993 observed reflections（I 2σ（I））.The compound shows potent anti-tumor activity in vitro.

Activity Screening Study on the Anti-tumor Effects of Extracts from Mahoniae caulis

[Objectives]To explore the anti-tumor activity of the extracts of petroleum ether,ethyl acetate,n-butanol and aqueous solution from Mahoniae caulis.[Methods]The extracts were extracted with petroleum ether,ethyl acetate,n-butanol and aqueous solution respectively,and then concentrated.The inhibitory effects of these extracts on the growth of three tumor cell lines in vitro were detected by CCK-8 method,and the IC 50 value was calculated.[Results]The four extracts inhibited the growth of the three tumor cell lines in vitro,among which the n-butanol extract had the best anti-tumor activity.The IC 50 values of the n-butanol extract on human gastric cancer(SGC-7901),human breast cancer(MCF-7)and human liver cancer(BEL-7404)cell lines were 0.23,0.25 and 0.58 mg/mL,respectively.[Conclusions]The ethanol extract of Mahoniae caulis under petroleum ether,ethyl acetate,n-butanol and aqueous solution had certain anti-tumor effect,and n-butanol extract had the best anti-tumor activity.

Synthesis and Anti-tumor Activity Study of Shogaol Analogues

As one of the largest global public health problems, cancer greatly endangers human health. Researchers have been committed to discovering anticancer drugs with high efficiency and low toxicity. Shogaol, a kind of ingredient from ginger with high anti-tumor activity, has attracted our attention. This study was an investigation on the composition and structure-activity relationship of different analogues of shogaol, with the purpose to screen for shogaol compounds which had higher anti-tumor activity and could be easily synthesized. We chose zingerone as the starting material to synthesize shogaol through four-step reactions without protective group, as a result, 31 analogues were synthesized for anti-tumor activity detect. The biological test results showed that： the missing of the unsaturated ketone and the phenolic hydroxyl group reduced the anti-tumor activity of shogaol; the site of phenolic hydroxyl had no significant influence on the anti-tumor activity, the indicating that the larger the steric hindrance of substituent, the lower the activity, but the higher the activity of cyclobutyl substituted derivatives.

Investigation of the Antimicrobial Activity and <i>in Vivo </i>Cytotoxicity of <i>Diospyros malabarica </i>(Desr.) Kostel. Fruit Extracts

Mankind is facing an unprecedented threat of existence due to the antibiotic resistance developed by bacteria. The unripe fruits of Diospyros malabarica (Desr.) Kostel. (family: Ebenaceae) can be considered as one of the natural sources to tackle this issue. The present study is designed to assess the antimicrobial activity of D. malabarica seed and flesh ex-tracts. Herein, D. malabarica extracts were prepared using polar solvents (i.e., water and 70% ethanol) and their antimicrobial activity as well as in vivo toxicity was investigated. Their antibacterial activity was investigated against gram positive (Bacillus subtilis) and gram negative (Escherichia coli DH5α, and Salmonella typhi) bacteria at different time points. All the extracts showed the highest antibacterial activity after 2 hours of incubation. The aqueous seed extract showed the maximum zone of inhibition (i.e., ~13 mm) against Bacillus subtilis with a minimum inhibitory concentration (MIC) value of 2 μg/μl. The an-tibacterial propensity was also confirmed through trypan blue dye exclusion assay, CellToxTM Green assay, and lipid peroxidation (LPO) assay. On the other hand, the etha-nolic seed extract demonstrated higher antifungal activity through inhibition of mycelial growth. All the extracts showed excellent hemocompatibility against both human and rat red blood cells (RBCs). They also did not show any toxicity to rat liver and kidneys. Taken together, this study demonstrates that the aqueous and ethanolic extracts of D. malabarica seed and flesh could be an effective source of natural antimicrobial agents with no cytotox-ic activity.

Purification and Characterization of Flammulin,a Basic Protein with Anti-tumor Activities from Flammulina velutipes

Aim To purify and characterize flammulin, a basic protein with anti-tumoractivities. Methods Ammonium sulfate, ethanol fractionation and column chromatography were used forseparation and purification. Electrophoretic analysis, amino acid analysis, and MS of flammulin werecarried out. Results Flammulin was purified to electrophoretic homogeneity and crystallized. With amolecular mass of 19891.13 Da, pI 8.9, λ_(max) = 276 - 278 nm, λ_(min) = 250 nm, flammulin wascharacterized by its lack of methionine. Fingerprint mapping of flammulin was determined by MALDI-MSfollowing in-gel protease digestion; no close matches were identified. Conclusion Flammulin waspurified to electrophoretic homogeneity, and its characteristics are discussed for the first time.

Lrp1 in osteoblasts controls osteoclast activity and protects against osteoporosis by limiting PDGF–RANKL signaling

Skeletal health relies on architectural integrity and sufficient bone mass, which are maintained through a tightly regulated equilibrium of bone resorption by osteoclasts and bone formation by osteoblasts. Genetic studies have linked the gene coding for low-density lipoprotein receptor-related protein1(Lrp1) to bone traits but whether these associations are based on a causal molecular relationship is unknown. Here, we show that Lrp1 in osteoblasts is a novel regulator of osteoclast activity and bone mass.Mice lacking Lrp1 specifically in the osteoblast lineage displayed normal osteoblast function but severe osteoporosis due to highly increased osteoclast numbers and bone resorption. Osteoblast Lrp1 limited receptor activator of NF-κB ligand(RANKL) expression in vivo and in vitro through attenuation of platelet-derived growth factor(PDGF-BB) signaling. In co-culture, Lrp1-deficient osteoblasts stimulated osteoclastogenesis in a PDGFRβ-dependent manner and in vivo treatment with the PDGFR tyrosine kinase inhibitor imatinib mesylate limited RANKL production and led to complete remission of the osteoporotic phenotype. These results identify osteoblast Lrp1 as a key regulator of osteoblast-to-osteoclast communication and bone mass through a PDGF–RANKL signaling axis in osteoblasts and open perspectives to further explore the potential of PDGF signaling inhibitors in counteracting bone loss as well as to evaluate the importance of functional LRP1 gene variants in the control of bone mass in humans.

Synthesis and cytotoxic activity of novel curcumin analogues

Five novel curcumin analogues bearing different substituents at 4-position of phenyl group were synthesized. Their structures were confirmed by NMR and HRMS spectrum. Their cytotoxic activities against six tumor cell lines were tested by the standard MTT assay in vitro. The results indicated that four analogues （1A-1C, 1E） with solubilizing moieties showed selective potent cytotoxicity against HepG2, HeLa and CT26 cell lines, and analogue 1A and 1C exhibited more potent cytotoxicity than curcumin against CT26 cell line. It was suggested that introduction of appropriate substituents to 4-position of phenyl group might be a potential option for structural modification of curcumin.

Synthesis and Primary Research on Antitumor Activity of Three New Panaxadiol Fatty Acid Esters

For making use of Ginseng resources that exhibit an antitumor activity and for finding new anticancer drugs, three new fatty acid ester compounds： 3/β-acetoxy panaxadiol （ Ⅰ ）, 3β-palmitic acid aceloxy panaxadiol （ Ⅱ ） , and 3β-octadecanoic acid aceloxy panaxadiol（ Ⅰ , Ⅱ, and m） were synthesized with panaxadiol, diacetyl oxide, palmityl chloride and stearyl chloride, and their structures were determined via MS, ^13C NMR, IR, TLC, and so on. The molar yields of the three compounds are 75.14%, 79. 08%, and 72. 57%, respectively. Meanwhile, the antitumor activity of the three new panaxadiol fatty acid ester derivatives and panaxadiol was compared by using the method of MTT. Tumor cell used was Vero cell line. Positive control was 5-FU, blank was an RPMI1640 culture medium, negative control was an RPMI1640 culture medium and the solvent for drugs to be tested. Compound Ⅰ has the strongest antitumor activity followed by panaxadiol; compounds Ⅱ and Ⅲ have similar and weakest antitumor activities. Furthermore, the antitumor activities of the panaxadiol fatty acid ester derivatives show positive correlation with the concentration of the test group, but show no relationship with the molecular weight of fatty acid. The methods that are used to synthesize the three compounds with high yields and strong antitumor activities are simple and show a great potential for meeting the needs of industrial manufacture of these drugs.