Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana x tubiflora

Five bufadienolides (1-5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana x tubiflora (Crassulaceae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC50 = 0.4 microM) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.     

Involvement of Na+, K+-ATPase inhibition in K562 cell differentiation induced by bufalin

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na⁺, K⁺ -ATPase prepared from K562 cells in vitro. N⁺, K⁺ -ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of ³H-bufalin and ³H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of ³H-bufalin was considerably higher than ³H-ouabain. The saturated level of ³H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of ³H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N⁺, K⁺ -ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin. © 1994 Wiley-Liss, Inc.     

Bufadienolides from Drimia robusta and Urginea altissima (Hyacinthaceae)

The bufadienolides, 6beta-acetoxy-3beta,8beta,12beta,14beta-tetrahydroxybufa-4,20,22-trienolide (12beta-hydroxyscillirosidin) (1) and 14beta-hydroxybufa-4,20,22-trienolide 3beta-O-[alpha-L-rhamnopyranosyl-[(1-->4)-beta-D-glucopyranosyl]-(1-->3)-alpha-L-rhamnopyranoside] (urginin) (2) were isolated from Drimia robusta and Urginea altissima, respectively.     

Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine ChanSu by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections

ChanSu (toad venom) is a traditional Chinese medicine for the treatment of serious liver and gastric cancers. The major cytotoxic compounds in ChanSu are bufadienolides. In this paper, a strategy combining qualitative LC/MS analysis and quantitative HPLC determination of major bufadienolides was used for global quality control of ChanSu crude drug. Majority of the bufadienolides in methanol extract of ChanSu were unambiguously characterized by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), and by comparing with pure compounds. In addition, eight major bufadienolides were simultaneously determined in one single HPLC run within 30 min with photodiode array detection (DAD). All compounds showed good linearity in a wide concentration range, and their limits of detection (LOD) were around 1 ng. Thus, > 95% of the bufadienolides in ChanSu could be characterized, and > 90% of them were quantitated. The established method is rapid, simple and sensitive, and could be used for the routine analysis of ChanSu crude drug and its preparations. This research sets a good example for the comprehensive quality control of traditional medicine.     

Effects of aplysiatoxin and debromoaplysiatoxin on growth and differentiation of normal human bronchial epithelial cells

The effects of aplysiatoxin and debromoaplysiatoxin on the clonal growth rate, cross-linked envelope formation and plasminogen activator secretion of normal human bronchial epithelial cells were studied. Neither compound was mitogenic over a wide range of concentrations (10^–13 to 10^–7M). Both aplysiatoxin and debromoaplysiatoxin inhibited clonal growth rate with 50% inhibitory concentrations of 3 × 10^–11M and 10^–10M, respectively. Both compounds induced the formation of cross-linked envelopes and increased plasminogen activator secretion with equal potency. These data are similar to those previously obtained with 12-0-tetradecanoylphorbol-13-acetate and teleocidin B and suggest that aplysiatoxin and debromoaplysiatoxin induce terminal squamous differentiation in normal human bronchial epithelial cells.Abbreviations TPA 12-0-tetradecanoylphorbol-13-acetate - NHBE Normal Human bronchial epithelial - ID₅₀ 50% inhibitory concentration (dose) - PA Plasminogen activator - CLE Cross-linked envelope - LHC Laboratory of Human Carcinogenesis     

Effects of sesquiterpenes and triterpenes from the rhizome of Alisma orientale on nitric oxide production in lipopolysaccharide-activated macrophages: absolute stereostructures of alismaketones-B 23-acetate and -C 23-acetate

The methanolic extract from a Chinese herbal medicine, the rhizome of Alisma orientale, was found to exhibit inhibitory activity of nitric oxide (NO) production in lipopolysaccharide (LPS)activated macrophages. Novel triterpenes, alismaketones-B 23-acetate and -C 23-acetate, were isolated from the active extract together with eight sesquiterpenes and eighteen protostane-type triterpenes. The absolute stereostructures of new triterpenes were characterized on the basis of chemical and physicochemical evidence, which included the chemical correlations with known triterpenes. The guaiane-type sesquiterpenes (alismol, orientalols A and C) and protostane- and seco-protostane-types triterpenes (alisols C monoacetate, E-23-acetate, F, H, I, L-23-acetate, and M-23-acetate, alismaketones-B 23-acetate and -C 23-acetate, alismalactone 23-acetate, and 3-methylalismalactone 23-acetate) inhibited LPS-induced NO production (IC50 = 8.4-68 microM). Other triterpenes (alisols A, A monoacetate, B, B monoacetate, E, G, K-23-acetate, and N-23-acetate and 11-deoxyalisol B) also showed the potent inhibitory activity, but they showed cytotoxic effects more than 30 microM (MTT assay). In addition, alismol and alisol F were found to suppress iNOS induction.     

A new and improved synthesis of 19-iodocholesterol 3-acetate

19-Iodocholesterol 3-acetate (VI) was synthesized in a single step by iodo group substitution for hydroxyl using either one of two different reagents:(1) carbodiimidonium methiodide (VIII) or (2) triphenyl-phosphine/N-iodosuccinimide (IX). The yields were as satisfactory as those obtained from the two step iodide replacement of a 19-hydroxy group via the 19-tosyloxy group.The principal intermediate, 19-hydroxy cholesterol 3-acetate (V), was derived in appreciable quantities, and relatively inexpensively, through the Pb (OCOCH₃) photolytic oxidation of the bromohydrin of cholesterol 3-acetate (⁴III) to the epoxide (IV) thence Zn reduction to the 19-hydroxy compound. A specially designed 12 liter flask was of aid in accomplishing the photolysis reaction. Dry column chromatography with the supportive puncture sampling was integral to achieving the good yields and high purity of 19-iodocholesterol (VIII).     

Selective inhibition of the cellular sodium pump by emicymarin and 14ß anhydroxy bufadienolides

Partial inhibition of the sodium pump (Na/K-ATP-ase) by a circulating inhibitor is known to occur in humans. The objectives of this study were to determine the effects of novel bufadienolides lacking an oxygen at C14 on sodium pumps in human erythrocytes and leucocytes, dog kidney and pig brain and to document the importance of the stereochemistry at C17 on the ability to inhibit these sodium pumps. 14α bufadienolides were weak inhibitors of all preparations studied. 3ß-OH,5ß,14ß bufadienolide produced near-total inhibition of dog kidney and pig brain Na/K-ATP-ase. Over the same concentration range, it maximally inhibited the sodium pump of erythrocytes by 70% and leucocytes by 47%. The inhibition profile induced in the leucocyte sodium pump deviated significantly from the simple sigmoidal relationship present in the other preparations over the 3 × 10^?5 to 1 × 10^?7 mol/l concentration range. Allo-emicymarin (17α) was confirmed to be a weak inhibitor of the sodium pump/ATP-ase compared with emicymarin (17ß) but both were weaker inhibitors of the leucocyte sodium pump than that of the other preparations. Molecules with the C14 in the ß configuration are more efficacious than in the α configuration. In the case of emicymarin, the attachment of the furone at C17 in the α configuration results in substantially weaker inhibitory activity than in the beta configuration, seen in most cardenolides and bufadienolides. Unlike ouabain and bufalin that show no specificity of action in these preparations, 3ß- OH,5ß,14ß bufadienolide selectively inhibits the activity of at least one low-prevalence subset of the leucocyte Na/K-ATP-ase.     

Resibufogenin and Cinobufagin Activate Central Neurons through an Ouabain-Like Action

Cinobufagin and resibufogenin are two major effective bufadienolides of Chan su (toad venom), which is a Chinese medicine obtained from the skin venom gland of toads and is used as a cardiotonic and central nervous system (CNS) respiratory agent, an analgesic and anesthetic, and as a remedy for ulcers. Many clinical cases showed that Chan su has severe side-effects on the CNS, causing shortness of breath, breathlessness, seizure, coma and cardiac arrhythmia. We used whole-cell recordings from brain slices to determine the effects of bufadienolides on excitability of a principal neuron in main olfactory bulb (MOB), mitral cells (MCs), and the cellular mechanism underlying the excitation. At higher concentrations, cinobufagin and resibufogenin induced irreversible over-excitation of MCs indicating a toxic effect. At lower concentrations, they concentration-dependently increased spontaneous firing rate, depolarized the membrane potential of MCs, and elicited inward currents. The excitatory effects were due to a direct action on MCs rather than an indirect phasic action. Bufadienolides and ouabain had similar effects on firing of MCs which suggested that bufadienolides activated neuron through a ouabain-like effect, most likely by inhibiting Na⁺/K⁺-ATPase. The direct action of bufadienolide on brain Na⁺ channels was tested by recordings from stably Na_v1.2-transfected cells. Bufadienolides failed to make significant changes of the main properties of Na_v1.2 channels in current amplitude, current-voltage (I-V) relationships, activation and inactivation. Our results suggest that inhibition of Na⁺/K⁺-ATPase may be involved in both the pharmacological and toxic effects of bufadienolide-evoked CNS excitation.     

Anti‐HSV‐1 and HSV‐2 Flavonoids and a New Kaempferol Triglycoside from the Medicinal Plant Kalanchoe daigremontiana

Kalanchoe daigremontiana (Crassulaceae) is a medicinal plant native to Madagascar. The aim of this study was to investigate the flavonoid content of an aqueous leaf extract from K. daigremontiana (Kd), and assess its antiherpetic potential. The major flavonoid, kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 1 ), was isolated from the AcOEt fraction (Kd‐AC). The BuOH‐soluble fraction afforded quercetin 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 2 ) and the new kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside‐7‐O‐β‐d ‐glucopyranoside ( 3 ), named daigremontrioside. The crude extract, Kd‐AC fraction, flavonoids 1 and 2 were evaluated using acyclovir‐sensitive strains of HSV‐1 and HSV‐2. Kd‐AC was highly active against HSV‐1 (EC₅₀ = 0.97 μg/ml, SI > 206.1) and HSV‐2 (EC₅₀ = 0.72 μg/ml, SI > 277.7). Flavonoids 1 and 2 showed anti‐HSV‐1 (EC₅₀ = 7.4 μg/ml; SI > 27 and EC₅₀ = 5.8 μg/ml; SI > 8.6, respectively) and anti‐HSV‐2 (EC₅₀ = 9.0 μg/ml; SI > 22.2 and EC₅₀ = 36.2 μg/ml; SI > 5.5, respectively) activities, suggesting the contribution of additional substances to the antiviral activity.     

Synthesis of feruloyl-myo-inositols and their inhibitory effects on superoxide generation

Ester compounds consisting of ferulic acid and myo-inositol, obtained from rice bran, were synthesized. The inhibitory effects of these feruloyl-myo-inositols on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (O2-) generation were examined using differentiated HL-60 cells. Among the derivatives tested, only 3,4,5,6-tetra-O-acetyl-1,2-di-O-[3-(4'-acetoxy-3'-methoxyphenyl)-2-pr openoyl]-myo-inositol (3) showed a distinct inhibitory activity.     

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na⁺ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 M veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P₄ formation from Ins 1,4,5-P₃.Abbreviations used Ins 1,4,5-P₃ inositol 1,4,5 trisphosphate - Ins 1,3,4,5-P₄ inositol 1,3,4,5-tetrakisphosphate - PMA phorbol 12-myristate 13-acetate     

Characterisation of Pectobacterium carotovorum causing soft rot on Kalanchoe gastonis-bonnierii in Malaysia

Soft rot disease can be found worldwide on fleshy storage tissues of fruits, vegetables and ornamentals. The soft rot Pectobacterium carotovorum subsp. carotovorum (Pcc) is an important pathogen of Kalanchoe spp. and other ornamental plants. The disease occurs on crops in the field, greenhouses and during transit, resulting great economic damages. The economic importance of crop loss by soft rot bacteria varies by severity of the disease and value of the crop. A destructive disease on Kalanchoe gastonis-bonnierii was observed in commercial ornamental plant greenhouses in Cameron highland and Melaka, Malaysia in 2011. Samples suspected to be infested with Pectobacterium spp. were brought to the laboratory. In pathogenicity test, a suspension of 10^6?CFU/ml of strains was able to cause soft rot on leaves and stems. A 434?bp banding pattern on 1% agarose gel was produced in polymerase chain reaction (PCR) amplification of pectate lyase encoding gene (Pel gene). PCR amplification of the intergenic transcribed spacer (ITS) (16S–23S rRNA) ITS region with G₁ and L₁ primers produced two main bands at about 540 and 570?bp. The ITS-PCR products were digested with RsaI restriction enzyme. For discrimination of the P. carotovorum subsp. carotovorum (Pcc) from P. carotovorum subsp. odoriferum (Pco), all isolates subjected to α-methyl glucoside test. All isolates were identified as Pcc based on phenotypic and molecular methods. This is the first report of soft rot disease caused by P. carotovorum subsp. carotovorum on K. gastonis-bonnierii, in Malaysia.     

Improved chromatographic purification of human and bovine type V collagen sub-molecular species and their subunit chains from conventional crude preparations Application to cell-substratum adhesion assay for human umbilical vien endothelial cell

Two human type V collagen sub-molecular species, designated [α1(V)]₂α2(V) and α1(V)α2(V)α3(V), were purified chromatographically from a commercially available preparation, in which cystine-rich collagenous contaminants were contained, with a column packed with Fractogel EMD SO₃⁻. From bovine crude preparations, the [α1(V)]₂α2(V) form free from the collagenous contaminants was purified. Type V collagen subunit chains were isolated from each type V collagen molecule by anion-exchange HPLC with a Bakerbond PEI Scout column. The highly purified human type V collagen molecules and their subunit chains were used to examine the inhibitory effect on human umbilical vein endothelial cell proliferation. It was confirmed that the α1(V) chain has inhibitory activity and it was found that the inhibitory effect of the [α1(V)]₂α2(V) form is stronger than that of the α1(V)α2(V)α3(V) form and that the α3(V) chain has no inhibitory activity.     

Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca⁺⁺-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha₁-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha₁-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha₁-adrenergic actions seems to occur at an early step of the alpha₁-adrenergic action. TPA (10^?11–10^?6M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha₁-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca⁺⁺ ionophore A-23187.     

Anti-tumor promoting effects of sesquiterpenes from Maytenus cuzcoina (Celastraceae)

Ten sesquiterpenoids (1-10), with a dihydro-beta-agarofuran skeleton, were isolated from Maytenus cuzcoina (Celastraceae). Their structures were elucidated on the basis of spectral analysis, including homo- and heteronuclear correlations NMR experiments (COSY, ROESY, HMQC and HMBC), and chemical correlations. The compounds have been tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compounds 1-3, 6 and 7 showed strong inhibitory effects on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA). Their structure-activity relationship is discussed.     

Effects of Peanut-Skin Procyanidin A1 on Degranulation of RBL-2H3 Cells

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m?z 599 [M+Na]⁺. Based on the results of ¹H-NMR, ¹³C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC₅₀ of 20.3 μM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca²⁺ influx from an internal store in RBL-2H3 cells.     

Synthesis and inhibitory activity of new benzimidazole derivatives against Burkitt's lymphoma promotion

As a continuation to our previous work concerning antitumor benzimidazoles, we have synthesized series of new derivatives of 2-(1-benzyl-2-methyl-1H-benzimidazol-5-ylimino)-3-(substituted)-thiazolidin-4-one (6a-e), 3-(2-methyl-1H-benzimidazol-5-yl)-2-substituted-thiazolidin-4-one (9a-f) and we have studied their inhibitory activity against the Epstein-Barr virus-early antigen (EBV-EA) activation introduced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Compound 6d was found to be significantly active and compounds 5a and 6e were also active.     

Syntheses of Jasmonic Acid Related Compounds and Their Structure-Activity Relationships on the Growth of Rice Seedings

Jasmonic acid (JA)-related compounds were synthesized, and their inhibitory activities on rice seedling growth were investigated. Three functions (C-1 CH₂COOH or CH₂COOCH₃, C-2 (Z)-2′-pentenyl or n-pentyl and C-3 ketone or hydroxyl) were essential for exhibiting inhibitory activity in this series of compounds. A dihydro-JA-related compound, 4-acetyl-nonanoic acid, also showed inhibitory activity similar to JA.     

Effect of 1,25-dihydroxyvitamin D3 on induction of scavenger receptor and differentiation of 12-O-tetradecanoylphorbol-13-acetate-treated THP-1 human monocyte like cells

We investigated the effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on the expression of scavenger receptors in human monocytic cell line (THP-1 cells) treated for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces their differentiation into macrophages. The capacity to degrade ¹²⁵I-labeled acetyl low density lipoprotein (LDL) was developed in accordance with macrophage differentiation. The treatment with 10 nM 1,25(OH)₂D₃ for 72 h inhibited the degradation of acetyl LDL by THP-1 macrophages in a dose-dependent manner, suggesting that 1,25(OH)₂D₃ inhibits scavenging function in macrophages. In order to clarify the mechanism of its inhibitory effect on degradation of acetyl LDL, we performed the ligand binding assay using ¹²⁵I-labeled acetyl LDL. Scatchard analysis revealed that 1,25(OH)₂D₃ decreased the number of scavenger receptors without changing the affinity for acetyl LDL. We next examined the effect of 1,25(OH)₂D₃ on the expression of scavenger receptor mRNA. The mRNA of type I scavenger receptor was first detected in THP-1 cells 4 days after the treatment with TPA, the mRNA level increased up to 6 days, and then decreased. The treatment with 1,25(OH)₂D₃ for 72 h dramatically decreased the mRNA levels after the acquisition of macrophage phenotypes as evidenced by nonspecific esterase staining. However, 1,25(OH)₂D₃ did not affect the activity of non-specific esterase nor the induction of interleukin-1β mRNA by lipopolysaccharide in THP-1 macrophages. These findings suggest that 1,25(OH)₂D₃ exclusively decreases the expression of scavenger receptors in TPA-induced THP-1 macrophages without affecting the basic cellular functions as macrophages. © 1995 Wiley-Liss Inc.