Isolation and identification of ent-kaurane-type diterpenoids from Rabdosia serra (MAXIM.) HARA leaf and their inhibitory activities against HepG-2, MCF-7, and HL-60 cell lines

A phytochemical investigation was conducted on Rabdosia serra leaf in this work. A new ent-kaurane-type diterpenoid named 6β,14α-dihydroxy-1α,7β-diacetoxy-7α,20-epoxy-ent-kaur-16-en-15-one, together with 6 known compounds were identified, including parvifolin G, effusanin E, lasiodin, nodosin, β-sitosterol, and stigmasterol. It was the first time that parvifolin G and effusanin E were found in R. serra. The assay of inhibition activity against HepG-2, MCF-7, and HL-60 cell lines indicated that 10 compounds (including rosmarinic acid, methyl rosmarinate and pedalitin which were isolated previously), except parvifolin G and stigmasterol, exhibited cytotoxicity against the tested tumour cells. The tumour inhibitory effects of ent-kaurane-type diterpenoids (except parvifolin G) were more effective than those of sterols and phenolics. Both 6β,14α-dihydroxy-1α,7β-diacetoxy-7α,20-epoxy-ent-kaur-16-en-15-one and lasiodin (IC₅₀ < 5 μM) displayed strong cytotoxicity against the tested tumour cells, indicating the potential for new chemotherapeutic drugs.     

Inhibition effect of procyanidins from lotus seedpod on mouse B16 melanoma invivo and in vitro

Significant growth inhibition effects of procyanidins from lotus (Nelumbo nucifera Gaertn.) seedpod (LSPCs) on mouse melanoma B16 were found both in vivo and in vitro. In vivo treatment with LSPCs inhibited tumour growth in C₅₇BL/6 J mice by 55.3% in terms of average tumour weight. LSPCs can significantly (P < 0.05) decrease lipid peroxidation (LPO) levels and increase the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in liver tissue. In vitro assay of anti-cancer activities suggested that LSPCs (25–100 μg/ml) possessed cytotoxicities against mouse melanoma B16 in a dose-dependent mode. Furthermore, LSPCs had significant (P < 0.05) stimulatory effects on mouse splenocyte proliferation. The prevention of tumour growth was exerted through diverse mechanisms, including cell-cycle arrest, induction of tumour cell death by apoptosis and increase of Ca²⁺ ions, together with stimulation of antioxidant enzyme activities and immunomodulatory activities.     

Effect of continuous milking and bovine somatotropin supplementation on mammary epithelial cell turnover

Objectives were to determine effects of continuous milking (CM) and bovine somatotropin (bST) administration on 1) mammary epithelial cell (MEC) proliferation, apoptosis, and ultrastructure during late gestation and early lactation, 2) expression of genes associated with proliferation, and apoptosis in mammary epithelial cells, and 3) milk yield and composition. Second-gestation, first dry-period cows were randomly assigned to either continuous bST throughout late gestation and early lactation (+bST; n = 4) or no bST (−bST; n = 4) administration. Within each animal, udder halves were randomly assigned to CM or a 60-d dry period (control) treatment. Daily milk yield and weekly milk composition were measured during the last 60 d of gestation in CM halves and from 1 to 30 d postpartum for both halves. Mammary biopsies were obtained at −20 ± 7, −8 ± 3, +1 ± 0, +7 ± 0, and +20 ± 0 d (mean ± standard error) relative to parturition. Prepartum half-udder milk yield was greater in +bST cows than in −bST cows (9.9 vs. 8.2 kg/d) and postpartum half-udder milk yields were dramatically reduced in CM halves compared with control halves (10.6 vs. 22.2 kg/d), regardless of bST treatment. Proliferation of MEC was reduced in CM halves at −8 d (2.7 vs. 5.4%). Apoptosis of MEC was elevated during early lactation for d +1 and +7 in control halves, but was only increased at d +1 in CM halves. Turnover of MEC was not affected by bST. Ultrastructure data indicated complete involution of the control half and lactation maintenance in CM glands (d −20). By d −8, control tissue contained alveoli in an immature secretory state, but CM tissue contained both lactating and immature alveoli. Postpartum ultrastructure parameters were similar between halves until d 20 when control tissue was composed of a homogeneous population of lactating alveoli, but CM tissue contained lactating, engorged, and resting alveoli. Expression of CCAAT/enhancer binding protein-β (CEBP-β), cyclin D1, and bcl₂ were up-regulated during late gestation, but did not differ between control and CM halves. Expression of α-lactalbumin was increased in CM halves during late gestation, but was not different in CM and control tissue after parturition. Other genes evaluated (bax, insulin-like growth factor binding protein 5, ATP-binding cassette 1, and p27) were not differentially expressed at any timepoints evaluated. Results indicate that CM reduced subsequent half-udder milk yield in primiparous cows through altered MEC turnover and secretory capacity. Negative effects of CM on the subsequent lactation were not alleviated by bST supplementation.     

Induction of apoptosis in U937 human leukaemia cells by the hexane fraction of an extract of immature Citrus grandis Osbeck fruits

The antiproliferative effect of an immature Citrus grandis Osbeck fruit extract was investigated using U937 human leukaemia cells. Maximum cytotoxicity was observed using the hexane fraction (HF) of the extract. Cell death was dose-dependent (IC₅₀ = ca. 60 μg/ml) and was characterised by chromatin condensation, apoptotic body formation, and DNA fragmentation. The induction of apoptosis was confirmed by caspase-3 activity assays and by immunoblotting using antibodies against Bcl-2, Bax, poly(ADP-ribose) polymerase (PARP), caspase-9, and caspase-3. The molecular mechanism underlying HF-induced apoptosis in U937 cells may involve a mitochondria-mediated signalling pathway, as demonstrated by an increase in the Bax/Bcl-2 expression ratio. Analyses of the HF by gas chromatography (GC) and GC-mass spectrometry (MS) tentatively identified 19 compounds, including γ-sitosterol (17.5%), 7-methoxy-8-(2-oxo-3-methylbutyl) coumarin (6.8%), stigmasterol (3.8%), and campesterol (3.4%). Together, our results provide the first evidence that the HF of an immature C. grandis Osbeck fruit extract induces apoptosis in U937 cells.     

Bovine alpha-lactalbumin stimulates mucus metabolism in gastric mucosa

Bovine α-lactalbumin (α-LA), a major milk protein, exerts strong gastroprotective activity against rat experimental gastric ulcers induced by ethanol or stress. To elucidate the mechanisms underlying this activity, the influence of α-LA on gastric mucus metabolism was investigated in vitro and in vivo. For the in vitro study, RGM1 cells (a rat gastric epithelial cell line) were selected for observation of the direct activity of α-LA on gastric mucosal cells and cultured in the presence of either α-LA or ovalbumin (OVA), a reference protein showing no gastroprotective activity. Amounts of synthesized and secreted mucin, a major component of mucus, were determined using [³H]glucosamine as a tracer, and prostaglandin E₂ (PGE₂) levels in the culture medium were determined by RIA. For the in vivo study, the thickness of the mucus gel layer, a protective barrier for gastric mucosa, was evaluated histochemically in rat gastric mucosa. α-Lactalbumin (3 mg/mL) significantly stimulated mucin synthesis and secretion in RGM1 cells and also increased PGE₂ levels in the culture medium. In contrast, OVA showed no enhancing effects under identical conditions. Neither indomethacin, a cyclo-oxygenase inhibitor, nor AH23848, a prostaglandin EP4 receptor antagonist, affected α-LA-induced enhancement of mucin synthesis and secretion. In vivo, oral administration of α-LA (300 mg/kg × 3 times/d × 7 d) increased the thickness of the mucus gel layer in rats. These results indicate that α-LA fortifies the mucus gel layer by stimulating mucin production and secretion in gastric mucus-producing cells, and that this enhancing effect is independent of endogenous PGE₂. Comparison of the efficacy of α-LA with OVA suggests that the activities observed in RGM1 cells are closely related to the gastroprotective effects in rat gastric ulcer models. In conclusion, α-LA stimulates mucus metabolism, and this action may be responsible for its gastroprotective activity.     

Technical note: Development of a challenge model for Streptococcus uberis mastitis in dairy heifers

A challenge model for experimentally inducing Streptococcus uberis mastitis in bred dairy heifers was developed. Qualifying heifers (n = 7) exhibited antibody titers of < 1:10,000 against Strep. uberis antigens and were free of intramammary infections (IMI). Two contralateral quarters of each heifer were assigned to receive an infusion of Strep. uberis (1,000 to 2,000 cfu); remaining quarters served as unchallenged controls. For a successful challenge and infection, 3 of 4 consecutive mammary secretion samples had to culture positive for Strep. uberis. Six of the 7 heifers were challenged successfully in both infused quarters with a mean dose of 1,080 cfu; once confirmed, infections were treated with a one-time infusion of nonlactating cow therapy. Before challenge, mammary secretion leukocyte counts averaged 8.4 × 10⁶/mL in all quarters. At 24 h after challenge, leukocyte count increased to 18.4 × 10⁶/mL in challenged quarters, peaking on d 5 at 24.3 × 10⁶/mL; unchallenged quarters remained at ≤ 10.4 × 10⁶/mL, but increased to 15.2 × 10⁶/mL on d 7 and then decreased. Before challenge, macrophages predominated (81%) in mammary secretions followed by lymphocytes (15.3%) and neutrophils (3.7%). By 24 h after challenge, neutrophils increased in challenged quarters and predominated for the duration of the trial (65.3 to 70%), whereas macrophages predominated in unchallenged control quarters (65.2 to 75.2%). The challenge model was successful in establishing Strep. uberis IMI in 85.7% of animals, and IMI were controlled (100% cure) by administering nonlactating cow therapy. All heifers calved free of IMI and antimicrobial residues, with milk production similar to that of herd mates and with somatic cell counts (SCC) < 200,000 cells/mL.     

Anticarcinogenic effect of phytic acid (IP6): Apoptosis as a possible mechanism of action

Several in vivo and in vitro studies provide convincing evidence for the anticarcinogenic properties of phytic acid (PA) (Inositol hexaphosphate, IP₆). The objectives of this investigation were to elucidate the effects of PA on suppression of colonic aberrant crypt foci (ACF) and to study the inhibitory effect of IP₆ on human colon carcinoma, CaCo-2 cell line. Fisher 344 male, weanling rats were divided into 3 groups of 15 each and were fed control diet (AIN 93 G-C) and C+(1 or 2 g/100 g PA in water) for 13 weeks. Rats received 2 s.c. injections of azoxymethane (AOM) in saline at 16 mg/kg body weight at 7 and 8 weeks of age. There was a 36% and 42% reduction in ACF in 1 and 2 g/100 g PA groups compared to control group (P<0.001). Cytotoxic effect of IP₆ was evaluated on Caco-2 cell line at concentrations of 0.25-4 g mol/l using lactate dehydrogenase (LDH) assay (LDH released from the cytosol of damaged cells into the supernatant), histone-associated DNA fragmentation assay using a cell death detection ELISA^® kit and microscopic analysis. The cells were treated with 0.25, 0.5, 1.0, 2.0, 4 g mol/l of IP₆ and incubated for 24 and 48 h. After 24 h of incubation, LDH release ranged from 13.55% to 44.70%. Histone-associated DNA fragmentation (an assay based on the quantitative sandwich-enzyme-immunoassay- principle directed against DNA and histones which allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates) was also dose dependent. CaCo-2 cells grown on slide flaskets in the presence of 0.25 g mol/l IP₆ showed membrane blebbing (zeiosis) characteristic of apoptitic activity. Results of this study indicate that IP₆ might exert anticarcinogenic activity through induction of apoptosis.     

Immunomodulatory and anticancer activities of phenolics from Garcinia mangostana fruit pericarp

The methanolic extract of Garciniamangostana fruit pericarp was partitioned into butanol and water fractions in this work. Three major phenolics were purified and identified as P₁ [1,3,6,7-tetrahydroxy-2,8-(3-methyl-2-butenyl) xanthone], P₂ [1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl-2-butenyl) xanthone] and P₃ (epicatechin). Strong antioxidant activities were detected for P₁–P₃. In vitro cell proliferation trials indicated that P₁ and P₃ exhibited good immunomodulatory activities when 7.5 μg/ml was used. Furthermore, P₁ and P₃ showed good cytotoxicities against human breast cancer cells (MCF-7) and human colon cancer cells (LOVO). P₁ exhibited the maximal cytotoxicity of 73.06% against MCF-7 cells and of 46.27% against LOVO cells when 62.5 μg/ml was used. The cytotoxicities of P₁, P₂, P₃ and paclitaxel against normal embryonic lung fibroblast cells (HELF) were in a decreasing order: paclitaxel > P₃ > P₁ > P_2. These results suggested that P₁ and P₃ could be used as a potential anticancer agent.     

The histamine H1 receptor is not involved in local control of mammary blood flow in dairy cows

Low concentrations of the essential amino acid histidine in circulation have been shown to increase mammary blood flow and it has been suggested that this effect is mediated by histamine. The hypotheses tested in this experiment were that interstitial histamine concentrations in the mammary gland are related to arterial His concentrations and that mammary blood flow is reduced by extracellular histamine via H₁ receptors. The hypotheses were tested by infusing saline or chlorpheniramine, a blocker of the H₁ histamine receptor, into the arterial supply of the mammary glands of lactating cows infused with 44 g h of amino acid mixtures with or without His for 10 h. Infusates were administered in a 2 × 2 factorial arrangement within a 4 × 4 Latin square to 4 multiparous Holstein cows in mid lactation. Exclusion of His from the infusate decreased protein content in milk from the infused udder half from 3.98 to 3.77%, and increased arterial α;-aminonitrogen concentration from 3.2 to 3.4 mM. Neither the decreased arterial His concentration nor the H₁ blocker affected plasma flow to the infused udder half. We conclude that histamine is not involved in the regulation of mammary blood flow. The H₁ blocker decreased milk production in the infused udder half from 4.6 to 3.5 kg without affecting protein, fat, and lactose percentages, suggesting an inhibition of milk ejection. Cows on chlorpheniramine ate less feed during the infusion than saline-infused cows, which resulted in lower arterial concentrations and mammary uptakes of acetate. The efficiency of plasma triacylglycerol uptake across the mammary glands was decreased by chlorpheniramine but net uptake of long-chain fatty acids was not affected. The mechanism by which an amino acid deficiency influences mammary blood flow does not involve histamine signaling through the H₁ receptor and remains unidentified.     

Effects of bovine somatotropin on beta-casein mRNA levels in mammary tissue of lactating cows

Bovine somatotropin (bST) increases milk production in lactating cows through its effect on nutrient partition and maintenance of mammary cell function. A positive relationship between bST treatment and abundance of β-casein mRNA in mammary tissues from lactating cows was hypothesized. In mammary tissue isolated from 14 midlactation Holstein cows, β-casein mRNA was 35.4% higher among 7 cows receiving continuous bST infusions at 29 mg/d for 63 d compared with tissue from 7 untreated control cows. To investigate whether increased β-casein mRNA resulted from a direct effect of bST on the mammary gland, explants of mammary tissue from other lactating cows that had not received bST were incubated with bST and prolactin in 2 experiments. Mammary explant cultures taken from 2 lactating cows that had not been milked for 48 h were supplemented with either prolactin or bST. Both prolactin and bST stimulated higher levels of β-casein mRNA in the mammary explants compared with their non-supplemented counterparts. Explant cultures from 4 additional lactating cows were prepared from rear quarter mammary tissue subjected to milking intervals of 6 h for right rear quarters or 20 h for left rear quarters. Both bST- and prolactin-mediated increases in β-casein mRNA were dependent on milking intervals. That is, levels of β-casein mRNA were increased by bST or prolactin supplementation in explants isolated from the mammary quarters biopsied 20 h after milking but not for those biopsied at 6 h after milking. Results are consistent with a potential role for bST in up-regulating or sparing β-casein mRNA levels in lactating bovine mammary tissue in a manner similar to prolactin.     

Coumestrol induces senescence through protein kinase CKII inhibition-mediated reactive oxygen species production in human breast cancer and colon cancer cells

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC₅₀ of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent K_i value of 7.67 μM. Coumestrol at 50 μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21^Cip1/WAF1 pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22^phox siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.     

Cytoprotective and pro-apoptotic activities of native Australian herbs polyphenolic-rich extracts

Three commercially grown native herbs unique to Australia, Tasmannia pepper leaf (Tasmannia lanceolata R. Br., Winteracea; TPL), anise myrtle (Syzygium anisatum Vickery, Craven & Biffen, Myrtaceae; AM) and lemon myrtle (Backhousia citriodora F. Muell, Myrtaceae; LM) as well as a reference sample bay leaf (Laurus nobilis L., Lauraceae; BL) were examined for potential cytoprotective properties. All native herbs exhibited greater cellular antioxidant activity as measured by the cellular antioxidant activity (CAA) assay than bay leaf and reduced the hydrogen peroxide (H₂O₂) induced death of hepatocellular carcinoma (HepG2) cells by 25–50%. All herb extracts reduced the proliferation of colon (HT-29; IC₅₀ = 0.75–1.39 mg/ml), stomach (AGS; IC₅₀ = 0.59–1.88 mg/ml), bladder (BL13; IC₅₀ = 0.56–1.12 mg/ml) and liver (HepG2; IC₅₀ = 0.38–1.36 mg/ml) cancer cells. No significant reduction of cell viability of non-transformed colon (CCD-18Co; IC₅₀ > 2.0 mg/ml) and mixed stomach and intestine (Hs 738.St/Int; IC₅₀ > 2.0 mg/ml) cells was observed. Flow cytometry analysis and the results of the cytokinesis block micronucleus cytome (CBMNCyt) assay conducted with respectively, promyelocytic leukaemia (HL-60) and colon adenocarcinoma (HT-29) cells suggest an increase in apoptosis following treatment with the herb extracts. The occurrence of apoptotic cells coincided with an increase in caspase-3 enzyme activity. The results of the CBMNCyt assay suggested no direct DNA damage in colon adenocarcinoma (HT-29) cells as a result of treatment with all extracts, applied at final concentrations of 0.5 and 1.0 mg/ml.     

Isomaltulose production by free cells of Serratia plymuthica in a batch process

Free cells of Serratia plymuthica were used to convert sucrose into isomaltulose and trehalulose reducing sugars. The effects of substrate concentration and temperature were observed in a batch process with flasks shaken at 150 rpm. The experimental design and response surface methodology analysis indicated that the conversion parameters had significant influence (p < 0.05) on sucrose conversion, and that a valid model was obtained after an analysis of variance (F_model = 10.48 > F_charted = 5.79) to obtain a response surface and isocurve. Conversion was favoured when a 30% sucrose solution and temperature of 25 °C were used, which resulted in a high conversion into isomaltulose – over 70% – and 7–8% trehalulose. Small amounts of glucose (5–7%) and fructose (5–8%) were formed in the reaction medium.     

Lack of toxicological effect through mutagenicity test of polyphenol extracts from peanut shells

The toxicological effect of polyphenols extracted from peanut shells was investigated in animal models. The safety data were needed to proceed with further clinical trials. The oral LD₅₀ of peanut shells polyphenols was determined to be higher than 15,000 mg/kg body weight. We also carried out a sperm abnormality test, a chromosomal aberration test and a micronucleus test in rats. The peanut shell polyphenols did not cause any abnormalities in the system. Furthermore, the administration of peanut shell polyphenols did not significantly alter changes in body weight or clinical signs. These results strongly indicated that peanut shell polyphenols did not induce mutagenicity. The results of this study suggested a lack of toxicological effect and supported the further use of polyphenol-rich extracts from peanut shells as a potential natural antioxidant.     

Composition analysis and immuno-modulatory effect of okra (Abelmoschus esculentus L.) extract

The aim of this study was to analyse the composition of okra (Abelmoschus esculentus L.) extract and investigate the effect of A. esculentus L. polysaccharides (AE-PS) on the maturation and function of dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs) in vitro. BMHC-derived immature DCs (BMHC-imDCs) were extracted from rats and treated with AE-PS. The hydrolysed okra extract contained 0.6% β-1, 3-d-glucan. AE-PS induced the presence of polymorphic nuclei and elongated protrusion in the BHMC-imDCs, indicating DC activation. Treatment with100 μg/mL of AE-PS increased the MHC class II and CD80/86 expression levels by 41% and 42%, respectively. Treated cells had reduced endocytosis activity. The secretion of IL-12 and IFN-γ increased significantly by 120% and 75%, respectively, when treated with 100 μg/mL of AE-PS. Moreover, IL-10 production was reduced by 66%. In conclusion, AE-PS exhibits stimulatory effects on rat dendritic cells and promotes the secretion of T_H1 cytokines.