Enhancement of Intestinal IgA Production by Ajoene in Mice

We investigated the effects of ajoene on intestinal IgA production. Ajoene (1.35, 4.5, and 13.5 µg/kg/d) was administered to mice for 4 weeks. The fecal IgA level in the 13.5 µg/kg/d group increased after 3 weeks. The intestinal IgA level also increased in a dose-dependent manner upon ajoene administration. An oil-macerated garlic extract, with 1500 µg/g of ajoene, enhanced the intestinal IgA production.     

Sterols and Asparagine in the Rice Plant,Endogenous Factors Related to Resistance against the Brown Planthopper (Nilaparvata lugens)

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 °C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 °C, but only 1 d at 37 °C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 °C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 °C, but only 1.2 d and 1.9 d for the respective activities at 37 °C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.     

大蒜化学成分的气-质联用分析

本文采用环己烷、乙酸乙酯和正丁醇对大蒜（Allium sativum L.）新鲜鳞茎的95%乙醇提取物进行萃取,并与水蒸气蒸馏法提取的挥发油成分进行比较,用GC-MS对其成分进行定性和定量分析.在环己烷萃取物中共检出112个成分,鉴定了38个化合物,占环己烷萃取物总量的80.08%;在乙酸乙酯萃取物中检出86个成分,鉴定了26个化合物,占乙酸乙酯萃取物总量的56.70%;在正丁醇萃取物中未检出挥发性成分.在水蒸气蒸馏法提取的挥发油中共检出109个成分,鉴定了29个化合物,占挥发油总量的83.58%.大蒜95%乙醇提取物的环己烷和乙酸乙酯萃取物及大蒜挥发油中皆以含硫化合物为主.在环己烷和乙酸乙酯萃取物中,阿霍烯的含量分别为生药的0.00395%和0.00145%,大蒜中阿霍烯含量达0.00540%.     

Molecular mechanisms of apoptosis induced by ajoene in 3T3-L1 adipocytes

Objective : Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic. Research Methods and Procedures : Mature 3T3‐L1 adipocytes were incubated with ajoene at concentrations up to 200 μM. Viability and apoptosis were quantified using an MTS‐based cell viability assay and an enzyme‐linked immunosorbent assay for single‐stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production was measured based on production of the fluorescent dye, dichlorofluorescein. Activation of the mitogen‐activated protein kinases extracellular signal‐regulating kinase 1/2 (ERK) and c‐Jun‐N‐terminal kinase (JNK) was shown by Western blot. Western blot was also used to show activation of caspase‐3, translocation of apoptosis‐inducing factor (AIF) from mitochondria to nucleus, and cleavage of 116‐kDa poly(ADP‐ribose) polymerase (PARP)‐1. Results : Ajoene induced apoptosis of 3T3‐L1 adipocytes in a dose‐ and time‐dependent manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of AIF from mitochondria to nucleus, and cleavage of 116‐kDa PARP‐1 in a caspase‐independent manner. Ajoene treatment also induced an increase in intracellular ROS level. Furthermore, the antioxidant N‐acetyl‐l ‐cysteine effectively blocked ajoene‐mediated ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP‐1. Discussion : These results indicate that ajoene‐induced apoptosis in 3T3‐L1 adipocytes is initiated by the generation of hydrogen peroxide, which leads to activation of mitogen‐activated protein kinases, degradation of PARP‐1, translocation of AIF, and fragmentation of DNA. Ajoene can, thus, influence the regulation of fat cell number through the induction of apoptosis and may be a new therapeutic agent for the treatment of obesity.     

Modification of API-2c and Its Reactive Site

The inhibitory activity of API-2c was reduced by carboxymethylation of methionine residues but not by chemical modifications of lysine and arginine residues. This indicates that API-2c requires methionine residue for inhibitory activity. Dissociation of API-2c-subtilisin BPN′ complex under mild conditions (pH-gradient dialysis) resulted in the conversion of API-2c to modified form (API-2C*).

The amino acid composition of API-2c did not change upon enzymatic modification. However, modified API-2c had two NH₂-terminal sequences (Asp-Ser-Pro- and Ile-Tyr-Asp-) and two COOH-termini (Phe and Met), and gave two bands on SDS-polyacrylamide gel disc electrophoresis after reduction of disulfide bonds by 2-mercaptoethanol. From these results, it is concluded that the Met-Ile bond located within a disulfide bridge in API-2c molecule is the reactive site for inhibition of subtilisin BPN′.