Investigating the gut microbiota modulation effects of raw garlic and allicin for cardiovascular disease protection - 16S rRNA gene sequencing of human and mouse fecal microbiota

Investigation of a cardiovascular protective effect of allicin in long-term L-carnitine-induced atherosclerosis ApoE knockout mouse model.Six-week-old ApoE knockout mice were randomly divided into experimental groups (i) control; (ii) control + allicin; (iii) carnitine; (iv) carnitine + allicin; and (v) carnitine + 3,3-dimethylbutanol (DMB). 1.3% L-carnitine in water was supplied for the carnitine intake group. Allicin treatment groups were orally administrated by 10 mg/(kg bw day) allicin in 0.5% carboxymethylcellulose (CMC). 1% of DMB in water were treated for the DMB group. Those ApoE knockout mice were sacrificed after 15 weeks as well as collected the feces sample.Investigation of a cardiovascular protective effect of allicin short-term L-carnitine feeding C57BL/6J mouse model.An 8-week-old male C57BL/6J after an adaptation period of 2 weeks, the mice have grouped and treated similarly to the long-term mice model as mentioned above and then the mice were sacrificed and collect the feces sample after two weeks of treatment.Human study-raw garlic containing allicin intervention.The healthy participants were recruited, the criteria as follows: (1) age more than 20 years old; (2) no exposure to antibiotics, probiotics, or carnitine supplements within the previous month; (3) have no history of chronic diseases including, diabetes mellitus, myasthenia gravis, chronic renal disease, hyperparathyroidism, epilepsy, and severe anemia; (4) Participants were excluded from the study if they reported recent gastrointestinal discomfort (such as abdominal pain or diarrhea). To screening the trimethylamine N-oxide (TMAO) producer, we use the oral carnitine challenge test (OCCT) method which previously exhibited better efficacy than fasting plasma TMAO to identify the TMAO producer phenotype. All of the participants fasted at least 8 hours before performing OCCT. 1500 mg of L-carnitine orally administrated to the participants. The blood and urine of participants were collected at 0, 24, 48, and 72 hours after carnitine intake, feces sample was collected during OCCT. Participants with plasma TMAO more than 10 micromolar after OCCT was defined as high TMAO producers and proceeded into the garlic juice intervention test. High-TMAO producers asked to consume 55 mL of raw garlic juice (48 mg of allicin equivalent) once a day during dinner for one week. After one week of raw garlic juice intervention, the second OCCT was performed as well as collected the fecal sample.All fecal samples were then aliquoted and stored at -80 degrees Celcius for further examination. Fecal genomic DNA was extracted using the QIAamp PowerFecal DNA Kit according to the manufacturer's instructions. Two-step polymerase chain reaction (PCR) workflow was performed for library preparation in accordance with procedures described in the Illumina 16S sample preparation guide. The 16S rRNA gene V3-V4 region was amplified and sequenced on the Illumina Miseq platform for paired-end sequencing (2 x 300 bps).