Allicin is considered responsible for most of the pharmacological activity of crushed raw garlic cloves. However, when garlic supplements and garlic foods are consumed, allicin bioavailability or bioequivalence (ABB) has been unknown and in question because allicin formation from alliin and garlic alliinase usually occurs after consumption, under enzyme-inhibiting gastrointestinal conditions. The ABB from 13 garlic supplements and 9 garlic foods was determined by bioassay for 13 subjects by comparing the area under the 32-h concentration curve of breath allyl methyl sulfide (AMS), the main breath metabolite of allicin, to the area found after consuming a control (100% ABB) of known allicin content: homogenized raw garlic. For enteric tablets, ABB varied from 36–104%, but it was reduced to 22–57% when consumed with a high-protein meal, due to slower gastric emptying. Independent of meal type, non-enteric tablets gave high ABB (80–111%), while garlic powder capsules gave 26–109%. Kwai garlic powder tablets, which have been used in a large number of clinical trials, gave 80% ABB, validating it as representing raw garlic in those trials. ABB did not vary with alliinase activity, indicating that only a minimum level of activity is required. Enteric tablets (high-protein meal) disintegrated slower in women than men. The ABB of supplements was compared to that predicted in vitro by the dissolution test in the United States Pharmacopeia (USP); only partial agreement was found. Cooked or acidified garlic foods, which have no alliinase activity, gave higher ABB than expected: boiled (16%), roasted (30%), pickled (19%), and acid-minced (66%). Black garlic gave 5%. The mechanism for the higher than expected ABB for alliinase-inhibited garlic was explored; the results for an alliin-free/allicin-free extract indicate a partial role for the enhanced metabolism of γ-glutamyl S-allylcysteine and S-allylcysteine to AMS. In conclusion, these largely unexpected results (lower ABB for enteric tablets and higher ABB for all other products) provide guidelines for the qualities of garlic products to be used in future clinical trials and new standards for manufacturers of garlic powder supplements. They also give the consumer an awareness of how garlic foods might compare to the garlic powder supplements used to establish any allicin-related health benefit of garlic.
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Do cooked garlic or acidified commercial garlic products have significant allicin bioequivalence? If so, how much of these foods would one need to consume to obtain the same ABB as raw garlic or a garlic powder supplement used in clinical trials?
The primary objective of this study has been to determine the problematic and hitherto unknown bioavailability of allicin from a variety of commonly consumed garlic supplements and garlic foods, in order to provide clinical trial researchers, manufacturers, and consumers with an improved knowledge basis when considering the possible health benefits of garlic products. The study has answered the following questions:
Although garlic supplements have been used in a large number of clinical trials, garlic is most commonly consumed as a food, usually as a cooked food and often as a commercial product suspended in an acid, such as whole cloves (pickled garlic) or small pieces (minced garlic), but no known clinical trials have been conducted with cooked or acidified garlic foods. However, the general public often wants to know if the results of clinical trials with supplements apply to garlic as a food, especially cooked garlic. In the U.S., cooked garlic is commonly prepared by boiling (soups) or roasting [ 40 , 41 ]. Because cooking or suspending garlic in acid fully inactivates alliinase [ 14 , 26 ], any allicin-related health benefits found with garlic supplements will probably be negligible in such foods. However, this logic assumes that the body itself—in the absence of active garlic alliinase—does not have the ability to metabolize alliin to allicin or allicin metabolites. Now that a method exists for determining the ABB of any garlic product, the validity of this assumption can be tested for alliinase-inhibited garlic foods.
The term “allicin bioavailability” is being used to represent the sum of three processes: enzymatic (garlic alliinase) formation of allyl thiosulfinates (mainly allicin) from alliin, usually in the gastrointestinal tract, followed by their absorption and metabolism to a quantifiable metabolite, AMS. Allicin absorption is known to be highly efficient [ 38 , 39 ], although partial metabolism to the rapidly absorbed intermediate, allyl mercaptan, may occur during absorption [ 17 ]. The term “allicin bioequivalence” refers to the metabolic formation of the allicin metabolite, AMS, from any S-allyl compound, without the assistance of garlic alliinase, including allyl polysulfides, alliin and possibly other S-allyl compounds, such as GSAC and SAC; the term is being used in particular for products in which garlic alliinase is inactive. Together, the terms are referred to as allicin bioavailability or bioequivalence (ABB).
The surprisingly low allicin release found under these standardized in vitro conditions indicates either that allicin release in the body from most garlic supplements is very low or that the U.S.P. dissolution test is not accurate. This dilemma highlights the need to determine the allicin bioavailability in vivo from garlic products, but attempts to do so have been troublesome. Allicin has been shown to be metabolized rapidly (half-life <1 min) to allyl mercaptan (allyl thiol) when added to whole blood [ 32 ], but neither allicin nor its transformation compounds ( ) nor allyl mercaptan were found in the blood, urine or stool after volunteers consumed a large amount (25 g) of chopped raw garlic [ 33 ] (p. 152). However, it has been known for some time that AMS is a component of human breath after consuming raw garlic and that its rate of appearance and decline in the breath indicated it to be a product of systemic metabolism [ 34 , 35 , 36 ]. Lawson and Wang [ 17 ] conducted studies on human breath AMS and showed that (a) the area under the 32-h breath AMS concentration curve (AUC) is linearly proportional to the amount of allicin consumed; (b) AMS is the main breath metabolite of allicin, accounting for at least 90% of the allicin consumed; (c) allyl mercaptan is a temporary intermediate in the formation of AMS from allicin; (d) allicin-derived diallyl disulfide and diallyl trisulfide are also metabolized mainly to AMS; and (e) AMS is an active metabolite, responsible for the ability of allicin to increase breath acetone levels. By the use of a sulfur-selective detector, the sensitivity of the method was improved for the consumption of small amounts of garlic [ 37 ]. Hence, a validated method for determining the bioavailability of allicin from any garlic product has been established.
Due to the abundance of alliinase [ 25 ], complete formation of allicin takes place in 0.5 min (5 min for the allyl methyl thiosulfinates) when water is added to garlic powder [ 14 ]. However, their formation in the body after consumption of garlic powder supplements is questionable because alliinase is inactive at pH 3.5 or below [ 14 , 26 ], a pH range commonly found in the stomach, although a moderate to high-protein meal can briefly raise the pH to 4.4 or higher [ 27 , 28 ], a range in which alliinase is active. Hence, many brands of garlic supplements have been enteric-coated to prevent disintegration in the stomach, and the U.S. Pharmacopeia (USP) has established a monograph for estimating allicin formation and release from such products under simulated gastrointestinal dissolution conditions [ 29 ]. However, 21 of 24 enteric brands subjected to this dissolution allicin release test yielded less than 20% of their allicin potential (the maximum yield of allicin from alliin upon activation of alliinase; the USP monograph refers to it as “potential allicin”), due to low tablet alliinase activity and to slow tablet disintegration [ 30 ]. Similarly, dissolution allicin release from non-enteric Kwai (Lichtwer Pharma, Berlin) tablets, the most commonly used garlic supplement in cardiovascular clinical trials [ 1 , 2 ], has also been found to be problematic: 44% for tablets made before 1993 and only 15% for tablets made from 1994–1997 [ 31 ].
The allyl thiosulfinates, of which allicin (diallyl thiosulfinate) is the most abundant and most studied member ( ), are enzymatic products of alliin, S(+)-allyl-l-cysteine sulfoxide, and alliinase. They are rapidly formed when raw garlic cloves undergo cell rupture ( ) or when dried and pulverized cloves (garlic powder) become wet [ 13 , 14 ]. The allyl thiosulfinates have been shown to be responsible for most of the pharmacological activity of crushed raw garlic cloves. Beginning in 1944 it was shown that allicin is responsible for the antibacterial activity of garlic and that selective removal of allicin also removed all activity [ 15 , 16 ]. Considerable evidence suggests that the allyl thiosulfinates, or their spontaneous transformation compounds (allyl polysulfides), or their common metabolite (allyl methyl sulfide, AMS), and , are responsible for most of the lipid-lowering, antioxidant, anti-atherosclerotic, and anticancer effects of whole garlic, as observed in animals and humans [ 13 , 17 ]. Both allicin and γ-glutamyl-S-allylcysteine (GSAC), as the source of S-allylcysteine (SAC) ( ), appear to be responsible for the hypotensive effects of garlic [ 18 ]. Indeed, no other compound has yet been shown to have significant activity at levels present in a normal human dose (3–5 g) of crushed raw garlic. The majority of the clinical trials on the possible cardiovascular effects of garlic have used supplements that are standardized on alliin or allicin potential [ 1 , 2 ].
Garlic supplements, mainly dried and pulverized whole clove supplements, have been used in a large number of controlled clinical trials since the mid-1980s, focusing primarily on serum cholesterol and blood pressure [ 1 , 2 , 3 ]. The effects on blood pressure have been moderately consistent for hypertensive subjects, while the effects on serum lipids have been inconsistent, even for persons with high baseline cholesterol levels [ 1 , 2 , 4 , 5 , 6 ]. Of the 23 qualifying trials on serum cholesterol with a garlic powder product, 43% found no effect [ 1 ]. Due to the inconsistencies, the trials have been the subject of several meta-analyses (nine on serum lipids and eight on blood pressure), with the overall conclusions being conservative [ 7 ]. Authors of the meta-analyses most frequently cite the high heterogeneity among the trials (high variation in dose, variable product types, identification of active compounds, standardization concerns, and unknown bioavailability) as the reason for caution in recommending garlic products for treatment of hypercholesterolemia and hypertension [ 1 , 4 , 5 , 7 , 8 , 9 , 10 , 11 ]. A recent review of the mostly in vitro antimicrobial effects of allicin concluded that determination of allicin bioavailability from various products is necessary before proper clinical studies can be conducted [ 12 ]. Hence, it is clear that attention needs to be given to the bioavailability and standardization of garlic’s active compounds under a variety of processing conditions, especially because of known or suspected potential issues with their formation, stability, metabolism, and detection in the body.
l(+)- and l(±)-S-allylcysteine sulfoxides (natural and racemic alliin), S-allylcysteine, diallyl disulfide, and diallyl trisulfide (each ≥98%) were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Allyl methyl sulfide and diallyl sulfide (each 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). γ-Glutamyl-S-allylcysteine (99%) was purchased from U.S. Pharmacopeia (Rockville, MD, USA). Allicin (98%) was prepared by oxidation of diallyl disulfide with hydrogen peroxide, followed by purification, as previously described [30]. Its concentration was validated with a second allicin standard prepared from alliin and crude alliinase [29]. Allyl methyl thiosulfinates, allyl-1-propenyl thiosulfinates, diallyl tetrasulfide, allyl methyl disulfide, allyl methyl trisulfide, and allyl methyl tetrasulfide were identified and quantified based on their relative retention times and relative extinction coefficients, compared to allicin and diallyl trisulfide, as previously described [19,21].
The control for 100% ABB was prepared by homogenization of 500 g of peeled raw garlic cloves (California Late variety, Garlic World, Gilroy, CA, USA; 39.5% dry weight), after addition of water at 0.60 mL/g, in an Osterizer blender at the highest speed for 2 min. This allowed alliinase to transform all of the alliin to known amounts of allicin and other allyl thiosulfinates. The homogenate was divided into 50-mL jars and stored at −80 °C, under which condition the allyl thiosulfinates have been shown to be stable for at least 24 months [37]. Prior to consumption, the homogenate was thawed overnight at 4 °C, at which temperature the allyl thiosulfinates have been shown to be stable for at least 3 days [30]. After thawing, the homogenate was further macerated with a high speed Polytron homogenizer (Brinkmann, Kinematica AG, Luzern, Switzerland) with a 12-mm generator for 1 min at speed 6 of 10. This made it possible for the viscous homogenate to pass through the modified tip (drilled to 3 mm, inside diameter) of a 10-mL disposable syringe. At the time of consumption, a specific weight of the homogenate was transferred by syringe into the bottom of one or more size 0 hypromellose (hydroxypropyl methylcellulose) capsules (vegetarian capsules). The bottom of the size 0 capsule rested inside the bottom of a size 00 capsule to prevent aqueous allicin from coming in contact with the throat. The capsule was then closed with the top of a size 00 capsule. Under these conditions, the outer capsule remained firm and tasteless for 6 min. The standard control was 1.4 g of garlic homogenate, which was made from 0.88 g of raw garlic (0.35 g dry wt).
All garlic supplements used in the study were purchased in 2009 at local stores or from online distributors and stored at ambient temperature. Only C1 was purchased directly from the manufacturer. All were tested by May 2011, within their claimed expiration dates (typically 2 years from the manufacture date, except 3 years for N1 and N4), with two intended exceptions (E6 and N4). identifies the garlic supplements used in the study and includes the claims for garlic powder content (sometimes called garlic extract content or garlic dry weight), the actual tablet or capsule weights (average of at least 10 units), standardization claims, if any, and the recommended dose. All labels stated that the products should be consumed with a meal, except for N3. gives the complete list of other ingredients for the supplements, with indications for the likely enteric coating agents when not specifically stated. The 13 supplements contained 51 different ingredients other than garlic powder.
Three common (purchased at grocery stores) and one less common (black garlic, purchased online) commercially prepared garlic foods were tested for ABB. The manufacturing procedures for all the products inactivates alliinase. Spicy Pickled Garlic (referred to as pickled garlic), manufactured by G.L. Mezzetta, Inc. (Napa Valley, CA, USA) consisted of whole raw cloves suspended in a medium of water, vinegar, crushed chili, and sodium bisulfite; cloves constituted 65% of the total volume. The pH of a 1:10 aqueous extract of the cloves was 4.3; the pH of the undiluted medium was also 4.3. Minced Garlic (referred to as acid-minced garlic), manufactured by Spice World, Inc. (Orlando, FL, USA) consisted of finely minced raw cloves mixed with a minimal amount of water (almost no standing liquid) and phosphoric acid; the pH of a 1:10 aqueous extract was 3.6. Chopped Garlic (referred to as oil-chopped garlic), manufactured by Christopher Ranch (Gilroy, CA, USA), consisted of finely chopped raw garlic mixed with soybean oil, olive oil (almost no standing liquid) and citric acid; the pH of a 1:10 aqueous extract was 4.3. The manufacturer’s web site calls it Chopped Garlic in Oil. Black garlic, manufactured in South Korea and distributed by Black Garlic, Inc. (Hayward, CA, USA), consisted of peeled whole cloves that were black (no additives).
Roasted. Garlic (California Early variety, Garlic World, Gilroy, CA, USA) was roasted at two different temperatures commonly used for cooking (actual air temperature): 160 °C for 30 min and 215 °C for 60 min. Three garlic bulbs were placed on a rack inside a pre-heated Nesco 6 L electric roaster oven (Walmart). Wire probes from a calibrated Fluke 52II digital thermometer (Fluke Corp., Everett, WA, USA) were used to measure the temperature (recorded every 5–10 min) of the air inside the oven and the temperature inside the cloves of two different bulbs. The average temperature inside the cloves, after the first 10 min, was 92 °C for the 160 °C preparation and 101 °C for the 215 °C preparation. Roasting caused 5% and 25% loss of total weight, respectively. After cooling, the cloves were peeled and the soft contents thoroughly mashed and mixed. All of the contents of the 160 °C cloves were soft, but about half of the weight of the 215 °C cloves was hard and unusable. Roasting decreased the moisture content of the cloves from 63% to 58% and 55%, respectively.
Boiled. Unpeeled garlic cloves (California Early variety, Garlic World, Gilroy, CA, USA) were boiled for 4 min or for 45 min, by adding 24 cloves (80 g for 4 min or 110 g for 45 min) from six bulbs to 1.5 L of boiling water. The water resumed boiling about 15 s after the cloves were added. After boiling, the cloves were cooled, peeled, and thoroughly mashed and mixed. Preliminary studies showed that the temperature inside the cloves reached 95 °C (boiling point 96 °C) by 4 min if the clove weights were under 5 g or by 6 min if the clove weights were 5.5 to 7 g. It was also shown that alliinase activity was completely inhibited in 2 min for cloves weighing under 5 g. Hence, for the 4 min preparation, only cloves weighing 1.2 to 5.0 g were used, while cloves for the 45 min preparation weighed 1.2 to 9 g. The total weight of the cloves boiled for 4 min did not change, while the weight of the cloves boiled for 45 min increased by 5%. To reduce variation, cloves from each of the six bulbs were split into the two groups used for boiling.
Diced (cubed) raw. Raw garlic cloves (California Early variety, Garlic World, Gilroy, CA, USA) were peeled, then placed on a plastic grid of 3-mm lines, and cut with a razor blade into cubes measuring approximately 3 mm on each side (40 cubes per gram). This size is similar to that of commercial minced garlic. Two cloves from each of the same bulbs used for the boiling preparations were used. The cloves were carefully peeled and cut in order to minimize allicin formation.
Protein-free, high-alliin garlic extract (PFHA). A garlic extract was prepared in which 97% of the soluble protein was removed, without loss of alliin. Garlic cloves (California Early variety, Garlic World, Gilroy, CA, USA) were peeled (100 g), boiled for 15 min in 1.5 L water (giving 14 mL water/g clove after evaporation loss), transferred to an Osterizer blender, homogenized at the highest speed for 1 min, filtered through cheesecloth, centrifuged at 1300× g for 20 min, subjected to 11 cycles of freezing at −22 °C and thawing, and finally centrifuged at 16,000× g. The soluble protein content decreased from 12.5 mg/g clove (before boiling) to 0.80 mg/g clove (before freeze-thawing) to 0.42 mg/g clove. The final clear extract contained 0.034 mg protein/mL.
Alliin-free, high-GSAC extract (AFHG). A garlic extract void of alliin and alliin-derived compounds was prepared to examine the effects of GSAC on breath AMS. Cloves (85 g), from the same 2-kg batch of bulbs that were used to prepare PFHA, were peeled, homogenized (2 min) in an Osterizer blender after addition of water at 14 mL/g (converted all of the alliin to allyl thiosulfinates), filtered through cheesecloth, boiled for 100 min, (causing all of the allyl thiosulfinates and resultant allyl sulfides to evaporate), and centrifuged at 1300× g for 20 min. Additional water was added as needed during boiling to make up for most of the evaporation loss. The soluble protein content decreased from 12.5 mg/g clove (before homogenization) to 2.4 mg/g clove (81% loss of protein). The final clear extract contained 0.25 mg protein/mL.
Alliin-free, high-SAC extract (AFHS). Product AFHG was treated with γ-glutamyl transpeptidase to convert 95% of the GSAC to SAC to learn if the two compounds have equal effects on breath AMS. A portion of AFHG (200 mL) was adjusted to pH 8.5 with 0.1 N NaOH, followed by the addition of 100 units of equine kidney γ-glutamyl transpeptidase (9.1 units/mg) (Sigma-Aldrich) and incubation at 37 °C for 3.5 h.
A total of 13 self-described healthy persons (6 female, 7 male) were recruited for the study in May–June 2009, all of whom continued throughout the 22 months of the study. Persons who had an intestinal disease, used tobacco, or frequently consumed alcohol, were excluded from the study.
The participant age range was 26–62 year (averages: total 35, men 34, women 36), with a body mass index (BMI) range of 22–35 kg/m2 (averages: total 26, men 28, women 25). The study was a series of bioassays; hence, it was not blind, nor was there a placebo or randomization Participants were always informed which garlic product they were consuming prior to consumption. Twenty-three types of garlic products were used in 43 tests, with usually 7–13 participants per test. A single set of bioavailability tests required two days to conduct; they were usually conducted two times per week and included 3–4 participants per set. Participants usually participated in two tests per month and were paid for each test. A record of all interactions with the subjects during a test, along with any report of side effects, was maintained on set consumption records. All bioavailability tests with garlic products and garlic extracts, along with the consent form and research protocol, were approved by the Western Institutional Review Board, Olympia, Washington, USA, on 18 March 2009 (study number 1105434).
All garlic supplements, the homogenized raw garlic (control), and the extracts, were consumed with either a standardized low-protein meal (LP) or a standardized high-protein meal (HP). The LP meal consisted of two slices of toasted white bread, 19 g of butter, 30 g of jam, a banana, and 200 mL of water; it contained 5.5 g of protein and 17 g of fat. The HP meal consisted of a whole wheat tuna sandwich and 200 mL of whole milk, including two slices of bread, 54 g of pressed canned albacore solid white tuna, and 34 g of garlic-free light mayonnaise; it contained 31 g of protein and 19 g of fat. The meals were isocaloric (460 kcal). The gastric pH is lowest before a meal (av 1.9) and quickly rises (av 14 min), depending upon the meal protein content, to a maximum pH of 4.4–6.7 after starting a meal [28,42]. Hence, the supplements were consumed immediately before the LP meal, to allow exposure to approximately the lowest gastric pH attainable when they are consumed with a meal—or immediately after the HP meal, to allow exposure to a substantially higher gastric pH. The control, in capsules, was always consumed immediately after the LP or HP meals to reduce the chance of stomach disturbance. For the control, gastric pH was not a concern, as alliinase had been activated before consumption. Garlic foods (cooked garlic, etc.) and their control were consumed inside the LP meal, with the garlic placed between the two slices of buttered bread and the jam omitted. The special extracts (clear aqueous liquids) were consumed with the LP meal.
It was essential to prevent dietary interference with the bioavailability test by restricting the consumption of garlic and onions for two days before each test and for the 1.5 days of each test. The restrictions were as follows: two days before a test, only modest amounts of garlic; one day before the test and during the test, no foods containing garlic or onion, with some exceptions. Some prepared foods listing garlic were found to contain too little garlic to cause breath AMS production: Kraft Miracle Whip, Pace Chunky Salsa, and Doritos Cool Ranch Chips. A small amount of cooked onion caused no interference. Prepared foods, such as ketchup, mustard, salad dressing, tomato sauce, and chips, which listed onion as a spice, but not garlic, caused no interference. Participants were discouraged from eating restaurant food unless they were sure garlic and onion were absent from their choices.
On the day of each test, participants were asked to not eat or drink until arriving at the facility, except, if necessary, up to 150 mL water one hour before arrival. After consuming the standard breakfast meal and garlic product, participants were asked to not eat or drink again until two hours later. However, if they felt discomfort during the two hours, small amounts of non-protein food were permitted. Participants were occasionally asked if they had adhered to these restrictions.
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Compliance with the diet restrictions was determined by the absence of AMS and the presence of no more than a modest amount of onion sulfides in a breath sample taken immediately before the test product was consumed. Non-compliance after the test began was monitored by a sudden increase in breath AMS concentration at 2–3 h after consumption of the subsequent meals consumed during the test. If non-compliance was determined, participants were asked to stop the test and repeat it at a later date.
Participants breathed into 1.2-L Tedlar® gas sampling bags (Grace Davison Discovery Sciences, Waukegan, IL, USA), fitted with a septum port, a barbed nickel-plated twist-valve, and a 4-cm length of Tygon® tubing, as a mouthpiece, attached to the valve. They were instructed to exhale a normal breath, starting at the top of the breath (avoiding the natural tendency to first take a deep breath), into the bag, then to empty the bag by flattening it, followed by exhaling a second breath until the bag was at least mostly full and closing the valve. After the flattening, the mouthpiece was placed against the tongue or lip to prevent air from entering the bag until the participant was ready to exhale the second breath. The speed at which one breathes into the bag was found to have no influence on the breath AMS concentration. Participants recorded their name and breath time on each bag.
Whole breath, rather than alveolar breath, was used throughout the study. Although alveolar breath has been shown to contain 18% higher AMS than whole breath [17], the procedure for sampling alveolar breath was considered too difficult for participants to use away from the laboratory, where most of the 17 breaths per test needed to be collected. Because all tests were conducted in the same manner and because only relative amounts are important, using whole breath was considered sufficient.
The average rate of disappearance of AMS from the Tedlar® bags was 0.4%/h (range 0.05–1.05%/h). The disappearance rate for each bag was determined over a 48-h period and only the better bags (<0.50% loss/h) were used for the breaths that needed to be stored overnight before analysis. Except for the overnight breaths, breaths were usually analyzed within 4–6 h after sampling. Following these guidelines, losses were considered insignificant and not corrected. Moisture accumulation in the bags was found to have no effect on the AMS concentration.
After an overnight fast, participants came to the facility, breathed into a bag and then consumed the standardized breakfast meal and the garlic product. After consuming the garlic product, participants left the facility with several empty bags and provided breath samples every hour for the next eight hours, then every two hours, except during sleep, until breath AMS was undetectable (typically 24–30 h), but not longer than 32 h. Participants returned to the facility to deliver breath samples and obtain more bags at about noon and 5 pm of the first day and at about 8 am and 2 pm of the second day.
Breath samples (5 mL) were injected directly (manually with a 5-mL gas-tight syringe fitted with a 0.63 mm side-port needle), one time, into a gas chromatograph (Agilent 6890) fitted with a model 5380 sulfur-selective pulsed flame photometric detector (PFPD, OI Analytical, College Station, TX, USA), PFPD zero-output setting of 10, and a 30 m × 0.32 mm × 4 μm SPB-1 Sulfur (bonded polydimethylpolysiloxane) capillary column (Sigma-Aldrich #24158). Helium was the carrier gas (1.6 mL/min, constant flow). The column temperature was programmed from 45 °C (0.2 min) to 200 °C (1.2 min) at 50°/min, with a hold time of 2 min, giving a retention time of about 4.0 min, a run time of 5 min, and a re-injection time of 11 min. The injection port contained a straight 4 mm borosilicate liner (Agilent #19251-60540) and was operated at 175 °C in the splitless mode, with a purge flow of 45 mL/min for 0.8 min. The detector was operated at 250 °C, with the following flow rates: hydrogen 9.5 mL/min, air 18.5 mL/min, helium (make-up) 5.5 mL/min. Due to injection of an overload volume of 5 mL and a rapid temperature gradient, the baseline did not become stable until 2.9–3.0 min after each injection. Because inserting the column into the detector requires disassembly of the detector and several hours of equilibration time, a 40-cm piece of the column (extension) was attached to the detector, with the other end attached to an Agilent Ultimate Union (G3182-61580). The remainder of the column was then attached to the extension at the union. This allowed for quick removal and replacement of the column for other uses of the gas chromatograph. The PFPD peak area is the square root of the detector response to sulfur. This detector gave about 20 times greater sensitivity (detection limit, 40 area units or 3 ng/L or 1 ppb at s/n = 2) than the FID (flame ionization detector) detector used in prior allicin bioavailability studies [17], which made it possible to measure the AUC after consuming small amounts of garlic products. AMS is normally absent from human breath [43].
The stock allyl methyl sulfide vapor standard (266 ng/L) was prepared by adding 13.0 μL of a solution of 88 μg allyl methyl sulfide/mL in methanol to duplicate 4.3-L glass jugs with lids fitted with septum ports. The concentration in the jugs reached stability by 2–3 h and remained stable for 24–30 h. Dilutions of this standard gave a curvilinear linear response down to 6 ng/L. Dilutions were prepared by adding various volumes of the stock (0.25–3 mL) and air (4.75–2 mL) to a 5-mL syringe and injecting the 5-mL. For volumes less than 1 mL, the stock was added to the 5-mL syringe using a 1-mL syringe. An air-blank was injected daily. When eight standard curves, created over a 14-month period (see examples in ), were applied to a single set of breath data, the resultant AUC was found to vary by only 5% (RSD, relative standard deviation), indicating the stability of the PFPD detector response.
Open in a separate windowThe AUC was measured in ng-h/L by determining the AMS concentration of each breath, based on AMS peak areas and interpolated concentration values (Spline Lowess regression) from the standard curve, followed by determining the AUC over 32 h. All of the calculations were determined using GraphPad Prism 5.0 (San Diego, CA, USA) software.
The ABB for each product was based on response factors (RF, RF2, RF3) and calculated as relative response factors (RRF, RRF2, RRF3). RF is the ratio of AUC to the μmol of alliin and alliin-derived dithioallyl compounds (AADD, see ) consumed. RF2 is the ratio of AUC to the μmol of total known S-allyl compounds (TKSA) consumed; TKSA is AADD plus GSAC and SAC (G/SAC). RF2 was especially useful when AADD was very low and G/SAC were dominant. RF3 is the ratio of AUC to the weight (g) of product consumed; it was used mainly for “aged” garlic products, such as black garlic, in which most of the alliin has been converted to unidentified S-allyl compounds. The relative response factors were calculated as 100% times the response factors for the products divided by the response factors for the standard control. When products contained alliinase activity (the supplements and diced raw garlic), allicin bioavailability was determined based on AADD. When the alliinase of garlic products had been inhibited (cooked, acidified), allicin bioequivalence was determined based either on AADD or TKSA. The minimum AUC that could be accurately determined was 15 ng-h/L, when there was a relatively sharp decline in AMS after Tmax was reached (typical when the product had active alliinase, such as with the control and N1). It was about 45 ng-h/L when there was a gradual decline after the Tmax (typical when alliinase was absent, such as with PHFA, AFHG, and alliin). When the product contained active alliinase, ABB as low as 0.7–11% could be determined when the AADD consumed was 320–20 μmol, respectively. When the product did not contain active alliinase, ABB as low as 2–32% could be determined when the AADD consumed was 320–20 μmol.
The formation and release of allicin from garlic tablets and capsules under simulated gastrointestinal conditions was determined according to the USP-NF (U.S. Pharmacopeia-National Formulary) dissolution method for delayed-release garlic tablets [29]. Using a model VK 700 dissolution apparatus (Agilent/Varian, Palo Alto, CA, USA) equilibrated at 37 °C, 2–6 tablets or capsules (enough to provide 20–30 mg alliin) were placed into each of two covered 1-L round bottom glass vessels containing 750 mL of 0.1 N HCl and paddle-stirred at 100 rpm for 2 h, after which 250 mL of 0.2 M Na3PO4 was added and the pH slightly adjusted, if necessary, giving 1000 mL at pH 6.80 ± 0.05. After stirring the buffered medium for 60 min, 1 mL was added to 0.05 mL of 210 mM (final 10 mM) carboxymethoxylamine (CMA) (Sigma-Aldrich) alliinase inhibitor, followed by high-performance liquid chromatography (HPLC) analysis of allicin. For tablets that had not completely disintegrated in 60 min of buffer, an additional 1 mL aliquot was taken upon complete tablet disintegration. Capsules were loosely wrapped with three winds of plastic-covered wire to keep them from floating. The time to achieve complete disintegration was determined by observation during the dissolution test. The percent dissolution allicin release (DAR) was calculated as the amount of allicin released during the dissolution tests divided by the amount of allicin released when pulverized tablets or capsule contents were incubated 30 min in water to allow complete allicin formation (allicin potential).
Garlic supplements (tablets and capsule contents) were mortar ground, extracted with 20 mM CMA alliinase inhibitor (50–100 mL/g) for analysis of alliin, GSAC, and SAC, or extracted with 70% acetonitrile (ACN)/30% 40 mM CMA (10 mL/g) for analysis of allyl sulfides. For water activation of alliinase to generate the allyl thiosulfinates, ground tablets or the contents of capsules were mixed with water at 50 mL/g, rotated rapidly for 10 min, centrifuged for 5 min at 1000× g, followed by addition of one volume of ACN (precipitates protein), centrifuged for 3 min at 16,000× g, and kept at 4 °C or lower until analyzed [37]. Commercial garlic foods were extracted with a Polytron homogenizer at speed 6 for 15 s, in the presence of either 20 mM CMA (10 mL/g) for analysis of alliin, GSAC, and SAC or in the presence of 75% ACN/25% water (10 mL/g) for analysis of allyl thiosulfinates and allyl sulfides. The control (raw garlic homogenate) was extracted with 20 mM CMA (20 mL/g) for analysis of alliin, GSAC, and SAC, or with 50% ACN (10 mL/g) for analysis of allyl thiosulfinates, or with 70% ACN (10 mL/g) for analysis of allyl sulfides. Diced raw garlic was extracted with a Polytron homogenizer (speed 6, 15 s) in the presence of 20 mM CMA (25 mL/g) for analysis of alliin, GSAC, SAC, and, after addition of 1 volume of ACN, for analysis of allyl thiosulfinates and allyl sulfides. Roasted or boiled garlic were extracted with a Polytron homogenizer (speed 6, 15 s) in the presence of water (25 mL/g) for analysis of alliin, GSAC, and SAC, or in the presence of 75% ACN (10 mL/g) for analysis of allyl thiosulfinates and allyl sulfides.
Alliin, GSAC, and SAC were analyzed by ion-pair HPLC (250 mm × 4.6 mm, Agilent/Varian Microsorb-MV 100-5 C18 250 mm × 4.6 mm column) at 208 nm by modification of the method of Arnault et al. [44]. Solvent A consisted of 20 mM sodium heptanesulfonate (ion-pair agent) (Sigma-Aldrich) and 20 mM sodium phosphate monobasic, pH adjusted to 2.1 with concentrated phosphoric acid. Solvent B was ACN. The column was heated at 38 °C with a flow rate of 1.0 mL/min. Gradient: from 0% B to 15% B in 5 min, then to 22% B by 15 min and hold until 17 min, then back to 0% B by 18 min and hold until 24 min. Typical retention times: l(−)-alliin 8.5 min, l(+)-alliin 8.8 min, isoalliin 9.1 min, γ-glutamyl-S-methylcysteine 11.0 min, S-allylcysteine 13.7 min, γ-glutamyl-S-allylcysteine 15.9 min, γ-glutamyl-S-cis-1-propenylcysteine 17.3 min, γ-glutamyl-S-trans-1-propenylcysteine 17.5 min, γ-glutamylphenylalanine 18.5 min. The allyl sulfides and allyl thiosulfinates (when their abundance was small) were analyzed, using the same column, isocratically with 70% ACN/30% water at 210 nm, as previously described [21] (1991 sulfides pub). When the allyl thiosulfinates were abundant, such as with the control, or when the dissolution allicin release and USP potential allicin (allicin potential) were determined, they were analyzed isocratically with 45% ACN/55% water at 240 nm, as previously described [37].
Alliinase activity, as μg allicin produced min−1 g−1 garlic powder (dry garlic matter), was determined as previously described [30]. Briefly, the capsule contents or mortar-pulverized tablets, without sieving, were added to water at a concentration of 1 g garlic powder content to 800 mL water, followed by immediate and vigorous shaking for 7–8 s and removal of 1 mL aliquots at 15, 30, 60, and 120 s to 0.05 mL of 210 mM CMA to stop the reaction. After analysis of allicin, the alliinase activity was determined from the time point giving the highest rate of allicin production. If the activity was found to be less than 500, it was redetermined at 200 mL/g garlic powder. The garlic powder content for tablets and capsules was based on label claims.
The protein content of extracts PFHA and AFHG were assayed according to the procedure of Bradford [45], using Brilliant Blue G (Sigma-Aldrich) in methanol as the binding dye and bovine serum albumin (Sigma-Aldrich) as the protein standard (standard curve 10–100 μg/mL).
To reveal any possible peptide-bound alliin or SAC, both AFHG extract and roasted garlic prepared at 160 °C (homogenized with 10 mL water/g) were incubated while stirring at pH 8.5 at 37 °C for one hour with a large excess of either protease from Bacillus licheniformis (9.6 units/mg, Sigma-Aldrich) or alkaline protease from Bacillus subtilis (400 PC units/mg, Bio-Cat, Inc., Troy, VA, USA), followed by one hour of incubation with porcine kidney γ-glutamyl transpeptidase (3.4 units/mg, Sigma-Aldrich). Aliquots were centrifuged at 16,000× g and directly assayed for alliin, SAC, and GSAC as described in Section 2.18.
Statistical analyses were conducted using Microsoft Excel Analysis ToolPak software (Redmond, WA, USA). Student’s t-Test (paired two sample for means; two-tail) was used to determine differences between groups. In a few cases, the two-sample t-Test was used when n was small (≤4). p-Values < 0.05 were considered to be significant. Data are presented as means ± standard deviation (SD).
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