Human, Animal and Plant Health Benefits of Glucosinolates and Strategies for Enhanced Bioactivity: A Systematic Review.

Glucosinolates (GSs) are common anionic plant secondary metabolites in the order Brassicales. Together with glucosinolate hydrolysis products (GSHPs), they have recently gained much attention due to their biological activities and mechanisms of action. We review herein the health benefits of GSs/GSHPs, approaches to improve the plant contents, their bioavailability and bioactivity. In this review, only literature published between 2010 and March 2020 was retrieved from various scientific databases. Findings indicate that these compounds (natural, pure, synthetic, and derivatives) play an important role in human/animal health (disease therapy and prevention), plant health (defense chemicals, biofumigants/biocides), and food industries (preservatives). Overall, much interest is focused on in vitro studies as anti-cancer and antimicrobial agents. GS/GSHP levels improvement in plants utilizes mostly biotic/abiotic stresses and short periods of phytohormone application. Their availability and bioactivity are directly proportional to their contents at the source, which is affected by methods of food preparation, processing, and extraction. This review concludes that, to a greater extent, there is a need to explore and improve GS-rich sources, which should be emphasized to obtain natural bioactive compounds/active ingredients that can be included among synthetic and commercial products for use in maintaining and promoting health. Furthermore, the development of advanced research on compounds pharmacokinetics, their molecular mode of action, genetics based on biosynthesis, their uses in promoting the health of living organisms is highlighted.

Glucosinolates: Molecular structure, breakdown, genetic, bioavailability, properties and healthy and adverse effects.

Glucosinolates are a large group of plant secondary metabolites with nutritional effects and biologically active compounds. Glucosinolates are mainly found in cruciferous plants such as Brassicaceae family, including common edible plants such as broccoli (Brassica oleracea var. italica), cabbage (B. oleracea var. capitata f. alba), cauliflower (B. oleracea var. botrytis), rapeseed (Brassica napus), mustard (Brassica nigra), and horseradish (Armoracia rusticana). If cruciferous plants are consumed without processing, myrosinase enzyme will hydrolyze the glucosinolates to various metabolites, such as isothiocyanates, nitriles, oxazolidine-2-thiones, and indole-3-carbinols. On the other hand, when cruciferous are cooked before consumption, myrosinase is inactivated and glucosinolates could be partially absorbed in their intact form through the gastrointestinal mucosa. This review paper summarizes the glucosinolate molecular breakdown, their genetic aspects from biosynthesis to precursors, their bioavailability (assimilation, absorption, and elimination of these molecules), their sensory properties, identified healthy and adverse effects, as well as the impact of processing on their bioavailability.

Supplementation of the Diet by Exogenous Myrosinase via Mustard Seeds to Increase the Bioavailability of Sulforaphane in Healthy Human Subjects after the Consumption of Cooked Broccoli.

SCOPE: Broccoli contains glucosinolate glucoraphanin, which, in the presence of myrosinase, can hydrolyze to isothiocyanate sulforaphane, reported to have anticarcinogenic activity. However, the myrosinase enzyme is denatured on cooking. Addition of an active source of myrosinase, such as from powdered mustard seed, to cooked Brassica vegetables can increase the release of health beneficial isothiocyanates; however, this has not previously been proven in vivo. METHODS AND RESULTS: The concentration of sulforaphane metabolite (sulforaphane N-acetyl-l-cysteine [SF-NAC]) in 12 healthy adults after the consumption of 200 g cooked broccoli, with and without 1 g powdered brown mustard, was studied in a randomized crossover design. During the 24-h period following the consumption of the study sample, all urine was collected. SF-NAC content was assayed by HPLC. When study subjects ingested cooked broccoli alone, mean urinary SF-NAC excreted was 9.8 ± 5.1 µmol per g creatinine, and when cooked broccoli was consumed with mustard powder, this increased significantly to 44.7 ± 33.9 µmol SF-NAC per gram creatinine. CONCLUSION: These results conclude that when powdered brown mustard is added to cooked broccoli, the bioavailability of sulforaphane is over four times greater than that from cooked broccoli ingested alone.

Sulforaphane Bioavailability from Glucoraphanin-Rich Broccoli: Control by Active Endogenous Myrosinase.

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or ß-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).

Bioactive capacity, sensory properties, and nutritional analysis of a shelf stable protein-rich functional ingredient with concentrated fruit and vegetable phytoactives.

Well-known health-protective phytochemicals from muscadine grape and kale were stably complexed with food grade protein (soy or hemp protein isolates) to create biofortified food ingredients for use in a variety of convenient, portable food formulations. The bioactive (anti-inflammatory) potential, sensory attributes and proximates of the prepared formulations were evaluated in this study. Anti-inflammatory properties of the protein-phytoactive ingredient particles were contributed by the polyphenolic content (muscadine-protein) or the combination of polyphenol, carotenoid, and glucosinolate content (kale-protein aggregates). Phytoactive compounds from the fortified matrices suppressed at least two biomarkers of inflammation; most notable with the expression of chronic pro-inflammatory genes IL-6 and Mcp1. Sensory analysis suggested both sweet and savory functional food applications for the biofortified ingredients. Proximate analyses determined that fortification of the soy protein isolate (SPI) with muscadine or kale bioactives resulted in elevated dietary fibers, total carbohydrates, and free sugars, but did not increase calories/100 g dry matrix compared to unfortified SPI. Overall protein content in the aggregate matrices was about 37% less (muscadine-SPI, kale-SPI and kale- HP50) or 17.6% less (muscadine-HP50) on a weight basis, likely due to solubility of some proteins during preparation and partial displacement of some protein mass by the fruit and vegetable phytoactive constituents.

Urinary 3,3'-diindolylmethane: a biomarker of glucobrassicin exposure and indole-3-carbinol uptake in humans.

BACKGROUND: Brassica vegetable consumption may confer a protective effect against cancer, possibly attributable to their glucosinolates. Glucobrassicin is a predominant glucosinolate and is the precursor of indole-3-carbinol (I3C), a compound with anticancer effects. However, objective assessments of I3C uptake from Brassica vegetables have not been successful. METHODS: We conducted a randomized, crossover trial to test whether 3,3'-diindolylmethane (DIM, a metabolite of I3C) excreted in the urine after consumption of raw Brassica vegetables with divergent glucobrassicin concentrations is a marker of I3C uptake from such foods. Twenty-five subjects were fed 50 g of either raw "Jade Cross" Brussels sprouts (high glucobrassicin concentration) or "Blue Dynasty" cabbage (low glucobrassicin concentration) once daily for 3 days. All urine was collected for 24 hours after vegetable consumption each day. After a washout period, subjects crossed over to the alternate vegetable. Urinary DIM was measured using a novel liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) method with [(2)H2]DIM as internal standard. RESULTS: Urinary DIM was consistently and significantly higher after Brussels sprouts feeding than after cabbage feeding, as evidenced by an average difference of 8.73 pmol/mg creatinine (95% confidence interval, 5.36-12.10; P = 0.00002). CONCLUSION: We have successfully quantified urinary DIM after uptake of I3C from food, and demonstrated that differences in glucobrassicin exposure are reflected in urinary DIM levels. IMPACT: Our LC-ESI-MS/MS-SRM method and the results of our study indicate urinary DIM is a measure of I3C uptake from Brassica vegetables, a finding that can be utilized in prospective epidemiologic and chemoprevention studies.

A secondary metabolite of Brassicales, 1-methoxy-3-indolylmethyl glucosinolate, as well as its degradation product, 1-methoxy-3-indolylmethyl alcohol, forms DNA adducts in the mouse, but in varying tissues and cells.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary metabolite of Brassicales species, and its breakdown product 1-MIM alcohol are mutagenic in cells in culture after activation by plant ß-thioglucosidase and human sulphotransferase, respectively. In the present study, we administered these compounds orally to mice to study time course, dose dependence, tissue distribution and cellular localization of the 1-MIM DNA adducts formed. We used isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry to quantify the adducts and raised an antiserum for their immunohistochemical localization. Both compounds formed the same adducts, N(2)-(1-MIM)-2'-deoxyguanosine and N(6)-(1-MIM)-2'-deoxyadenosine, approximately in a 3.3:1 ratio. 1-MIM glucosinolate primarily formed these adducts in the large intestine, with a luminal-basal gradient, probably due to activation by thioglucosidase from intestinal bacteria. 1-MIM alcohol formed higher levels of adduct than the glucosinolate. Unlike after treatment with the glucosinolate, luminal and basal enterocytes were similarly affected in caecum, and liver and stomach were additional important target tissues. Maximal adduct levels were reached 8 h after the administration of both compounds. The hepatic DNA adducts persisted for the entire observation period (48 h), whereas those in large intestine rapidly declined due to cell turnover, as verified by immunohistochemistry. Hepatic adduct formation was focused on the periportal hepatocytes with concomitant depletion of glycogen, p53 activation and p21 induction. Adduct formation in caecum was associated with massive apoptosis, p53 activation and p21 induction, in particular after treatment with 1-MIM alcohol. It remains to be studied whether similar effects occur in humans after the consumption of Brassicales species.

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

SCOPE: Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin. METHODS AND RESULTS: Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin. CONCLUSION: The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

Bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design.

Broccoli consumption may reduce the risk of various cancers and many broccoli supplements are now available. The bioavailability and excretion of the mercapturic acid pathway metabolites isothiocyanates after human consumption of broccoli supplements has not been tested. Two important isothiocyanates from broccoli are sulforaphane and erucin. We employed a cross-over study design in which 12 subjects consumed 40 g of fresh broccoli sprouts followed by a 1 month washout period and then the same 12 subjects consumed 6 pills of a broccoli supplement. As negative controls for isothiocyanate consumption four additional subjects consumed alfalfa sprouts during the first phase and placebo pills during the second. Blood and urine samples were collected for 48h during each phase and analyzed for sulforaphane and erucin metabolites using LC-MS/MS. The bioavailability of sulforaphane and erucin is dramatically lower when subjects consume broccoli supplements compared to fresh broccoli sprouts. The peaks in plasma concentrations and urinary excretion were also delayed when subjects consumed the broccoli supplement. GSTP1 polymorphisms did not affect the metabolism or excretion of sulforaphane or erucin. Sulforaphane and erucin are able to interconvert in vivo and this interconversion is consistent within each subject but variable between subjects. This study confirms that consumption of broccoli supplements devoid of myrosinase activity does not produce equivalent plasma concentrations of the bioactive isothiocyanate metabolites compared to broccoli sprouts. This has implications for people who consume the recommended serving size (1 pill) of a broccoli supplement and believe they are getting equivalent doses of isothiocyanates.

The impact of loss of myrosinase on the bioactivity of broccoli products in F344 rats.

In vitro, animal, and epidemiological studies all show that broccoli products containing sulforaphane, the bioactive hydrolysis product of glucoraphanin (GRP), lower risk for cancer. As a result, GRP-rich extracts are appearing on the market as dietary supplements. However, these products typically have no hydrolyzing enzyme for sulforaphane (SF) formation. We evaluated safety and compared efficacy to other broccoli preparations. Four daily doses of 0.5 mmol GRP/kg BW, given by gavage to adult male F344 rats, caused temporary cecal inflammation that was essentially resolved four days later. A similar dose dispersed in the diet caused no inflammation. To compare efficacy, we fed rats 20% freeze-dried broccoli (heated or unheated), 3.5% broccoli seed meal, or 4.3% semipurified GRP, each balanced within an AIN93G semipurified diet, for 4 days. Diets lacking myrosinase (semipurified GRP and heated broccoli florets) caused upregulation of NAD(P)H-quinone oxidoreductase 1 (NQO1) in colon but not liver. Surprisingly, broccoli seed, rich in myrosinase and GRP, also caused NQO1 upregulation in colon but not liver. In contrast, unheated broccoli florets caused upregulation in both colon and liver. These data suggest that GRP supplements may not exert systemic effects. We hypothesize that within whole broccoli additional components enhanced sulforaphane-dependent upregulation of NQO1 in liver.

Nutrients and phytochemicals: from bioavailability to bioefficacy beyond antioxidants.

The effect of any dietary compound is influenced by the active bioavailable dose rather than the dose ingested. Depending on the individual predisposition, including genetics and medication, a bioavailable dose may cause different magnitudes of effects in different people. Age might affect the predisposition and thus the requirements for nutrients including phytonutrients (e.g. phytochemicals such as flavonoids, phenolic acids and glucosinolates). These are not essential for growth and development but to maintain body functions and health throughout the adult and later phases of life; they are 'lifespan essentials'. Major mechanisms involved in chronic, age-related diseases include the oxidant/antioxidant balance, but the latest research indicates indirect effects of dietary bioactives in vivo and adaptive responses in addition to direct radical scavenging.

Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study.

Broccoli sprouts are widely consumed in many parts of the world. There have been no reported concerns with respect to their tolerance and safety in humans. A formal phase I study of safety, tolerance, and pharmacokinetics appeared justified because these sprouts are being used as vehicles for the delivery of the glucosinolate glucoraphanin and its cognate isothiocyanate sulforaphane [1-isothiocyanato-(4R)-(methylsulfinyl)butane] in clinical trials. Such trials have been designed to evaluate protective efficacy against development of neoplastic and other diseases. A placebo-controlled, double-blind, randomized clinical study of sprout extracts containing either glucosinolates (principally glucoraphanin, the precursor of sulforaphane) or isothiocyanates (principally sulforaphane) was conducted on healthy volunteers who were in-patients on our clinical research unit. The subjects were studied in three cohorts, each comprising three treated individuals and one placebo recipient. Following a 5-day acclimatization period on a crucifer-free diet, the broccoli sprout extracts were administered orally at 8-h intervals for 7 days (21 doses), and the subjects were monitored during this period and for 3 days after the last treatment. Doses were 25 micromol of glucosinolate (cohort A), 100 micromol of glucosinolate (cohort B), or 25 micromol of isothiocyanate (cohort C). The mean cumulative excretion of dithiocarbamates as a fraction of dose was very similar in cohorts A and B (17.8 +/- 8.6% and 19.6 +/- 11.7% of dose, respectively) and very much higher and more consistent in cohort C (70.6 +/- 2.0% of dose). Thirty-two types of hematology or chemistry tests were done before, during, and after the treatment period. Indicators of liver (transaminases) and thyroid [thyroid-stimulating hormone, total triiodothyronine (T3), and free thyroxine (T4)] function were examined in detail. No significant or consistent subjective or objective abnormal events (toxicities) associated with any of the sprout extract ingestions were observed.

Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model.

Cruciferous vegetables, such as Brassica, which contain substantial quantities of glucosinolates, have been suggested to possess anticarcinogenic activity. Cutting and chewing of cruciferous vegetables releases the thioglucosidase enzyme myrosinase, which degrades glucosinolates to isothiocyanates and other minor metabolites. Cooking of cruciferous vegetables inactivates the myrosinase enzyme, allowing intact glucosinolates to reach the large intestine, where they can be degraded by the indigenous microflora into isothiocyanates. This local release of isothiocyanates may explain the protective effect of cruciferous vegetables on the colon epithelium. However, little is known about the amounts and identities of glucosinolate metabolites produced by the human microflora. The production of allyl isothiocyanate from sinigrin was investigated in a dynamic in vitro large-intestinal model, after inoculation with a complex microflora of human origin. Sinigrin and allyl isothiocyanate concentrations were analysed in the lumen and dialysis fluid of the model. Peak levels of allyl isothiocyanate were observed between 9 and 12 h after the addition of sinigrin. The model was first set up with a pooled and cultured human microflora, in which 1 and 4% of, respectively, 1 and 15 mM sinigrin, was converted into AITC. However, the conversion rate was remarkably higher if different individual human microflora were used. Between 10% and 30% (mean 19%) of the sinigrin was converted into allyl isothiocyanate. The results of this study suggest that allyl isothiocyanate is converted further into other, yet unknown, metabolites.

Glucosinolates: bioavailability and importance to health.

Epidemiological studies suggest that brassica vegetables are protective against cancers of the lungs and alimentary tract. Cruciferous vegetables are the dietary source of glucosinolates, a large group of sulfur-containing glucosides. These compounds remain intact unless brought into contact with the enzyme myrosinase by pests, food processing, or chewing. Myrosinase releases glucose and breakdown products, including isothiocyanates. These highly reactive compounds are potent inducers of Phase II enzymes in vitro. Isothiocyanates also inhibit mitosis and stimulate apoptosis in human tumor cells, in vitro and in vivo. To understand and exploit such effects it is important to determine the routes of absorption of glucosinolate breakdown products, their metabolism, and delivery to systemic tissues. Glucosinolates can be gained or lost by vegetables during storage. They may be degraded or leached during processing, or preserved by thermal inactivation of myrosinase. Glucosinolates are broken down by plant myrosinase in the small intestine or by bacterial myrosinase in the colon. Isothiocyanates are absorbed from the small bowel and colon, and metabolites are detectable in human urine two to three hours after consumption of brassica vegetables. Interpretation of epidemiological data and exploitation of brassica vegetables for human health requires an understanding of glucosinolate chemistry and metabolism, across the whole food chain, from production and processing to the consumer.

Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans.

Broccoli sprouts are a rich source of glucosinolates and isothiocyanates that induce phase 2 detoxication enzymes, boost antioxidant status, and protect animals against chemically induced cancer. Glucosinolates are hydrolyzed by myrosinase (an enzyme found in plants and bowel microflora) to form isothiocyanates. In vivo, isothiocyanates are conjugated with glutathione and then sequentially metabolized to mercapturic acids. These metabolites are collectively designated dithiocarbamates. We studied the disposition of broccoli sprout glucosinolates and isothiocyanates in healthy volunteers. Broccoli sprouts were grown, processed, and analyzed for (a) inducer potency; (b) glucosinolate and isothiocyanate concentrations; (c) glucosinolate profiles; and (d) myrosinase activity. Dosing preparations included uncooked fresh sprouts (with active myrosinase) as well as homogenates of boiled sprouts that were devoid of myrosinase activity and contained either glucosinolates only or isothiocyanates only. In a crossover study, urinary dithiocarbamate excretion increased sharply after administration of broccoli sprout glucosinolates or isothiocyanates. Cumulative excretion of dithiocarbamates following 111-micromol doses of isothiocyanates was greater than that after glucosinolates (88.9 +/- 5.5 and 13.1 +/- 1.9 micromol, respectively; P < 0.0003). In subjects fed four repeated 50-micromol doses of isothiocyanates, the intra- and intersubject variation in dithiocarbamate excretion was very small (coefficient of variation, 9%), and after escalating doses, excretion was linear over a 25- to 200-micromol dose range. Dithiocarbamate excretion was higher when intact sprouts were chewed thoroughly rather than swallowed whole (42.4 +/- 7.5 and 28.8 +/- 2.6 micromol; P = 0.049). These studies indicate that isothiocyanates are about six times more bioavailable than glucosinolates, which must first be hydrolyzed. Thorough chewing of fresh sprouts exposes the glucosinolates to plant myrosinase and significantly increases dithiocarbamate excretion. These findings will assist in the design of dosing regimens for clinical studies of broccoli sprout efficacy.

Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli.

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 micromol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 +/- 12.7% and 10.2 +/- 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 +/- 13.9 and 6.9 +/- 2.5 micromol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.

Ascorbigen and other indole-derived compounds from Brassica vegetables and their analogs as anticarcinogenic and immunomodulating agents.

Searches for the natural compounds that determine the anticarcinogenic properties of a cruciferous-vegetable diet, revealed the products of alkaloid glucobrassicin biotransformations; among these, ascorbigen, an indole-containing derivative of L-ascorbic acid, was found to be the most abundant. Study of chemical properties of ascorbigen showed that it is capable of different transformations in acidic (including gastric juice) and slightly alkaline (including blood) media. The stable and unstable products of ascorbigen transformation determine the biological properties of the compound. The most important product of ascorbigen transformation in gastric juice is 5,11-dihydroindolo[3,2-b]-carbazole, with a binding affinity to the Ah receptor only 3.7 x 10(-2) lower than that of tetrachlorodibenzodioxin. This compound may be responsible for modifying P450 enzyme activities. Ascorbigen and its analogs are available synthetically. Their biological evaluation showed that some of the compounds of these series are immunomodulators. The most active is N-methylascorbigen, which demonstrates therapeutic effects (inhibition of tumor growth, protection of animals from bacterial and viral infections). The immunomodulatory activity of natural ascorbigen may be an additional factor of importance for the anticarcinogenic properties of a cruciferous-vegetable diet.