Rhonda: Hello, everyone. I am sitting here with Dr. Jed Fahey who is an assistant professor and a clinical biochemist here at Johns Hopkins. Jed played a huge role in discovering that broccoli sprouts contain very high amounts of the precursor to sulforaphane called glucoraphanin. Since then, he has made huge contributions to the field and has studied sulforaphane, and glucoraphanin, and broccoli sprouts, and from everything from cancer to the microbiome to the brain. So I'm very excited to have a conversation with him today. So, Jed, maybe you can sort of kick it off by telling people a little bit about what glucoraphanin and sulforaphane is and some of the history behind discovering it.

Jed: Sure. I'd be happy to. So, sulforaphane is a small molecule that was discovered in broccoli by Paul Talalay and Yuesheng Zhang, his student at the time, in 1992 or so. They published on it. And it was of interest in particular because it was a huge inducer of protective enzymes in people. Of course, it wasn't known that this occurred in people at the time, but in cell cultures and in animals, they showed that it up-regulated the protective enzymes, cytoprotective enzyme system known by some as the phase II enzyme system.

And, Paul then, who was one of the grandfathers of chemoprotection and has...it really helped make people stand up and pay attention to the fact that you could potentially prevent diseases like cancer. Paul asked me to join the group in 1993, and the challenge was, can you get broccoli with more sulforaphane? I came from a background in plant biotechnology, and, of course, I said, "Yes, we should be able to." And we started trying to breed and cross broccolis to get higher levels of sulforaphane.

Quickly, it became obvious that it was very difficult to predict how much sulforaphane a broccoli plant would have based on things like smell, touch, color and you pretty much had to run it through an HPLC to measure levels of sulforaphane. What's more is we realized that sulforaphane is not what's present in the intact plant. So what's present in the plant is something called glucoraphanin and that is a thioglucose conjugated molecule, not to get too heavily into the biochemistry of it, but it's a bigger molecule, it's a precursor and it's very stable. Sulforaphane is not at all stable. It's highly reactive. And so at any rate, it turned out that the intact broccoli plants had glucoraphanin at various levels ranged all over the map, couldn't figure out exactly how much one had without doing chemical analyses.

And we were going out to the field in the eastern shore of Maryland. And eventually, winter came along, and we couldn't go out to the fields and get broccoli anymore. So we started growing them in incubators in the labs starting from seeds. And lo and behold, it turns out that broccoli sprouts had much higher levels of the precursor of sulforaphane, glucoraphanin, than did the mature plants, the heads of broccoli that you buy in the market. So we then determined that if you grow broccoli sprouts all the same way, which you pretty much do if you are a home sprouter or a commercial sprouter, you add a certain amount of light, you add fresh water, you grow them for a certain amount of time at a certain temperature. So if you did that with a whole slew of different cultivars, that's the different sub-varieties of broccoli, you got a range of different activities, different amounts of glucoraphanin.

Anyway, bottom line is by selecting the appropriate genotypes, the appropriate broccoli genetics, if you will, we identified some varieties that had very high levels of glucoraphanin. Paul and I made a conscious decision at that time that we were going to promote the use of broccoli sprouts, not broccoli seeds. Because it turns out the seeds had the highest amount on a per gram basis of glucoraphanin. But at the time, no one had eaten broccoli seeds. You know, they weren't green. They didn't have the sort of cachet or the appearance of eating healthy green vegetables. So we focused on sprouts, which had much higher levels than the mature plants, although lower levels than the seeds.

And it turns out that then there is an enzyme that the plant tissue contains called myrosinase. And that enzyme converts glucoraphanin, the precursor, which is stored in vacuoles in the plant cells to sulforaphane. And typically in nature, the plant does that as a protective mechanism. If an insect starts chewing on the leaf of a broccoli plant, for example, it breaks open cells, right? And so those cells then release their glucoraphanin and the enzyme that's present at the same site hydrolyzes glucoraphanin and forms sulforaphane. And sulforaphane repels those bugs or is in some cases toxic to those bugs. So they go...and they fly away or they crawl away. But it turns out that sulforaphane is also a foreign compound for our cells. But in the process of being recognized and chucked out of the cell, if you will, it up-regulates the protective enzymes in those cells. And so that's why it's so special.

Rhonda: Like a hormetic effect, like a hormetic effect where...

Jed: Yeah. So clearly, if all you ate was sulforaphane, you'd be in trouble.

Rhonda: Yeah.

Jed: But the same with just about anything you can suggest. So it gears up or cranks up the protective mechanisms of the cells.

Rhonda: And one of those, I guess, one of the main protective mechanisms would be the Nrf2 pathway.

Jed: Yeah. So I have a feeling that sulforaphane would be just be another interesting phytochemical if at almost the same time, we, and I use the term "we" loosely because I was only very peripherally involved with this. But a number of people at Johns Hopkins and in Japan discovered, in Japan and England, I should say, but a small number of people discovered this Nrf2 pathway and really fleshed out all the details of it. And this happened in parallel to the interest in sulforaphane and broccoli.

And it turns out that the Nrf2 pathway is an extremely important pathway for up-regulating the protective enzymes and protective proteins including perhaps the heat shock proteins in cells so that they can protect themselves against various insults. It's an integral part of protection against a variety of chronic diseases. And as I say, Tom Kensler and Paul Talalay, Albena Dinkova-Kostova all at Hopkins, Masi Yamamoto in Japan, John Hayes in England, excuse me, and I'm certainly leaving out a few people, but small number of people initially put this pathway on the map. And it turns out that the Nrf2 pathway controls something between 3% and 5% of our cellular proteins. So it's very important.

Rhonda: Wow. I didn't realize it's that much.

Jed: Yeah. And why is it important? It's important because it recognizes molecules like sulforaphane through a system, a biochemical system that I hesitate to explain without props and graphs, and also because I'm not the world's expert on this, certainly. But it recognizes sulforaphane or other similar molecules as they enter cells in the cytoplasm of the cells. Then there's actually a chaperone protein that's in the cytoplasm. It's called Keap1. That molecule when it binds to sulforaphane, or vice versa, changes in conformation and it releases Nrf2, which then migrates to the nucleus and turns on or up-regulates the antioxidant response element which is responsible for the transcription, for initiating transcription of a whole series of protective genes or genes in coding for a bunch of protective enzymes.

And this happens very quickly, this protective response, and it's quite efficient. So to get back to sulforaphane, so sulforaphane was discovered and then everybody started searching for the mechanism by which it acted. The Keap1-Nrf2 mechanism was discovered, and they sort of both developed a following, if you will, in parallel. And it turns out that sulforaphane is still probably the most potent activator of Nrf2 to be found naturally in from the natural world. There have been synthetic activators that are more potent, that have been produced, been made chemically, but sulforaphane still sort of takes the cake in terms of its protective ability, and ability to up-regulate protective enzymes. You know, when I give lectures to students, I frequently make, point out to them how they're doing this glucoraphanin into sulforaphane conversion, so I will do it for this webinar.

When you chew on a red radish, you're familiar with the fact that the first sensation is cool and crunchy and then within 20, 30 seconds, you develop heat, you start tearing, your nose starts running, has a lachrymating effect. So what you are doing is you're acting like that insect that I told you lands on a leaf of a broccoli plant. You're breaking the plant cells by crunching on the radish, you're releasing a compound that's very, very similar to glucoraphanin. It's actually called glucoraphenin in radishes. It happens to be more of a lachrymator. It's got a more mucus-inducing effect in people. And you're letting myrosinase in those radish tissues act on glucoraphenin and form sulforaphene and some other related isothiocyanates. That's the broad name for that category. So it happens that fast when you chew on fresh vegetables that contain this system.

Rhonda: Does sulforaphane also activate Nrf2?

Jed: It does, but to a lesser degree than sulforaphane.

Rhonda: So many of these isothiocyanates also activate Nrf2, but not to the same degree as sulforaphane?

Jed: They do. And just in case you're about to ask me, you find this system, it's been dubbed the mustard oil bomb by some, 20 or 30 years ago. You find this system almost exclusively in cruciferous vegetables, or brassica vegetables or cole crops, they're sometimes called, depending upon where you come from, where you hail from. In Eastern Europe, they typically call them cole crops. So this happens to be a very large family of, I don't know, 500 or 600 genera, many, many thousands of species.

Rhonda: Yeah, I know, like, maybe 10 of them.

Jed: Well, we know maybe 20, but, I mean, there are very many of them. And they grow worldwide although the brassica or the cold crops that we're familiar with in the United States are all temperate climate crops. They're common to areas where there's a cold winter or a freeze in the winter. There are some interesting relatives we can talk about later or now if you like which are tropical. Moringa is the one which has gotten most attention recently and we've been interested in for about 20 years. It's a relative of broccoli, but it's a tropical plant. It's actually a tree. And it, too, has this system of glucosinolate, myrosinase, and isothiocyanate. The former being the storage form in the plant, the latter being the biologically active form.

Rhonda: Right, okay. Yeah, maybe we can get to that little bit later, but talk a little bit more about the sulforaphane in the broccoli sprouts. I wanted to ask you something because you mentioned, it really piqued my attention, as you said that broccoli seeds actually had higher amount of glucoraphanin than the sprout. If I were to say take those broccolis, because I sprout, If I were to take those broccoli seeds, add a little water and blend them in a blender, would that activate myrosinase within the seed, the crushing of the seed?

Jed: You bet.

Rhonda: So I could actually get a higher, more potent amount of sulforaphane theoretically if I were to just take the seeds and because... Man, that would be easier than sprouting in a way.

Jed: Yes. That's true. So, interestingly when we discovered that, and this was published in 1997, so it's ancient history. It's probably before you were born, right? So when we discovered that broccoli sprouts in seeds were such a potent source of sulforaphane or its precursor, we weren't aware that anybody had eaten broccoli seeds. People eat all sorts of seeds, poppy seeds and rapeseed or canola seed, in fact, are used to express oil and they're a close relative. But no one had eaten broccoli seeds. And when we tried them, they're quite bitter. But interestingly, if you bake them just gently, don't scorch them, if you bake them, they get their very nutty taste and they're sort of pleasant tasting. The problem is if you bake them, you kill the enzyme, you kill myrosinase. Because it's a...

Rhonda: Right. So that's something important, retention.

Jed: Yeah. So there actually have been some epidemiologic studies, and I'm getting off topic a bit, but some epidemiologic studies suggesting that people who eat certain amounts of cruciferous vegetables have reduced risks of various cancers. Breast cancer and lung cancer are high on the list of cancers that have been studied. Excuse me. And it turns out that if you eat raw cruciferous vegetables, you're a bit better protected, again, based on the epidemiology. This has not been the subject of interventions of randomized clinical trials, for example. But it looks like the protective effect is greater if you eat raw vegetables. But now, a lot of people don't like to eat raw cauliflower or broccoli or...well, radishes, they do. So at any rate, you asked about the content in broccoli seeds and indeed, they have much more glucoraphanin and they have plenty of active myrosinase. But I have a feeling, I've never made a smoothie from raw broccoli seeds. I have a feeling it would be pretty bitter.

Rhonda: I'll let you know, because I'm going to try it.

Jed: I thought you might.

Rhonda: You made a pretty important point for people and that is that the myrosinase enzyme is heat-sensitive and as you pointed out, most people like to cook their cauliflower and broccoli or slightly steam it and their trade-off for that is maybe it tastes a little better, but also it inactivates the myrosinase enzyme.

Jed: Correct.

Rhonda: So they're not actually getting as much sulforaphane from the glucoraphanin as they would have if they were to just chew on that raw broccoli. But what about the myrosinase enzyme? Is the stability the same throughout all the cruciferous family? So, like, if you look at the broccoli sprouts versus the mature broccoli or kale?

Jed: So myrosinase appears to be about as stable throughout the family. There are differences, subtle differences to the protein composition of myrosinase. This is in different cruciferous vegetables, to be sure. Subtle enough so that I can’t even describe them to you. I mean, they're minor differences, but they certainly must confer increased or decreased stability or catalytic ability on those molecules. Interestingly, if you ingest glucoraphanin or glucosinolates without myrosinase at all, you then can count on the bacteria in your gut, in your intestines, primarily, to make that conversion. And sometimes scientists think they're so darn smart. Just to back up just a little bit, we observed that when you delivered just glucoraphanin to people, just give them one dose, one reasonable amount that which they would get in a serving of broccoli, and then we collected their urine for 24 hours. We asked them to collect their urine for 24 hours, so you get a full 24-hour collection which pretty much spans the period during which the enzyme myrosinase acts, sulforaphane goes into cells, gets recognized, turns on Nrf2, gets spit back out, and then gets excreted in the urine.

So by collecting a 24-hour urine, we can get a very good idea of how much bioavailability there is, how much metabolism and availability. And it turns out that if we looked at 100 different people, and we did, after giving them one dose of glucoraphanin, their bioavailability, the amount they gave back to us in their urine was all over the map. It ranged from very, very little, but always something to 40% or 50%, or even 60% or 70% of what we gave them. But the mean was pretty low. It was about 10%. So most people converted, and metabolized, and excreted only about 10% of what they were given. So, when you give sulforaphane itself, the end product, the active ingredient, if you will, we get more, like, 70%, 75%, 80% bioavailability. Still a bit of variability person-to-person, but since we're up sort of near maximum, it appeared to be not as great. Well, it was not as great in terms of a percent of the whole. So that sounds a little convoluted. But essentially most sulforaphane came back at us in the urine and was available. So, in our infinite wisdom, we thought if we co-deliver myrosinase, if we give these people active myrosinase in addition to glucoraphanin, they're all going to give back 70% to 90% of the metabolites and we'll get rid of the variability person-to-person. Didn't happen that way.

Rhonda: Really?

Jed: Really. It didn't happen that way. What did happen is that we moved the bar up so that instead of 10% bioavailability for 70%, we had about 35% or 40%. So the average moved up substantially. It was about three or four times more, but there was still quite large person-to-person variability. So we have to sort of step back a little bit and say, "Yes, this is due to intrinsic myrosinase activity in the gut because certainly, we hadn't done anything to get rid of that. That was still acting on these molecules.

But also just innate mammalian genetics, differences between you and me and the way we process these metabolites once they're formed, differences in absorption, differences in distribution, in metabolism. So there's a lot we still don't know. There's a whole lot we still don't know. But as a result of this sort of cocky thinking that we could abolish this variability and at least have a very predictable amount delivered to people, we did learn that we can greatly increase bioavailability by delivering it with myrosinase. And, in fact, there are now companies that are selling glucoraphanin plus myrosinase. You have to be careful if you buy those supplements because it's not a given that they're going to be as stable as multivitamins and things that you can just leave on the shelf for years.

So you need to pay attention to the expiration dates on those products that have live active enzyme. I'm not in the supplement business, but I've become more and more familiar with it. Most supplement makers, it appears, don't like to sell stuff that has to be refrigerated or put in a freezer. I think it increases their cost. And so most of the supplements that I'm aware of are made to be shelf-stable, but I think it won't... If you were to buy a supplement that has active myrosinase or that says they have active myrosinase, I think it wouldn't hurt to put them in a refrigerator, treat them as you would treat a probiotic supplement.

Rhonda: And you've actually measured the content of glucoraphanin and/or sulforaphane in some of these supplements that are found on the market. Is that correct?

Jed: We have. We have.

Rhonda: And you found that there's only a really small amount of supplements that actually contain what they say they contain?

Jed: Yes.

Rhonda: I think one of them you mentioned was a French company that has actually sulforaphane in it, Prostaphane?

Jed: Yes. So I've actually been told that there...and I think it was someone in the industry that told me this, that there are something, like, 1,000 supplements that claim to have broccoli sprouts or broccoli extracts in them that are on the market now. I certainly it would take me all of my waking hours to try to vet that statement and validate the content of each of them. But, in our lab, we have looked at a number of supplements. Some of those that are more prominent or that you see advertised or some of those that we've been aware of because we know people that have asked us about them. And many of them 20 years ago, there were a bunch of supplements that said they have sulforaphane and sulforaphane cures cancer because John Hopkins says so. This is highly inaccurate and they should have been taken to court. But you can't make statements like that. Nobody has shown that any of these supplements prevent cancer or cure cancer. We certainly hope to be part of the group of scientists who are able to put some teeth behind that evidence and, eventually, I think one day it may be shown that in people, cancer can be prevented by taking something like sulforaphane, but that has not been proven to date.

So anyway, to get back to your question, we have looked at a variety of supplements. Twenty years ago, when they started coming out, there were many supplements that we could demonstrate were not even broccoli sprout or broccoli. It had no broccoli in them yet they said they were broccoli supplements. People were selling what they called broccoli seeds and when you actually grew them out and grew plants from them, you grew cauliflower or you grew canola or rapeseed from them or something else. Or in some more egregious cases, we found stuff that was sold as broccoli seeds that was really alfalfa seeds. So a lot of liberties were taken, a lot of shysters were on the market. I think that's cleaned up somewhat now, but now there are a ton, and as I say, I was told a 1,000, but many, many, many supplements that say they have sulforaphane or glucoraphanin in them.

We have analyzed a small number of them, those that are made by labs or companies that we've read and heard good things about that haven't been challenged by the FDA for sanitation problems or for mislabeling. And there are supplements that contain glucoraphanin alone, there are a few now that contain glucoraphanin plus myrosinase and there are a few that contain sulforaphane. We are publishing this, should be out in the next week or two, a paper in which we actually look at the bioavailability of sulforaphane from a French supplement that is called Prostaphane. There's been at least one publication that looked at its ability to change the PSA trajectory in men who have had prostate cancer.

Rhonda: And that's a biomarker for prostate cancer?

Jed: It's a biomarker for prostate cancer return rate. And so those tablets, they're tablets that should be refrigerated to prolong the life of the sulforaphane in them. The bioavailability of the sulforaphane from those tablets was essentially identical to that from powders that we made in the lab by extracting broccoli sprouts and treating them with myrosinase and freeze-drying them.

Rhonda: That's very impressive.

Jed: So we were delighted to see that they worked. Yeah. Unfortunately, they're not being sold in the U.S. I hope that one of the supplement companies can... I hope that somehow or another business, the business of supplements gets them to this country. I mean, we haven't really talked much about the clinical trials we've been involved with, but I can tell you that about a year and a half ago, two years ago, we finally reached our limit in terms of the ability of our center that's called the Cullman Chemoprotection Center here at Johns Hopkins. We reached the limits of our ability to produce broccoli sprout extracts for clinical trials, essentially, for other people's clinical trials. We are a small group and we've done a number of small clinical trials here, but we've had many, many people come to us looking for something they can use in a clinical trial, people with expertise in areas different than ours like asthma and allergy and COPD and a variety of psychological, neuropsychiatric conditions.

And so we've supplied them with powders, freeze-dried extracts that we made. I'd like to say we made them all at Johns Hopkins. We actually had to go all the way across the country to the largest freeze-drying factory in the world, it's called Oregon Freeze Dry, and do the extraction, hundreds and hundreds of gallons, do the extractions there and use their massive freeze-drying facilities, then do all the quality assurance, and bring them back to Baltimore. In many cases, we had them put into gel caps so that they were easier to dose. And repeated quality assurance and microbial testing and on and on and on. And then all of the paperwork and documentation that goes along with that. I'm saying that, in a sense, to complain, but to say that as a basic research operation, we were becoming overwhelmed with being a little too helpful, I guess you could say, to people who had very interesting clinical trials that we wanted to see happen. Because they were working with the stuff that we had discovered and work with.

So a couple of years ago, I went to a small number of supplement companies and said, "Help," and challenged them to take the supplements that they were making that we had tested and verified contained what they said they had and to offer to supply them for clinical trials that we were approached about partnering on. And not only to supply the material free of charge, but to supply all the documentation and paperwork that would go along with an application to an institutional review board or to the FDA, in fact, for an investigational new drug application for permission to work on that disease with that compound. And a few companies did come forward. One of them was called Nutramax and they have a product called Avmacol, which they sort of took a page from our playbook and they made something with glucoraphanin and myrosinase.

And so that's actually been used in a number, quite a large number of clinical trials now. And so I don't think... We've tested it in people, of course. I don't think there's anything ready to be published yet, but those publications will be coming along very shortly.

Rhonda: And that actually does contain the glucoraphanin and myrosinase and you've validated that?

Jed: Yes, we would certainly not recommend something to anybody without checking it. And we've checked every batch that they've put on the market because we don't want to be in a position... I don't want to have egg on my face, and have recommended to a friend that this is a product worth using and then have it turn out to be a dud.

Rhonda: So you mentioned the sulforaphane one, which is the Prostaphane, the Avmacol, which has the glucoraphanin plus the myrosinase, and then there's the one that just has glucoraphanin. I think you mentioned it was by Thorne?

Jed: They're one of a number of companies that have a decent...I shouldn't say that have a decent one. They're probably if you put this on a webinar, there are probably going to be 10 companies that say, "We have a decent one, too." I'm sure there are others, but we've tested...

Rhonda: You've tested, and that's important.

Jed: Yes, we've tested a product by Thorne, which is a medium or a large-size supplement maker, and their product is called Crucera-SGS.

Rhonda: But that only has glucoraphanin in it?

Jed: Right.

Rhonda: And this actually brings me back to...I kinda want to get back to this and that is the microbiome. And so if you're taking a supplement that only has glucoraphanin and does not have the myrosinase enzyme there, then you're relying on what you possibly potentially have in a species of bacteria in the gut, which maybe you can illuminate what that species of bacteria is, or if there's ways we can increase it, probiotics. Obviously, wiping out the gut bacteria. So people with chronic antibiotic use may not be able to convert glucoraphanin very well, the sulforaphane, I would presume.

Jed: Right. You must have read one of our papers. So in fact, we did show that, this was a number of years ago now, we showed that if volunteers took a Fleet enema, self-administered enema, which reduces the number of bacteria, certainly in their large intestine, by, I don't know, five or six orders of magnitude, and took a preoperative antibiotic course, in other words, something that one would take prior to having intestinal surgery, they essentially wiped out most of the bacteria in their intestines. And those people went from whatever their level of conversion of glucoraphanin to sulforaphane and its metabolites was, which was, as you heard earlier, was 10% to 70% to nothing. So they lost their ability to convert glucoraphanin to sulforaphane.

And then over the course of a couple of weeks, I think, as I recall, we followed them, one or two subsequent challenges with glucoraphanin two weeks and four weeks later, I think. They gradually regained their ability to do that reaction. Clearly residual bacteria or new colonizers in their guts had myrosinase activity. So we've looked at hundreds and hundreds of people's ability to do this conversion and nobody can't do it. So everybody that's living and breathing appears to have myrosinase-producing bacteria in their gut. But as we said earlier, obviously, it's at very different levels.

Just as clearly, although we don't do bacterial sequencing here, and this is a new and very exciting field of the microbiome, but it's very clear that there are certain strains or certain species of bacteria that do have myrosinase activity. Others have shown that there are, I believe one of them is enterococcus, there are lactobacilli that have myrosinase activity. There are Bifidobacterium, I believe, that have it. And again, this is not my field of expertise, so I may have misspoken. But the bottom line is that there are a number of species of bacteria that do this, but certainly not all bacteria in your gut.

This whole, incredibly fascinating field of the microbiome has certainly shown that each of us has different populations and different ratios of different types of bacteria in our guts. And certainly, to the degree that this is true, that's going to affect the amount of myrosinase activity in our guts. I can tell you that we have done, we haven't published this, but we've done studies with mice in which we've challenged them with glucoraphanin on a regular basis over time. And the goal was to try to push them by natural selection, to try to push them to have more bacteria with more myrosinase activity. The fact that we haven't published it means we didn't move the needle well enough or reproducibly enough to have a good story to tell, but certainly that's what we believe might happen.

Having said that, we look at real people and we query them as to their habits. There is no evidence to date that people who say they eat a pound of broccoli a day, that's a little bit of an exaggeration, but people who eat a lot of broccoli don't necessarily convert glucoraphanin better than people who don't eat much at all. So that speaks to some other selective pressure on the gut on myrosinase-containing bacteria.

Rhonda: Yeah. I would think that even just eating a healthy meal in general when you're eating a diet high in vegetables and fermentable fibers and things that are growing Bifidobacteria and lactobacillus and all these species of bacteria that are commensal bacteria, that even that itself would potentially then increase your chances of being able to have more myrosinase because you're having more of this commensal microbiome, but I guess we don't know.

Jed: You know, we don't know. I think both of our hunches are correct. But the proof is in the pudding. This whole issue of probiotics...

Rhonda: Yes. That was going to be my next question. That would be interesting to view.

Jed: Well, it's fascinating. You know, in Europe, there is a lot, I think the regulatory climate in Europe for probiotics is a bit more receptive than in the U.S. You probably know more about it than I do. But I know that the use of probiotics seems to be increasing in the U.S., and studies by those who are expert in the field of probiotics are mixed.

Rhonda: Well, it's the same case as the glucoraphanin sulforaphane supplements. A lot of the probiotics in the market, there is a lot of dead bacteria in them...

Jed: A lot of garbage, yeah.

Rhonda: ...a lot of garbage. I mean, this is ubiquitous in all supplements. You know, I think there was even a study showing something like 70% of, like, a lot of these supplements on the market are not actually what they say they are, they don't have the concentrations they say. But, yeah, I think that there is a lot of mixed data and that comes down to not having quality probiotics and also the numbers. That's another problem, too low numbers of....

Jed: Right, 10 billion per...

Rhonda: Yeah, that's nothing. You know, it's like a drop in the pool. It's not going to even move the needle. But there is a probiotic out there that I've seen at least two dozen publications including clinical trials in humans and then there's more mechanistic studies in animals using VSL #3. It's called, they're sachets and they have 450 billion probiotics per sachet and they have a mixture of lactobacillus and Bifidobacterium and some other ones but showing efficacy in treating IBS, Crohn's. You know, so I think you can find quality probiotics, but it's just, again, it's the same thing that you've seen in your field, is finding the right one.

Jed: Absolutely. I think the issue of the numbers of cells, I mean, to a non-scientist consumer, "billions" sounds like a huge number. Well, yeah, it is, but it's still...you know, you can fit a billion bacteria on a pin, probably, well, maybe a teaspoon. Anyway, the issue there, though, is a lot of the issue is survival. So if you boast about having billions of bacterial cells of a certain type in yogurt, for example, and you're not on proton pump inhibitors, so you have an acid stomach and you send that yogurt through your stomach, how many of those bacteria actually make it through the acid environment of the stomach? So, I mean, people have studied that certainly, but there are studies, as you say, with a small number of specific strains showing effects. I think some of the more striking effects have to do with childhood diarrhea and there are even studies showing effects on asthma.

Rhonda: Effects on the brain. It's incredible.

Jed: Yeah. So I'm absolutely a believer that what's goes on in the gut matters to the rest of the body. There is no question about it. I think, as you say, a lot of the studies suffer from the fact that what's being given probably couldn't have been expected to be efficacious anyway because it just died or didn't get there. But speaking of probiotics, wouldn't it be cool if someone came up with a myrosinase containing strain and put it in a probiotic formulation so that you could take that along with your cruciferous vegetables?

Rhonda: So cool.

Jed: I believe that there are companies that are working on that now because certainly, it's well enough known what strains of bacteria have myrosinase activity. There may be issues of getting that activity maintained when you grow up vats full of bacteria to freeze-dry them, to put them in a formulation. But I hope it's only a matter of time before we see formulations that contain myrosinase and that are safe. I mean, obviously, safety of these probiotic formulations is something that is a concern to the regulators and it should be. So we'll see. I don't think we're quite there, but I hope we are getting there.

Rhonda: You actually showed something very interesting about the effects of sulforaphane on a certain type of microbe species, H. pylori.

Jed: So H. pylori, it's full name is Helicobacter pylori. Helicobacter pylori is a very interesting organism in that it grows primarily or exclusively in the stomach. And it occupies that niche in a very unique fashion. It apparently has an enzyme that is called urease, but an enzyme that allows it to neutralize the pH in a little micro-environment around the bacterial cells in the stomach. We shouldn't use the term "gut" because we also use the term "gut" to refer to the intestines.

So the stomach is very acid, extremely acid, that's part of the way it does its job. And Helicobacter tunnels into the mucus layer inside the stomach and, with enzymes, creates this little zone of neutral pH which allows it to thrive, otherwise it would be killed as are any other, or almost any other bacteria that enter the stomach. And it's a very interesting critter, if I can use the term. Can I call it "critter"?

Rhonda: Yeah.

Jed: It's a very interesting critter because some people, some very well-respected and well-known microbiologists, for example, Martin Blaser at NYU, maintains and I happen to drink this Kool-Aid, I happen to believe this, that Helicobacter has been around all during the evolution of humankind. Certainly, for tens of thousands of years, people have had Helicobacter in their systems. And something like 55% of the world's population at the moment has Helicobacter in their systems. In some areas, recently in Japan and certainly some areas in Africa and Asia, almost all people have Helicobacter, and some areas of South America. And it's a colonizer. It's a commensal symbiotic organism, it would appear. It may actually confer benefit on its host, in other words, you, the person.

If levels of Helicobacter get too high, if the number of bacterial cells per square millimeter or per square inch or per square mile or however you want to quantify them gets too great, then they start to have clearly pathological effects. They can cause ulcers. They do cause ulcers and that can eventually lead to stomach cancer. I believe the last estimate I saw was that the World Health Organization considers Helicobacter to confer, I think, a three or four to six fold increased risk of stomach cancer if you are colonized. But the question is if you are colonized with Helicobacter, can you reduce the levels of colonization, keep it down to a low roar, as it were. In other words, let a few of them hang around in your stomach as long as they don't overrun the system?

And so the prescription for someone who has Helicobacter in this country and I believe in Europe, I'm not a gastroenterologist, but, is to wipe it out. So to treat it with so-called triple therapy, three separate antibiotics and kill it so you can't find it anymore. And I should say about 15% of people who are given that treatment either can't take it or it doesn't work. So an alternative that many people find appealing is a dietary approach to controlling Helicobacter. In other words, and we don't have answers to these questions, but if so many people around the world are colonized and it's been with us for so long and humankind is still humankind, are there perhaps benefits to having it? So, for example, it's been shown, I believe this was Martin Blaser who showed this a number of years ago, that there is an inverse correlation between Helicobacter infection and childhood asthma. So it may protect, due to stimulation of the immune system, other mechanisms. It may protect against certain diseases, but we just don't know enough.

So maybe reducing the levels of Helicobacter in the stomach, again, keeping it to a low roar where it can't cause or where it doesn't cause major inflammation, doesn't give you ulcers may be enough to reduce the risk of its causing stomach cancer at some point in the future. If it doesn't cause inflammation and there are only a few cells here and there that are sort of hanging on, maybe that's okay. So that can be done by a dietary approach. And with that in mind, we actually looked at the ability of sulforaphane to kill Helicobacter. Of course, if it had wiped out all the Helicobacter in people who are infected, I would have been happy with that also. That would have certainly been an interesting finding.

What we ultimately found, and this was done with collaborators in Japan in a 50-person trial, we found that Helicobacter can reduce the levels...sorry, the sulforaphane or broccoli sprouts, actually, fresh broccoli sprouts were able to reduce levels of colonization in infected people or colonized people and were able to reduce markers of inflammation in those same people. That work grew from an observation that I made with a different colleague, a French colleague who was visiting the U.S. on sabbatical, Alain Lozniewski. So he and I discovered and published in about 2002 that in vitro, in a test tube, sulforaphane was very capable of killing Helicobacter. Not only did it kill natural strains, but it killed strains that he had re-cultured from some of his patients. He's a gastroenterologist, and it killed singly and doubly antibiotic-resistant strains. So, as you know, antibiotic resistance in bacteria of all sorts in all settings is a huge problem and Helicobacter is no different. Once Helicobacter in people starts seeing a bunch of antibiotics, some of them develop resistance including resistance to two of the commonly used antibiotics that are used to treat it. So the fact that sulforaphane was equally effective in killing them was, we thought, quite significant. And interestingly, sulforaphane is not as potently antibiotic against a whole variety of other bacteria.

Rhonda: So do you know why the H. pylori is so sensitive to it?

Jed: You know, we don't. We thought we had some clues and, actually, Dr. Lozniewski's colleague in France had done some work on that which was never published. This colleague, for reasons that we need not get into here, is no longer in the business of science and so that paper never got published and we never really finished the proof. So we're not sure why it's so potently antibiotic. One thing I can tell you is that in the quest for that answer, quite recently, Kitty Stephenson who works here with us and I started looking at the ability of sulforaphane to inhibit urease, which is that enzyme that I told you that neutralizes the pH in the mucus of the stomach. And we found that, indeed, sulforaphane is quite an effective inhibitor of that enzyme.

But, so again, we thought we had a really eureka moment, but it turned out that wiping out that enzyme wasn't sufficient to kill Helicobacter because strains... How do I put this? Strains of Helicobacter that had been engineered by others to not contain urease were still killed by sulforaphane. So it wasn't...so the urease... So inhibiting urease might be important from a disease prevention standpoint, but it's not how the molecule killed the bacteria. Sorry to present such a complicated story, but that's the way things roll in this business.

Rhonda: That's science. Yeah, for sure. But the also very not surprising, to me, results of it lowering inflammation is one of my obsessions with sulforaphane, generally speaking. I'm very interested in anything in my diet, in my lifestyle that I can do that will lower the amount of systemic inflammation that I have in my body. And it's been shown, and you've shown this and others have shown that even broccoli sprout extract powder given to people can lower C-reactive protein levels by as much as 20%. Other inflammatory cytokines, IL-6, can be lowered by something similar. You know, so it's having a robust and measurable effect in people that's been repeatable in several different studies that I have seen.

But one of the reasons I'm so interested in this is because, well, inflammation really plays a role in a lot of diseases like cancer, but it really seems to be a driver of the aging process. And I don't know if you've...I think I mentioned this to you briefly, but I actually think that sulforaphane may be a anti-aging compound. It's one of the reasons I'm obsessed with sprouting and taking it. I think that actually not only is it preventing these diseases like cancer, but also may be actually delaying the aging process by activating Nrf2, which then activates all these anti-inflammatory genes, activates the antioxidant genes including all the glutathione-related enzymes. I don't know if you've seen the study, but I think I also mentioned to you that I would love to see some lifespan studies done in animals. I know those are not easy to do. They take a long time. But there was a study that was done in this red flower beetle. Have you seen this study?

Jed: No, no.

Rhonda: Okay. So let me tell you. So there's a red flower beetle. Yes, it's a bug, but they have an Nrf2 gene that's homologous to humans, also the FOXO gene, as well. So the scientists fed these red flower beetles different doses of broccoli sprout extract and the doses ranged from, it was low to high, I can't recall, but it was a dose response. And what they found is that at, I think it was at the highest dose, it extended the lifespan of these beetles by 15% and when they exposed these beetles to a high oxidative stress all the time by keeping them in a warmer environment constantly, it extended their lifespan by 30%. And it was totally dependent on Nrf2. So if they knocked down Nrf2, the lifespan extension went away. So I was, like, this is a great teaser, right? Because if it's happening and, I mean, obviously, it's a bug, but it's the same gene, they have a homologous gene and if it's extending this lifespan of this critter that has the same, similar gene that we have, then I feel like there is potential there.

Jed: That's fascinating. No, it wasn't for me...so I have ...

Rhonda: I will send you the paper.

Jed: Please. I have to ask you, is this a red beetle that likes flowers or a beetle that likes red flowers?

Rhonda: It eats red flowers.

Jed: Okay. Okay. Well, that's absolutely fascinating. Look, I can give you some anecdotes and tell you of some false starts that we were involved with and I say "false starts" because since they haven't resulted in publications their work is not finished. But we have talked with colleagues at the National Institute of Aging about, for example, looking at centenarians. There is a Baltimore centenarian project. So there are collections of people who have made it to the ripe old age of 100 and more. Aren't they a beautiful cohort to look at the Nrf2-induced genes in? And I would be surprised if this hasn't been done already. We've talked about it for years, and I know we've talked about doing it in animals. But doing precisely, as you suggest, here is a cohort of very old people who are quite healthy. Do they have some... You know, everybody wants to know is there something magic about that? But are the Nrf2-inducible cytoprotective genes part of that equation? And we suspect that they might be. The prolonged lifespan experiments will pretty much have to be done in mice. And again, there is someone at the...there's a colleague that I know we've talked to at the Institute of Aging who was interested in doing that. I'm not sure if that ever happened. I had a high school student who wanted to do a project here a number of years ago and we got her working on nematodes, Caernorhabditis, which is a common experimental vehicle for those interested in looking at lifespan because its lifespan is so short and it cycles quickly.

Rhonda: I did a lot of work on C. elegans when I was...before I went to grad school, lifespan studies.

Jed: Well, we ought to get together and do this experiment again if it hasn't been done. You know, one of the problems with sulforaphane research nowadays is I can't keep up with it all. No one can really keep it up with it all because there are two or three papers a week coming out on sulforaphane and quite a few more on the Nrf2 pathway. I mean, if you try to be a renaissance person and know all the literature in this field, it's going to smoke you because there are thousands, there are, I think, a couple of thousands of papers a year on Nrf2. So it's very difficult to keep track of them all and it's possible that someone has done those lifespan experiments with C. elegans. Again, we started, we didn't have much muscle in it. We had a high school student who then had to go back to this...

Rhonda: Any preliminary data? I didn't see anything in the literature, by the way. I looked for C. elegans. I didn't see anything with sulforaphane that was published.

Jed: No, we don't have any preliminary data from that student. We also had, I'm looking at my shelf for the binder. It may not still be here. We had a colleague who was a bona fide expert on C. elegans who was going to do some studies and I'm not sure if that ever happened or if those were ever published. So, yeah, sorry, I wish I could jump up and down with great...

Rhonda: But do you agree with me here? I mean, just knowing what we know about how sulforaphane is, as you mentioned, the most potent dietary, that we know of activator, of the Nrf2 system, and knowing what we know, we do know that Nrf2 does play a role in delaying aging, in brain aging, in tissue aging. It's literally lowering inflammation and reducing oxidative stress. These things cause aging.

Jed: Right. So let me come back at you with a short story about progeria, a disease of extreme aging or extremely accelerated aging and let me also come back at you on the issue of brain inflammation, neuroinflammation, because there is evidence in both of those cases and that actually does speak to the aging issue. I mean, I think we'd both like to see someone do a study on centenarians or on C. elegans and just have a dramatic finding that lifespan is extended. Maybe that's coming. So the progeria study is that we got a very small grant to look at a very terrible condition called progeria or Hutchinson-Gilford's progeria syndrome. This is a disease that is characterized by accumulation of a protein called progeria, which is actually a mutant protein that has a...again, I'm not sure if your audience is ready for this, but a farnesylation...a farnesyl tag on it.

Rhonda: Basically a marker that's on top of the...

Jed: Yeah, sort of. So essentially, in a normal cell, you have a cell membrane, you have some cellular architecture, you have a nucleus, and that nucleus holds its structure, maintains its structure due to a number of reasons including the presence of proteins called lamins. And these lamins form a network that helps give it elasticity and shape and blah, blah, blah. And so one of the things that happens in the pathway to making these lamins is you have a molecule, a sort of sticky molecule put on the protein at one point and then taken back off at a later point. Why? I don't know. That's how cells do it.

And so in progeria, actually, Francis Collins, the head of the NIH made this discovery, I think, back in the early '90s. There's a mutation which allows this sticky protein to persist and so that accumulates on the inside of the nucleus and it causes a bunch of phenotypes, a bunch of symptoms which result in dramatically increased aging so that kids who are born with it don't live past their teens. It's a uniformly fatal disease and it's characterized especially by cardiomyopathy, and stroke, and the diseases that many older people die of.

Rhonda: Happening in the teens?

Jed: Happening in the teens.

Rhonda: Right. So it's definitely accelerated aging.

Jed: Yeah. So very characteristic phenotype. The cells of those people are also characteristically misshapen, the nuclei have all these problems with them, outcroppings and blebbings. They don't look like nice sort of oval spheroid nuclei. They're misshapen. And so we're studying cultured cells from a progeria where control donors, tissue donors because there are only a few hundred kids with progeria in the United States, period, and hundreds in the world.

But anyway, it's a terrible disease. We're trying to help a little bit. Others have shown that sulforaphane has an effect in reversing the phenotype in sulforaphane. Karima Djabali in Germany showed this a couple of years ago. And we said that we were going to look at a number of other isothiocyanates from different plants because sulforaphane, while it was effective in reversing this phenotype, also was fairly...it was toxic at slightly higher levels. So the therapeutic index or the window in which you could operate and use it as a drug, let's say, was very narrow. So what we're doing is looking at other isothiocyanates, very similar molecules, which we know also activate Nrf2 to see if there are some that have much greater thresholds of therapeutic windows.

Rhonda: And this is specifically in the context of progeria has this effect?

Jed: Specifically, in the context of progeria.

Rhonda: And that's characteristic of any sort of hormetic plant compounds, that there is a very small therapeutic window.

Jed: Or not. I mean, there may be... Right.

Rhonda: There's a window.

Jed: There's a window, exactly. And we want to find windows that are large. So why do I mention progeria in terms of aging? Well, obviously, it's a disease of acute aging or accelerated aging. Many people seem to think that by understanding a bit more about how we can reverse this phenotype in people with acute aging, that this may also be applicable to normal aging and to perhaps slowing down normal aging. It turns out that all of us have some small amount of progerin, this protein in our systems. And I believe the evidence is that it increases somewhat as we age, not certainly to the acute levels that you find in kids with progeria.

So we're using sulforaphane to explore this. Since we got our grant, there's a very interesting and, I think, quite important publication from Tom Misteli's group at the NIH showing that, in fact, Nrf2 is very intimately related to this process in progeria. And that what happens is that progerin, the sticky protein that I told you is on the inside of the nucleus, actually binds Nrf2 as it enters the nucleus to do its signaling thing and it gloms it up against the inside of the nucleus, so to speak, so that perhaps one among the chores of sulforaphane or among the functions of sulforaphane are to increase the amount of Nrf2 that's coming into the nucleus, certainly, but it may also enhance clearance of mutant proteins and even progerin from the nucleus. So there's a huge amount that we don't know there, but it's an exciting...

Rhonda: Very exciting.

Jed: It's been exciting for us just studying aging.

Rhonda: Yeah. It sounds like even just the mutant progerin would, seems like it's stopping Nrf2 from being activated in that...

Jed: It's sort of holding it up at the gate.

Rhonda: ...essentially, it's stopping it, right. And so that's possibly part of the pro-aging effect, as well. I was reading somewhere, maybe you can confirm this, that sulforaphane does increase the activity of Nrf2, like, Nrf2, I think it's something like 60%. Like, Nrf2 is activated every 180 minutes or something like that. And sulforaphane bumps that up a 60% increase in it being activated. So I don't know if that's...

Jed: Yeah, I can't comment on that. Sorry. But I mean...

Rhonda: But it's activating it more often for sure?

Jed: Correct, correct. Yeah.

Rhonda: And that's, for me, what I want. Another quick question I wanted to ask you...

Jed: It up-regulates its production. So if you look at RNA levels for Nrf2 or for Keap1, its tether protein that's present in the cytoplasm. After treatment with sulforaphane, you see those levels going up. So 60%...

Rhonda: I think that was a translocation of the nucleus. That was the increase that it was doing.

Jed: Okay, yeah. Unfortunately, I can't comment on that, just don't know.

Rhonda: Yeah, I mean, that's okay. I'll talk about it at another...

Jed: But I'm sure it's true, I believe you.

Rhonda: Well, it was from a publication. So if the publication was accurate. But so we're talking about aging and also brain inflammation and, I mean, obviously, there's been some interesting studies on sulforaphane in the brain.

Jed: Yeah. So this is an area where we're actually spending a lot of our time now, is partnering on clinical trials that are looking at just that. So it turns out that the neurologists and those who have been studying the brain and diseases of the brain for a long time in, I guess, the fairly recent past have determined that inflammation is a huge component of a number of those conditions. Schizophrenia, autism, Alzheimer's are among them. And so we can we can speculate as to how that inflammation has an effect. As far as I'm aware, you don't see increases in brain volume. It's not that kind of inflammation, but markers of inflammation clearly are up in people with these conditions and can be reversed in some cases with anti-inflammatory drugs.

And so people have come to us recently with the question, "Okay, we know sulforaphane reduces inflammation. You know, can it possibly help with autism, Alzheimer's and schizophrenia?" The three that we're actually looking at. I should back up, though, and say that sulforaphane, and isothiocyanates like it, have many effects. They do affect many pathways. We talked about Nrf2. I may have made it seem like that was the best thing since sliced bread, and the only thing. It's not the only thing. We also mentioned antibiosis, selective antibiosis against Helicobacter. What we haven't talked about yet is the fact that sulforaphane actually inhibits the NF-kappaB pathway, which is one of the main inflammatory pathways in the body. And there's even some, as it's called, crosstalk between the Nrf2 pathway and the NF-kappaB pathway, so the inflammatory and cytoprotective pathway.

Sulforaphane also, sorry, up-regulates the so-called heat shock response. And I'll try to tie these together in a second. But there are a number of other pathways in which it's active, the mTOR pathway is another. So with all of these biochemical pathways that sulforaphane targets, many of them seemed to come together in a few of the neurodegenerative or neurodevelopmental diseases. And so autism was really the first one that I guess I can say was put in our lab or came to our attention.

So Dr. Andy Zimmerman, a colleague at...who was at the time at Harvard Medical School and the Mass General Hospital came to Paul Talalay back, I don't know exactly when, 2008 or 2009 or 2010 or somewhere in that range. And Andy Zimmerman had shown previously, this was published in 2007, that the so-called fever response of children with autism was real. He sort of codified it and got it in print. Apparently, psychiatrists and caregivers had been commenting anecdotally for a long time that some of their charges, their kids or the people they're giving care to who had autism, when they got a fever, they got better. Their symptoms reversed or relapsed. So autism is characterized by a number of things including repetitive motions, not making eye contact, social and behavioral impairment, if you will.

And so a lot of these characteristics got a lot better when kids had fevers. And so back to Dr. Zimmerman, he knew that we and others had shown that sulforaphane was effective in up-regulating the heat shock response. And so his question to Paul was, "Hey, why don't we see if sulforaphane also helps autism because in half of the kids, when they get a fever, the symptoms go away or they don't go away, but they rela-,..."

Rhonda: Improve.

Jed: "They improve and that's likely related to this heat shock response. Wouldn't it be interesting if sulforaphane has an effect?" As all of us got thinking about it, there were clearly a number of other mechanisms by which sulforaphane could be acting including reduction of inflammation and enhancement of the antioxidant enzymes. You know, more effective clearance of oxidative, reactive oxygen and reactive nitrogen species.

Rhonda: Right. I never thought about heat shock protein playing a role in autism. That's very interesting. I mean, neurodegenerative diseases for sure, but I didn't... And so that's an interesting connection he was making that I wouldn't have made, but I can see how he is making it. It's very interesting.

Jed: Yes. So the paper is 2007, Curran, C-U-R-R-A-N, is the first author. We'll get you a copy. So yeah, it's a fascinating potential connection. And so that was sort of...that was what got the collaboration started. So we supplied broccoli sprout extract, and they, up in Boston, looked at, I guess it was about 44 subjects, all men, all young men, boys, men. As you probably know, autism is about four to one, male to female in terms of its incidence in this country, anyway. And less than or more than 1 in 100 kids now are born with autism. So it's a huge problem. I don't need to go into detail about why it's such a big problem, but, because you're an expert on autism among other things.

Rhonda: Not really.

Jed: But, well, you've studied it. So at any rate, we did this trial. It was published in 2014. We, for various reasons, biomarkers of inflammation of the Nrf2 pathway and heat shock response were not evaluated in blood from those subjects. But what we showed was a rather dramatic reduction in many of the symptoms of autism in about half of the subjects compared to placebos. Those who were given placebo instead of broccoli sprout extract in which there was no detectable change.

Rhonda: I think it was, like, a 37% improvement in, if I remember correctly from your paper. Yeah, it was very, very robust from a small amount.

Jed: It was dramatic. Right, the n was small, the number of subjects was small, but it was a dramatic improvement. I am so sad that we never got the biomarker data from those subjects, although theoretically, it's still available. As a result of that trial, a lot of people got interested in the possibilities. And so Dr. Zimmerman and his team, including the Cullman Chemoprotection Center here, have a follow-up grant from the Department of Defense to study a younger cohort, male and female, boys and girls, about 50 subjects. But a similar trial design and it is going to be...it is biomarker-rich. So we've already done a pilot study with 10 subjects in which we evaluated... We refined our ability to collect samples and to process the biomarker samples. We're collecting blood from all of them. And this is a trial design where, half of them, half of the subjects are getting, actually, Avmacol, one of the supplements, the dietary supplements, the one with glucoraphanin and myrosinase, half of them are getting that, half of them are getting a placebo for, I think, it's 15 weeks, for about the same amount of time as we gave subjects in the previous trial.

Biomarker-rich, then there's a washout period, and then everybody goes on the sulforaphane product, the Avmacol, for another 15 weeks. Dr. Hua Liu and I...she's doing most of the biomarker work here at Hopkins. She and I were just up in Worcester, Massachusetts. The team is now at UMass Medical Center in Worcester, Massachusetts. They've processed about a third of the subjects. So we're about a third of the way through completing the trial. And we're very excited, obviously, about what we may find and about getting to the task of processing these biomarkers.

Rhonda: Very exciting. And what was the dose difference from the first trial? The first trial, the dose of...

Jed: Well, it's a little complicated, but if you bear with me, the first trial delivered sulforaphane-rich broccoli sprout extract.

Rhonda: Okay.

Jed: Okay. Remember that's 70% bioavailable, okay. This trial's delivering glucoraphanin plus myrosinase. It's calibrated to deliver about the same amount as the previous trial. So between 100 and 150 micromoles per subject per day.

Rhonda: Is there a reason why you're not doing a dose response or trying higher doses, as well, to see if there's a more robust effect?

Jed: The next trial will do that.

Rhonda: Okay, so that's in the pipeline?

Jed: Well, it's in this pipeline.

Rhonda: Okay.

Jed: Yeah. So, I mean, look, after a trial like this, especially one where we were not able to publish biomarker results, there are many people saying, "Wow, that's really interesting, but it's got to be repeated." So we and others are trying to repeat it just essentially as closely as possible to the way it was designed. You know, it does make sense and it's unfortunate. If these trials weren't so damned expensive, I mean, we could try all...

Rhonda: It's expensive and long to do.

Jed: We could try all conditions.

Rhonda: I know, I imagine. It's very, very exciting.

Jed: It's exciting, but it's very expensive, its labor-intensive, and it also...you know, you've got the hopes, and fears, and desires, and tears of a lot of people involved. These are conditions that hurt to see people going through. And so to do a whole bunch of trials with a whole bunch of conditions might also make the people who are suffering from these conditions really think that this is...that we already have the answer. This is why I'm so afraid of some of the cancer prevention trials that are being done. You know, people get on their high horse and they don't know what they're talking about.

You know, again, one of the most frequently heard things, for me, is, "Oh, scientists at Johns Hopkins say this cures cancer." I mean, I've heard that said about so many things, not just, certainly not just our work. It ain't true. We would love for it to be true, but same thing with autism, I guess if it were cheap and easy to do and nobody cared about the subjects involved, there'd be a lot of trials, a lot of them would be lousy trials, and a lot of people would have their hopes raised only to maybe 10 or 15 years down the road find out that, yeah, those trials weren't really done that well. So all of the oversight, all of the self-criticism and the peer criticism is probably worth it because I think it does serve a purpose. Anyway, back to the story, so, enough philosophizing. So we have this follow-up trial underway. Interestingly, there are four other, additional autism trials using all using Avmacol, and this is if it doesn't work, I'm to blame because I identified it as something that looked like it was the best of what was out there, and I got the company to agree...

Rhonda: I mean, this is something that's available for people right now.

Jed: It's available, and we know that it produces sulforaphane and we know that it's a decent product and it has been through all sorts of quality assurance. But so when people who came to us and said, "We'd like to do an autism trial. We'd like to model it after Andy's original trial, essentially, try to replicate the findings. We want more of that sulforaphane that you produced for it." And my answer has had to be, "I don't have any more. And I'll show you, as my witness, I'll show you our freezer and show you that we don't have any more in our clinical freezer. So we just can't produce any more." And so we had to suggest that people switch to something commercial.

So four other studies, one of them has finished its patient accruals at UCSF, and we're in the process now of evaluating biomarkers, and they're using metabolomics to evaluate biomarkers. They're looking at small molecules produced by the various metabolic pathways that are either induced or up-regulated or not and hope to be able to make some correlations with symptom reduction and biochemistry. There is a trial just starting at the University of North Carolina, there's a trial at Rutgers. I'm not sure how far along they are, pretty far along, I think. And all three of those trials are about the same order of magnitude as our original trial, 20 to 50 patients or subjects.

The other trial is in China and there are, I'm going to get this wrong, there are either 120 or 180 subjects. And that's just starting to study drug or supplement is they're...and this is at a school for autistic kids in Changsha, China. And you can read the descriptions of most of these trials, I think all of them, on clinicaltrials.gov, which is the government's database for clinical trials. So, again, all these studies are looking at biomarkers of inflammation, as you say, IL-6 is one of the key markers that people are looking at, COX-2, TNF-alpha. The supposition is that those markers are going to go down. The supposition is that markers of Nrf2 activation are going to go up, and heat shock protein markers are going to go up. We'll see.

Rhonda: Well, I mean, it's been shown in people that don't have autism that are given sulforaphane at least, I guess, it may depend on the dose, but it has been shown. The heat shock protein, that really caught my attention. I came across it when I was reading about sulforaphane and how it can be neuroprotective for Alzheimer's disease, Parkinson's, and even Huntington's. These are all diseases of protein aggregation of which heat shock proteins play a major role in repairing and preventing, both. They do both. So I was very surprised. I guess not that, it wasn't that shocking once I found out that sulforaphane activates, because it is a stress response pathway, heat shock proteins do respond to stress like heat stress. So I guess I wasn't that shocked, but I was a little surprised at first to see that it plays a role. And possibly, that's how it's helping prevent and protect against some of these neurodegenerative diseases.

Jed: Very possible, yeah, yeah. You know, this is another case of studies that didn't happen. There were grand plans for a large multicenter study in Europe looking at the effects of sulforaphane on autism. PI was in Spain, highly regarded, the study didn't get funded and it didn't happen.

Rhonda: Autism or Alzheimer's disease?

Jed: Sorry, Alzheimer's, Alzheimer's, yeah. I mentioned schizophrenia, in terms of schizophrenia, we now have two studies that are just starting. Again, they've all been approved. They've been through FDA approval and institutional approval, one in Baltimore at the Sheppard Pratt Hospital, which is a psychiatric hospital in Baltimore, and the other in China. Again, the idea is to look at remission or reduction of symptoms of the diseases of schizophrenia and look at a variety of biomarkers in blood and perhaps, urine.

Rhonda: Were you involved in that pilot trial that was published in... There was a small trial in schizophrenic people.

Jed: This was the [inaudible 01:25:56]?

Rhonda: Yes. Yeah.

Jed: No, I know about it and I've...

Rhonda: So is this based off of that or is this sort of where...? No?

Jed: No. So they only had 10 subjects in that trial. This was Dr. Hashimoto's work, I think he was the corresponding author on that. I've talked with him, familiar with the work. So, I mean, based on publication precedent or history, I guess, you could say we're following off his work. But the way this actually evolved, I think, is that the principal investigators saw the autism work and heard us talking about anti-inflammatory responses in heat shock effects and so on and became interested in doing the trial. Certainly, they and others in the schizophrenia community are familiar with the paper you're talking about. And there's also some very interesting animal modeling in schizophrenia, some from the same group. So, yeah, I mean, I think all signposts are pointing in the direction...

Rhonda: That's cool. There's definitely a common denominator with oxidative stress and inflammation in both autism and schizophrenia, but also in depression. And this is another brain dysfunction, I guess, if you want to call it that, but inflammation has been shown now to play, actually, a major causal role in depression. And there's been some animals studies that you may have seen where the sulforaphane, broccoli sprout extract in sulforaphane has shown to be even as good as fluoxetine, which is Prozac, in alleviating, like, all these different methods of stress they have to make an animal depressed, and then they give it Prozac or broccoli sprout extract and it works just as well, which is extremely interesting.

Jed: I totally agree. We won't talk about the way those experiments are done here because I think they can be sort of...

Rhonda: Inhumane? Some of them? Some of them.

Jed: They're distressing to hear about, yeah. But needless to say, it's better to do them on mice than on people. Yes. In fact, just last week, I was at the Stanley Medical Research Institute, its annual meeting in Baltimore and there was a lot of talk about inflammation and depression. They're focusing on bipolar disorder and schizophrenia. And in fact, they funded the study at Sheppard Pratt that's just starting, as I say. So, yeah, I mean, there is so many conditions that we hope may respond even if only partially, and the fact that it can be a dietary approach is, I think...

Rhonda: Wouldn't that be cool?

Jed: That would be...

Rhonda: I mean, if people could, instead of getting on something with side effects possibly takes broccoli sprouts or some sort of broccoli sprout extract or supplement that's out there that's really effective, that would be so...because it's just, it's so good for you. The sulforaphane, the broccoli sprouts are so good for you. So I just would be really excited to see that happen.

Jed: Well, you've reminded me that I need to give a little lecture about supplements versus food and the lecture, I guess, goes sort of like this. In this country, a lot of people take supplements, right? A lot of people eat Big Macs and don't exercise, and there's nothing...there seems to be very little that those of us who call ourselves nutritionists or nutritional biochemists or health communicators or whatever we call ourselves, there's very little we can do to reverse that. Some people are making some progress and that's good, but when I came here 23 years ago, I thought part of my job was to get people to eat healthier diets, diets rich in fruit and vegetables. I still think that's my job, part of my job, but I see how well it works, and a lot of times, it just doesn't work.

And so there're many people who are going to watch this webinar I'm sure like to think that they're going to eat a healthier diet and adopt a healthier lifestyle, but may not do it. And a lot of those people probably are going to wind up looking for supplements, and if they see a broccoli sprout supplement and hear us talking about, it's likely they'll buy, they'll buy one and they'll take that and continue to sit on the couch and watch TV and eat French fries. I mean, let's keep fighting it, but I'm not sure how successful we'll be in the long run, but now, so let's move this lecture to another part of the world. Let's move the lecture to the so-called developing world, a term I don't like because it implies that we're developed and I don't think we are especially after what just happened in November in this country, but we won't talk about politics.

So you go to the developing world where people can barely afford live on a couple of dollars a day, can't afford a course of antibiotics to treat, for example, Helicobacter infection, where diseases are...sorry, conditions like autism may be a death sentence, blindness in children may be a death sentence because if they can't see, they may run out in front of a car or a bicycle or fall in a lake. I mean, the tragedies that befall people without a so-called developing world social structure to catch them are legion.

But as long as those people are still eating, the possibility that we can, by suggesting a single alternative plan, for example, that they may be able to have an effect on autism or Helicobacter colonization or the risk of various cancers, for example, or asthma, or air pollution injury.

I think there's a very real possibility that we can make a difference. I mean, when you talk about things like cancer prevention, if I invent a magic potion, I'm not going to see the results of it in my lifetime because people are going to have to be taking it for 10s, and 20s and 30s of years before the effect is manifest epidemiologically. And those trials are far too expensive to do as an intervention when you're looking at a population and asking "Does this prevent cancer in someone who's not at high risk for that particular cancer?" Anyway, I'm digressing. Go back to the developing world.

So if you can get someone to change one of their foods or change a few of their foods, or grow something different, then I think the potential to have a real effect is quite impressive. We've worked the numbers, we've published a few times on, so the cost of prevention, and when you compare the cost of a dietary change, even in this country, to the cost of some things like automobile seat belts and statins and a variety of other preventive approaches, a dietary change is way down on the list in terms of cost. Take that to the developing world, I think it's even a more impressive bang for your buck.

And I say that because we're talking about broccoli sprouts and we've been talking a lot about supplements. When you go to the tropics, which is where most of the developing world is, there are plants, and I think I mentioned moringa earlier. Moringa, it happens to be a tropical tree that grows everywhere. It's a weed, and it's also full of an isothiocyanate that is, in many cases, more active than...it may be better than sulforaphane in many situations. Better or worse, but it's sort of on the same level of efficacy of sulforaphane. I'm mentioning that because we've studied it here and I've been involved with actually trying to promote moringa for nutritional reasons. And I've been very fascinated by the potential pharmaceutical or pharmacologic potential of moringa. But it's but one example and there are others that will cite you various other examples of plants that can grow in developing areas of the world, can grow in the dryland tropics, or the wet rainforest tropical regions that could, theoretically, really be game changers not only nutritionally, but in terms of disease prevention in those populations.

And I think we really need to spend, I guess, the preaching part of this, I think we all need to spend more time, and attention, and money on some of those plants. And they may actually have benefit for those of us in the West anyway. I mean, I'm not suggesting that we would want to bring tropical moringa here and try to get people all over the U.S. to eat it because it's got various pharmacologic value to it nor am I suggesting...I certainly wouldn't suggest that we try to cover the world, the equatorial world, with broccoli sprouts because they just won't work. Broccoli is a temperate climate plant and it's too...you know, sprouts are too fragile to import to the tropics or export to the tropics, I think. But the point is that there are plants in various regions of the world that could be adopted, or engineered, or taken over and we should really explore them a bit more, I think.

Rhonda: So I have a couple of questions. So the moringa, does it activate some of the same pathways, different pathways that sulforaphane does?

Jed: It does...

Rhonda: There's a lot of crosstalk... It does?

Jed: Yeah, yeah. So the isothiocyanates have this N double-bond, C double-bond S-group. Again, non-chemists won't care, but that's the active part of the molecule that is responsible for most of its activity, we think. The other part of the molecule that's hanging off that is responsible for solubility and permeability, and contributes to its biological activity. I think it's not as relevant in terms of the activity of the molecule. And so all of the isothiocyanates from cruciferous plants and from moringa all have that NCS group, which is what's responsible for binding to the Keap1 molecule. There are two sulfhydryl groups on reactive cysteines on that Keap1 molecule, and our colleague Dinkova-Kostova has shown that. And the isothiocyanate binds there and causes a change in conformation of that protein. So if you have a different isothiocyanate that maybe is bulkier, maybe it's less effective in getting in that position on the molecule, but, yeah.

Rhonda: Okay. And can we cultivate moringa in places in the United States?

Jed: We can, and it's being cultivated in southernmost Florida and I think even in Southern Arizona and Southern California. It doesn't tolerate frost. So you have to protect it from frost, but, I mean, it's like oranges and lemons and citrus fruit in that respect. So it can be grown in the States. And there are people...I'm actually on the scientific advisory board of a company that's producing moringa in Ghana in Africa. They produce in women's cooperatives and they produce in a responsible way, and they are very attentive to cleanliness of the product that's coming back and they have something that they sell in the U.S. They put it in bars, and they put it in drinks, and they sell the powder plain. There a number of companies that do that.

Rhonda: I've seen it at Whole Foods.

Jed: Yeah, yeah.

Rhonda: Moringa, yeah.

Jed: Yeah, I think in fact...

Rhonda: It was in health food stores.

Jed: Yeah, yeah. There are also some really lousy companies that are producing moringa. There's a multilevel marketing company that's producing it and making outlandish claims. So as with dietary supplements...

Rhonda: Do you have like a list of...do you know which ones are more reliable based on...

Jed: Well, the company that I'm fond of is called Kuli Kuli.

Rhonda: Kuli Kuli?

Jed: K-U-L-I K-U-L-I, yeah. And I think that...

Rhonda: And they make in the United States or they have...

Jed: No, they produce it outside of the United States. They bring it in.

Rhonda: Oh, they are importing, okay.

Jed: Yeah, there probably are companies that are producing it in the U.S. I'm sure there are. As with nutritional supplements, you have to be very careful of claims that are made and what's really in it. So there's a company, I think the biggest company that's been identified with moringa. I'm blanking on the name of the company, but they made a drink and they're a multilevel marketing company and they made many, many irresponsible claims. They invoked the name of Johns Hopkins even in some of their advertising and frankly, I hope they're not doing well. So you just, you have to be careful.

Rhonda: Okay. Another question just to get back to some of what you were initially saying about how this plant, which is growing in some of these tropical regions in more developed nations and how it can be very powerful in cheap dietary intervention that may make a huge difference in people's health in those nations. It just came to my mind knowing what I know about sulforaphane and how sulforaphane is very powerful, and this has been shown in multiple clinical studies, to immediately start to detoxify air pollutants that were exposed to, like, benzene, alkaline, but benzene is a big one. Like, I mean, you start to excrete benzene by, like, 60% after just 24 hours of taking broccoli sprout extract. And I'm thinking about developing countries, there's been a few studies coming out recently that the air pollution and some of these things are a big problem over there, and it's been shown to cause very high stroke risk in young people even because these things are inflaming the blood vessels. It's not just benzene, it's particulate matter and things like that, as well. But still, what's very interesting, if moringa would also be activating these phase II detoxification enzymes, which would be getting rid of some of this compounds and even playing a powerful role in just reducing inflammation in the body and preventing people from having strokes at such young age. So you're right. I think it is very important. You know, people here in the U.S., there's a very large population of people that are interested in aging well and not degenerating and I'm part of that group.

Jed: We all are.

Rhonda: But there is also people that have serious risks that are living in countries where they don't have the luxury of going to a health food store and eating kale smoothies and things like that.

Jed: Exactly, exactly.

Rhonda: So that's really cool that you're involved in that research and advocating for that, as well.

Jed: Well, I think, I actually got involved with a not-for-profit back in the early 2000s that was advocating the use of moringa from a nutritional perspective and I've sort of stayed in touch with that group ever since. And there was a lot of effort to try to get some clinical trials based on its nutritional benefits. It's very high, the leaves are very high in protein, way higher than kale, or broccoli, or most other leafy green vegetables.

Rhonda: Yeah, that's interesting.

Jed: Yeah. And it's also the leaves stay on the trees longer than just about anything else in cases of drought. But there was not the uptake to do a clinical trial, and now there is so much sort of dogmatic promotion of moringa for its nutritional value in those areas of the world that it's so widely used now that I think it's going to be difficult to get some of the rigorous Western-style clinical trials done that we might desire because everybody is just assuming that it's nutritionally, it's a panacea.

Rhonda: And where is it being used mostly?

Jed: Really, all over the tropics, certainly in the Philippines, in large swaths of West Africa and South Africa and in India. I mean, its origin was in northern India near the Pakistan border and it spread around the world since. It spread a long time ago, but it spread around the world from those points of origin all over.

Rhonda: Do people just eat the leaf? Like, how are they preparing? What are they doing? Do they make moringa smoothies?

Jed: Well, some people do. It's interesting, but moringa, because it's sort of a ratty-looking tree, I mean, it's a 20, 30, 40-foot tall tree, but it's sought of ratty-looking and it's sort of hardscrabble. Yeah, it's not gorgeous and the leaves... It was called the horseradish tree or the drumstick tree. Drumstick tree because it has long seed pods that look like drumsticks. Horseradish because it's stringent.

Rhonda: Does it taste like horseradish?

Jed: Yeah, it's harsh. It's sort of like eating Japanese radish, daikon. So, because...and I can give you some to try if you'd like later.

Rhonda: I would, actually. Love to try it.

Jed: Okay, yeah. Because of that harsh taste... Actually, I have some on my shelf right over there. Because of that harsh taste and because of the way it sort of grows like a weed, it's regarded in a lot of areas as a famine food. And it has also been sort of a last resort in cases where people are starving. And from what I understand from my social and behavioral science colleagues, many times, plants like that sort of get a stigma attached to them. So people with little excess money, people who are doing okay may reject what are thought of as famine foods as sort of low class when they don't need them.

So getting people to voluntarily adopt a food like that may be difficult and has proven to be difficult in some areas, and apparently that's one of the reasons. However, it's quite widely eaten, the dried powdered leaves are very easy to store so they maintain their high protein content for a long period of time. That is not the case with things like spinach and kale and some other green leafy vegetables. I mean, imagine drying iceberg lettuce leaves and powdering them and then having them later, yuck.

So in the tropics, dried powdered moringa leaves are found in a lot of areas and are used in food. It's funny, the "Hopkins Magazine" just did a little profile on our involvement with moringa and they quoted me as talking about the first time I had cooked moringa in Africa, which was in Ghana at an international moringa meeting and it was moringa leaves... So I'd had them powdered and I'd had the supplements made from them back in the U.S. But this was in 2006, I think. There was a stew and it looked sort of like saag, like Indian saag, like spinach stew, had some chunks in it. And I was offered some at this meeting on the shores of the Atlantic, a beautiful scenery under sort of tiki huts. And I ate it and then I asked my hosts what the chunks were and it was rat. So I was a little grossed out by my first sampling of moringa made as it's eaten locally, but I actually...

Rhonda: Tastes like chicken?

Jed: Tastes like chicken? Yeah, exactly. Exactly.

Rhonda: Wow, I don't know if I could stomach that, especially after experimenting on rats. So these leaves are high probably in also other micronutrients, folate, magnesium.

Jed: They are. They are.

Rhonda: You know, but what's the glucosinolate that is stored in the moringa, what is it called?

Jed: It's called glucomoringin.

Rhonda: Glucomoringin? That's a name I'll have to remember.

Jed: Yes, the lengthy scientific term is 4-L-alpha-rhamnosyloxypyranosyl-benzyl glucosinolate, so there.

Rhonda: Wow. I'll go with Glucomoringin.

Jed: So, you probably prefer the shortcut, yeah.

Rhonda: And what's the active... Oh, that's the isothiocyanate?

Jed: That is the glucosinolate and it's hydrolyzed by myrosinase that's present in moringa leaves to moringin, or for 4-L-alpha-glucopyranosyloxy benzyl isothiocyanate. So it's got a big, honking sugar group, a rhamnose sugar group, and a benzyl group attached to this NCS.

Rhonda: Okay.

Jed: So it's actually, structurally, it's very different because it's got a lot of excess chemical baggage. So in terms of steric hindrance getting to a protein, getting to a molecular site, it's quite different. But as I said before, it's actually more active in some assays and less in others.

Rhonda: Than sulforaphane.

Jed: Than sulforaphane.

Rhonda: It's very interesting and it kind of brings me to just another quick question because you mentioned daikon and that's something that I've seen in several studies where there seems to be some particularly stable form of myrosinase in daikon. Is that true?

Jed: Yeah.

Rhonda: You know where I'm going with this? Can you eat daikon or add some... Is it found in mustard powder? Is that...

Jed: It is. It is.

Rhonda: Okay. Can you sprinkle mustard powder on your broccoli and increase the bioavailability? That's what I was getting at.

Jed: I think so. And so where you're, I think you're trying to draw me out on the issue of myrosinase and where it is, and how it is, and what the complications are. So myrosinase is a simple enzyme. It's a protein. It turns out that there are companion proteins that have slightly different activities or that modify the products of myrosinase. To make a complicated story even more complicated, the glucosinolate gets converted by myrosinase, not directly to an isothiocyanate, but to an unstable intermediate that then rearranges and forms the isothiocyanate.

So there's an opportunity for other enzymes to come in and sort of re-direct the pathway. And one of those enzymes is actually present in broccoli and at normal physiologic gut pH, temperature and without an excess of iron ions and so on and so forth. There's not a lot of diversion from isothiocyanate to these other compounds, but most of the other compounds are not necessarily bad for you, but they don't have the same phase II enzyme-inducing activity so you're essentially wasting or losing some of the precursor.

Rhonda: Are we talking about sulforaphane nitriles?

Jed: Sulforaphane nitriles...

Rhonda: So they're not bad. They're just not...

Jed: They're not effective, right.

Rhonda: Okay, because that was another question I had.

Jed: Well, there are some other compounds called indoles that we can talk about in a minute and they're present in the broccoli heads. They're not present to any large degree in broccoli sprouts. So indoles from broccoli form, we're going all over the map here, but I'll come back. I'll come back. So indoles from broccoli form something called indole-3-carbinol or I3C, which is omnipresent in health food stores or supplement sites, or DIM, D-I-M, diindolylmethane.

These compounds have gotten sort of a mixed review from a health perspective because it's been shown they can polymerize or form dimers or tetramers that actually resemble dioxin, the potent toxin. And so in animal studies, now I haven't updated my brain on this for a number of years, but when I last did about four, five years ago, there were an equivalent number of animal studies showing a cancer-preventive effect of indole carbinol, I3C, to those which showed that it actually promoted cancer. And some of these were even in the same animal model and what it turned out was that the preventive effect depended on whether you gave I3C before or after you gave a carcinogen.

So as a lab animal, you're in a cage, you eat what you're given, and then the way these experiments are done is a carcinogen is administered usually for once or for maybe three or four days either before giving the protective compound or after giving the protective compound. And then you follow that animal out or those animals out for many, many weeks until cancers develop and you count tumors, and you determine that there was protection or not. It depended on when vis-à-vis the carcinogen, they got the protection. That's great for an animal...well, it's not so great for the animal, but it's great for an animal experiment, but you and I get our carcinogens continuously, one might imagine, right? Whether it's from sunlight or aflatoxins in our food...

Rhonda: Or benzene.

Jed: ...or benzene, yeah. So we get our carcinogens continuously and we do get to choose when we eat our protective compounds if you want to look at it that way, I suppose, or else, we're eating our protective compounds all along if we are eating a protective diet.

I think I'm going to be able to get back to what you originally asked me, but we were quite happy when we determined that in broccoli sprouts, there were essentially no indole glucosinolates, therefore, there was no indole-3-carbinol or diindolylmethane that we would have to worry about. We wouldn't have to do this sort of mental arithmetic and think "It's unbalanced, is this a good thing or not?" Now, the epidemiologic studies still say that eating more broccoli or more cruciferous vegetables is good for you from the perspective of a bunch of different cancers, etc., etc.

Rhonda: Across the board.

Jed: Across the board, yeah. So I don't think that I3C, indole-3-carbinol, is bad for you. A lot of women take it for menopausal problems, it's invoked in the estrogen cycle, and I can't remember off the top of my head exactly what the indication is, but I don't think it's bad for you. We don't worry about it with broccoli sprouts because it's just not a factor. So back to your...we got off track, but you asked about myrosinase. So all the cruciferous vegetables do have myrosinase. Many of them have these other accessory proteins or additional enzymes that will direct the conversion of glucosinolates not just to isothiocyanates, but to some other stuff.

And, interestingly, daikon or Japanese radish doesn't have... I'm not sure that I can say it doesn't have all of them, but it doesn't have the main enzymes, epithiospecifier proteins, they're called, that do some of this redirection to alternative products. So, in fact, if you were to eat cooked broccoli, so just, let's get hypothetical now in the kitchen. So if you were to eat cooked broccoli and you were to want to get as much of a sulforaphane benefit as you could from it, you might add grated daikon or ground up daikon seeds even at very low level to facilitate the conversion. In other words, add live enzyme to facilitate the conversion and you wouldn't have the complication of that other enzyme.

Actually, this has been published. My colleague Yuesheng Zhang who was one of the people who discovered sulforaphane originally, he was the one who actually pointed this out to us many, many years ago and it was subsequently published by others in Illinois. But the fact that broccoli has this epithiospecifier protein means that you're not getting complete conversion to sulforaphane if you use the broccoli enzyme. So for reasons that had nothing to do with knowledge of that fact when we first started making broccoli sprout extracts rich in sulforaphane, I was adding in a very small amount of seven-day-old daikon sprouts to the boiling kettles once they cooled down to catalyze the conversion. And the reason I was doing it is because once we boiled broccoli, we kill the native enzyme, and I wanted to add something that had the biggest bang for its buck of myrosinase and I had found that daikon sprouts had a hell of a lot of myrosinase activity. It was really just sort of a greedy...well, I was actually doing it because I wanted as little contamination of the taste and the other compounds present in daikon sprouts. So I was able to add 1% or 2% by weight daikon sprouts to the broccoli mix to get complete conversion. And as I say, Yuesheng Zhang, who's now at the Roswell Park Cancer Institute, then pointed out to me that, "Hey, did you know that we found the epithiospecifier protein isn't in daikon sprouts?" So, anyway, a long story, but...

Rhonda: So that's really a useful little hack for people including myself.

Jed: Yeah. But daikon sprouts are pretty harsh-tasting. I mean, if you don't...

Rhonda: You can just do a little bit, right?

Jed: But you can do a little bit, yeah.

Rhonda: And what about the mustard seed?

Jed: Mustard seed has isothiocyanates. It has glucosinolates, rather. Sinigrin is the name of the glucosinolate it's rich in. It produces a compound called allyl isothiocyanate and it has myrosinase. And so, yes, you can grind up mustard seed and use that, too.

Rhonda: Mustard powder and put it on your broccoli after you...

Jed: Yeah. I don't know. I suppose most mustard powder is okay, but it depends on how long it's been stored. I mean, as long as it develops a bite when you eat it, it should have active myrosinase.

Rhonda: Okay, yeah. I guess the other thing would be taking some of these supplements like glucoraphanin, taking it, eating it with your cruciferous may even enhance because the cruciferous, the raw cruciferous theoretically would have myrosinase. So if you're taking your supplement with the cruciferous, you may also be getting the biggest bang for your buck, as well.

Jed: Yeah, yeah. Well, that's what this...Nutramax, the supplement company, was trying to do, I think, is to develop to co-deliver myrosinase from a known source, known purity and potency with glucoraphanin. But it's a supplement, not a food. Yeah, I think if I were shunning all supplements, yeah, I would eat broccoli or broccoli sprouts. Well, if you eat fresh sprouts, they're fine. Oh, you mentioned that your freeze broccoli sprouts.

Rhonda: I wanted to ask you about that.

Jed: So as you freeze them, this is interesting, you freeze them, you take them out of the freezer. If you put them in a blender and make a smoothie right away, then the myrosinase is going to act in that liquid, and so you're going to be getting plenty of sulforaphane, I think. If you let those sprouts thaw out, there's a bunch of juice that's going to run out. They're going to look like a slimy mess, right? So, by the time you see that juice and either pour it off, or pour it in your container, probably most of the myrosinase activity has occurred and sulforaphane has probably started to bind to proteins and macromolecules in that vegetable mess. And because what happens is when you freeze the plant tissue, when you thaw the plant tissue, I guess, you've broken down all the cell, many of the cells. So the lignin, the structure on the outside of the plant cells is still there, but the cells are trashed.

Rhonda: Is that why freezing increases sulforaphane, it's just breaking it?

Jed: Well, ice crystals are forming, yeah, it allows the enzyme and the substrate to come into contact, form sulforaphane. So it's probably okay, but you have to capture all the juice or the easiest thing is just throw it in the blender right away, which is probably what you do. But it's interesting because friends in industry, and in fact, we were wrestling with this. We have a freeze dryer. We have a beautiful freeze dryer that we were actually given by a Japanese sprout company as sort of thanks for what we've done that allowed them to build a business in broccoli sprouts. And this freeze dryer has trays. And you can put, say, five pounds of sprouts on the trays. But what you have to do is you have to freeze the sprouts first and then put them in the freeze dryer, pull a vacuum, and it winds up pulling off all the moisture. If you stick the trays in a freezer and freeze them and then move them into the freeze dryer, basically what you've done is allowed all the cells to break the thaw as you're pulling a vacuum. And when we looked at those freeze-dried sprouts, they had no glucoraphanin, and no myrosinase, and no sulforaphane to speak of because it had all reacted and then it was just a mess. So there is value in thinking a little bit about how that enzyme behaves before your start making a thing. You can actually freeze dry broccoli sprouts very nicely as long as you deep freeze them, or quick freeze them and maintain them in a frozen state when you go to dry them.

Rhonda: And that will retain the sulforaphane. The myrosinase.

Jed: The myrosinase is not coming in contact with the glucosinolate then.

Rhonda: Okay.

Jed: Yeah. It's a complicated story. Probably wasn't worth telling.

Rhonda: Well, it kind of made me think of something else and that is I've noticed when I've made my broccoli sprout smoothie, after blending it, if I let it sit in my cup for a prolonged period of time, it tastes different, like there is...and I don't know if that's the sulforaphane or the sulforaphane nitriles or what chemical reaction is going on there. But it tastes different than if I just blend it up and drink it immediately.

Jed: I think it's probably the sulforaphane. So when you blend it and drink it immediately, you probably haven't given enough time for all of the glucosinolates to be converted to sulforaphane.

Rhonda: Do you have any idea how long, what the temporal sort of timeline in minutes?

Jed: Probably a couple of minutes to, yeah... I mean, so when we did these conversions in big vats, 600... I think there were 600...no, 200 or 300-gallon steam kettles at Oregon Freeze Dry, we added daikon sprouts, homogenized and let the stew sit for 2 hours to get complete hydrolysis. You can do the calculation of sort of the half-life of the enzyme and how fast it works. And so it certainly, it takes a matter of many minutes. So in your particular case, with your broccoli sprouts, maybe it was 10 minutes or maybe it takes a half hour to get completely converted.

So probably what you were doing is chugging the not-so-nasty-tasting glucosinolates.

Rhonda: It does get more foul-tasting, as you had said, It does.

Jed: Yeah, exactly. And so the conversion is happening on the way down the tubes and then, when it gets to your intestines, probably more happens. And I should mention, speaking of intestines, we have no idea where most of the myrosinase activity is. Most of the bacteria in your guts are in your large intestine. But I wouldn't rule out the fact that a fair amount of conversion occurs in the small intestine, too.

Rhonda: Really?

Jed: Yeah. And I say that based on the fact on the pharmacokinetics that we see because we know that...we see the metabolites of sulforaphane occur, appear in the blood within 10 minutes of ingestion.

Rhonda: That's pretty fast.

Jed: And when you talk to a gastroenterologist about gut transit time, it stands to reason that it takes more than 10 minutes to get, or 15 minutes, to get all the way to your large intestine and undergo a chemical reaction. So, of course, it's very difficult to access the small intestine of a person, experimentally. It's been done, but...

Rhonda: Do you know how long? So, for example, if I drink my broccoli sprout on Monday, I'm going to activate Nrf2 while I have some of these metabolites glutathione, things like that. How long does that reaction occur before then? Like, so I've been taking this broccoli smoothie when we're not traveling and we're home. We're getting it, like, probably five days a week. You know, do you have to take it every day or do you think that's sort of overkill? Does it last longer? Do you have any idea?

Jed: I think you are perfectly healthy. You're good. No, I think you should be fine. Again, we don't know enough, but it's apparent certainly in animal systems and in the limited number of cases where it's been looked at in people. It's not like taking a drug. It's not like taking an aspirin, which is going to be flushed from your system and the pharmacokinetics look like this and they are there, it appears, its metabolites appear and it's gone. What you are doing when you take sulforaphane is you're up-regulating a bunch of enzymes. And those enzymes have a rather long, comparatively, a rather long half-life meaning they don't lose their punch. They don't get deactivated or get broken down all that fast. And their half-lives are on the order of days, maybe even weeks for some of them, but certainly a few days is a reasonable guess for many of them. So once you boost all these enzymes, they stay up-regulated, in other words, more active for a period of time and then it goes away. So that reminded me I wanted to come back to the issue of probiotics. It seems there was something we wanted to talk about probiotic-wise that we didn't get to.

Rhonda: I think we were just trying to figure out whether or not taking probiotics would potentially increase the bacteria that contain myrosinase in the gut. I mean, the conclusion that I came to was it's possible depending on the type of probiotic you're taking, but obviously, general gut health and good gut health, and you're feeding your bacteria the right foods so that they flourish, as well, is important. Not taking antibiotics.

Jed: Yeah, I think we covered that.

Rhonda: Yeah, I think we covered quite a bit. I am so thankful that we got to speak today because, I mean, I could just keep asking you so many questions because I'm very interested in sulforaphane, as you can tell.

Jed: Well, indeed. And I'm just wondering if there's anything we were thinking about before we sat down together that we sort of missed and, yeah, I've got one. We missed something. We missed something. Urinary health. Well, urinary health meaning, really, bladder health, I think. So we've been involved with about 25 separate clinical trials looking at sulforaphane or glucoraphanin. And in fact, you can... I'm happy to give you a list of them you can post with this webinar and there are about twice as many clinical trials that we plus whoever else has been working on it around the world has done. So 55 or 60 clinical trials, all told. Almost all of the trials, in fact, all of the trials that have been published to date, and when I counted them the other day, they were about 30, I think, publications on clinical trials with sulforaphane or broccoli sprouts, all of them have had positive effects to one degree or another with one exception and that was, unfortunately, a large trial on COPD, Chronic Obstructive Pulmonary Disease, that we were involved with that just didn't show an effect. We think we know why now. Hindsight's always great, of course. We think those lungs were probably just in such bad shape that they couldn't have responded.

But one of the trials that you won't see a publication on, that I'm very sad did not get to completion and did not really accrue enough patients and I hope we can find collaborators to do a trial on this, is bladder cancer prevention. And, again, I go back to my old friend Yuesheng Zhang at Roswell Park Cancer Institute in Buffalo. He made the observation many, many years ago that, gee, when you think about how sulforaphane courses through the body, it winds up being excreted in the urine as either sulforaphane free and clear, or the majority of it comes out as its conjugates with glutathione, one of the main antioxidant peptides in the body. Glutathione or acetylcysteine sulforaphane, a variety of sort of antioxidant, glutathione-derived conjugates. And all of them have some activity in up-regulating Nrf2, also. So what happens? Go in here, you eat sulforaphane or you ingest it or you can even put it on your skin. We've done a number trials showing protection against ultraviolet radiation. It gets metabolized, it gets excreted. How does it get excreted? In the urine. What happens to urine? It hangs around in the bladder. So what you wind up having is a high concentration, relatively high concentration of both sulforaphane and its active metabolites in the bladder bathing that bladder epithelium and it's only, obviously, periodically released.

We think it would be the perfect place to demonstrate protection against cancer or cancer prevention. And so we actually had permission for and started and only accrued, I think, only enrolled, I think, one or two subjects a trial in which we were to look at just that. And these were people, where we were looking at secondary reappearance of bladder cancer. So they had had a tumor removed that had cystectomy and we were looking at the health of the bladder and levels of Nrf2 in bladder tissue that was acquired by biopsy.

So that was the design of this study. As I say, it didn't happen. We're actually trying to get one off the ground with dogs now. Dogs are a little different because they sort of pee when they want to. So there's probably not the same constant reservoir of sulforaphane and its metabolites in the bladder, but certainly to a degree, there will be. It's also harder to collect the 24-hour urine on a dog and to do some of the interventions. But so we are trying to get a canine bladder cancer prevention trial off the ground. And as I say, we'd love to see it done in humans because it's...if there was a gimmie, that's a gimmie. I mean, it's got to work there if it's going to work anywhere.

Rhonda: The epidemiology is so strong showing, I mean, I can just, study after study after study showing cruciferous intake raw broccoli it's showing...and preventing bladder recurrence, as well. People that are eating these foods, it's not a clinical trial, but it's just associative data. And there's been animal studies with bladder cancer where they give them some carcinogen and sulforaphane in it. So I agree with you. I mean, it would be really nice to see an actual clinical trial done in humans and it does make perfect sense. Prostate cancer is another one that seems to also sort of go hand in hand. I mean, the prostate and prostate inflammation seems to be lowered and one study is showing that men with prostate cancer that were given pretty relatively high doses of sulforaphane, it slowed their doubling rate of the PSA by 86%. So, I mean, that's pretty significant.

Jed: Yeah, yeah. So there are two prostate cancer studies...well, there are probably more. But Joshi Alumkal at OHSU did one and Bernard Cippola in France using, actually, the supplement. So Cippola's study used, where he showed a dramatic reduction in the trajectory of PSA numbers was in, used... What's it called?

Rhonda: Prostaphane?

Jed: Prostaphane, thank you. Yeah, the French supplement. And Alumkal's study used our homemade broccoli sprout extract. You mentioned the animal studies. We actually were partnered on three or four animal studies with Yuesheng Zhang and Rex Munday in New Zealand. And it was extremely impressive to see the difference in bladder cancer.

Rhonda: Right, tumor size.

Jed: Tumor number, tumor size and number.

Rhonda: I mean, it was. I saw the publication, extremely robust.

Jed: Yeah. Nice, gross color pictures, too.

Rhonda: Yeah, I'm convinced, yeah. You know, and the other thing is smokers. Smokers get bladder cancer and that, you know...I think, first of all, people that smoke should quit smoking first and foremost, but if they don't quit smoking, I mean, I think they should be consuming broccoli sprouts like nobody's business because they're accumulating so much benzene and all sorts of carcinogens.

Jed: I used to know a seedsman, a guy who ran a seed company and he smoked like a chimney, smoked cigarettes, and when he learned about the broccoli sprouts story, he would eat a handful of broccoli, toast a handful of broccoli seeds and eat them. And he claimed that he was doing that to counterbalance the cigarettes. I couldn't advise him on that. Well, you mentioned benzene a number of times and we didn't talk about the China studies but with our colleague, Tom Kensler, and many others, we did a series of studies over the years starting in about 2002. Interestingly, it started out in an area of China, a subsistence farming area just north of Shanghai where there was a lot of aflatoxin exposure. And so we were looking at liver cancer risk, looking at biomarkers of liver cancer and excretion of aflatoxin adducts vis-à-vis intake of sulforaphane.

Over time, as those studies progressed, there was a series of studies. The liver cancer risks started to come down because they were exporting their contaminated grain to other parts of China from this region, and with globalization, they were importing more and more food. So their diet was becoming less aflatoxin-rich. But at the same time, of course, air pollution was going through the roof. And so the most recent of those studies we published in 2014 showed, really, a dramatic enhancement of the clearance of benzene, and acrolein, and other pollutant conjugates in lockstep with broccoli sprout extract consumption.

Rhonda: It was a 60% increase in excretion, which is just phenomenal. And it convinced me, like it definitely is a problem not only in China, but...

Jed: India.

Rhonda: India, yeah. I mean, but we've got air pollution here in the U.S. and I live next to a busy street. And I see all sorts of things accumulating in my place, you know. So I'm taking that for my benzene excretion, as well. So it's very powerful and it was just within 24 hours.

Jed: It's a dramatic effect and there... So that's an interesting public health dilemma. You can tell someone not to smoke. You can sort of say in the back of your mind, I would never say this out loud, but in the back of your mind, you can say, "They had it coming. The idiots wouldn't stop smoking." We never say things like that. It's not politically correct. But with air pollution, you've got...I mean, people are trapped whether it's highly educated people like you that are living on the street where you know there's pollution or people in the developing world where, you know...

Rhonda: You have to breathe.

Jed: You got to breathe.

Rhonda: You have to breathe and air pollution is...

Jed: You got to eat, and breathe, and sleep, and we can go on.

Rhonda: Yeah. it's becoming... It's an increasing problem and I've seen just more and more studies. You know, a lot of people have focused on the effects on respiratory inflammation and those sorts of, which are very obvious. But like I said, the other health disease risk that's really becoming evident with air pollution is actually heart attacks and stroke because it's causing... It's low-level chronic inflammation. You know, and that's, of course, where sulforaphane shines. You know, I mean, it's inhibiting NF-KappaB pathway, like you said, activating all these anti-inflammatory pathways. So it seems like a no-brainer. It would be nice to get something like that into China and India, broccoli sprout. Well, I guess in India, they've got moringa.

Jed: True, true. So if I were doing a sort of a similar sister study to the broccoli sprout air pollution study in India, I would want to do it with moringa, I think. Just like we're actually trying to get two new autism trials off the ground in the tropics, one in Bangladesh and one in Israel, and moringa thrives in both places. Of course, in Israel, I can get broccoli sprouts easily also because there's electricity and refrigeration. But on the other hand, we may have a better evaluative network of psychiatrists that can sort of help with the evaluation. So yeah, pick your plant, pick your poison, and there's a pairing to be made. I'm sorry we talked a lot about sulforaphane and broccoli sprouts that makes us seem like a one-stop-shop, but in a way, you can only be expert on a few things in this world, right?

Rhonda: Right. Well, the cruciferous family as you mentioned, there's over...was it 500 different genuses or something like that? I mean, most people are familiar with broccoli, cauliflower, kale, Brussels sprouts, cabbage, mustard greens.

Jed: Yeah, mustard greens, yeah, Kohlrabi, rutabaga, watercress, wasabi.

Rhonda: That's right. Watercress, wasabi. But broccoli sprouts are unique in that they actually have higher amounts of glucoraphanin, and that was sort of...

Jed: Very true. One thing that I always tell my class...the classes that I teach when we talk about phytochemistry. You know, it's, of course, easy for me to drone on about what we've been talking about for the last hour or so. But it's important for people to realize that there are many, many, many other biochemical mechanisms that other plants and other phytochemicals target. And so there are plenty of other good vegetables and fruits and there are plenty of reasons for eating them that don't have anything to do with Nrf2, and they could be the subject...I hope they're the subject of another webinar that you do with someone else that knows about them, for instance, the berries.

Rhonda: Right, and the cyanines and... Absolutely, I'm interested in all of them, but I'm particularly interested in broccoli sprouts so I'm extremely excited to have this conversation with you.

Jed: I'm going to have to have frozen sprout smoothies one of these days.

Rhonda: You know, like I said, you add a little bit of ice, too, also just so it's like... Something about the cold, like, helps negate the bitterness that, like...

Jed: The flavor, the head doesn't come out.

Rhonda: So but I think it's also very mental. I do notice a mental effect, as well, like, there's something kind of nootropic about it where all of a sudden, you're just more focused, and I don't know. It could be completely placebo.

Jed: So tell me, some people...many people that I've heard will talk about certain drinks or nutritional products or supplements and say it gives them more energy. Other than eating carbs, eating sugar, eating a Clif pack, gel pack, or something, I don't get that. I don't get that with broccoli sprouts, for example, and I've heard people say, "I've got more energy from eating broccoli sprouts." Do we have a good answer to that someplace?

Rhonda: I don't know if we do. You know, I think that the reducing of inflammation...inflammation drains your energy. I mean, it's a very energy consuming process. When you're activating immune cells, that requires ATP immune. So I do think that we can make an argument for it, but how immediate that effect is, I don't know.

Jed: It's a little more long-term.

Rhonda: You know, I'm not sure. I do know that drinking...I don't know, have you had a smoothie with the broccoli sprouts before?

Jed: Yeah, I'm not a...

Rhonda: You haven't noticed anything?

Jed: No, I never get more energy. Really, I mean, I've never found a food that gives me more energy. I suppose...I mean, sugar, I guess, does, but, yeah, sugar does.

Rhonda: Sugar does.

Jed: But that's not really ideal either.

Rhonda: No. Well, unless you're eating fruit, and berries and things like that are good.

Jed: Yeah. If we're going down a path that's going to get us nowhere, though.

Rhonda: Well, I would like to mention for people that want to follow up on your research and all your publications and things like that, you have a website called chemopreventioncenter.org.

Jed: Chemoprotectioncen-,...

Rhonda: Oh, chemoprotectioncenter.org

Jed: Let's call it up and be sure.

Rhonda: Yeah, it's chemoprotectioncenter.org, I have it right here.

Jed: Okay, yeah.

Rhonda: So you want to tell us a little bit about...so that's your website?

Jed: Sure, that's the website of our group here. This is, we're speaking in the Cullman Chemoprotection Center, which is a center devoted to the study of plants and phytochemicals for prevention of chronic disease and other ailments. And we have a website. There's a donation box on it. We are always eager to solicit donations from people who think like we do. We are funded from grants and the largess of wonderful, interested people.

We have a list of our publications on this website, most of them are clickable. You can at least get to the abstract. We're happy to send any of our publications that the general public can't get, can't find by clicking. Some of these are copyright-protected and we can help you out with them. And our website says a little bit about our mission. So we encourage you to...

Rhonda: Great. Again, that's chemoprotectioncenter.org. And you're also on Twitter. You're not very active, but you do have a Twitter handle. That Twitter handle is @jedosan, or J-E-D-O-S-A-N. So if you decide to become more active on Twitter and tweeting things, then...

Jed: What's Twitter? We also are on Facebook, The Chemoprotection Center.

Rhonda: Okay, great. Chemoprotection Center is also on Facebook. Excellent. Well, thanks again, Jed. I've really, really enjoyed this conversation. I've learned a lot.

Jed: As have I. As have I. Thanks.