Vitamin B2 very effective against Candida strains in vitro and in vivo

[Mauritio]

Vitamin B2 has been shown to have anti-bacterial effects (even against anti-biotic-resistant bacteria) and anti-endotoxin effects.

As it seems B2 also has potent anti-fungal effects. The researchers first looked at its effects in vivo finding that :
" The formation of C. albicans hyphae and biofilm was inhibited by RF. Mechanistically, RF disrupted membrane and cell wall integrity, as well as promoting reactive oxygen species and pyruvate accumulation."

B2 was actually more effective than the common anti-fungal fluconazole!
"Compared with the control group, 1× MIC of RF inhibited more than 90% of C. albicans, Candida krusei, and Candida parapsilosis grwoth between 2 and 24 h (Fig. 1B), and the antifungal activity of 1× MIC of RF was more effective than that of the FCZ group (64 μg/mL)."

Riboflavin also strongly inhibited early biofilm formation, less so mature biofilm.

The expression of biofilm-related genes was strongly downregulated:

Another mechanism of action was the accumualtion ROS inside the fungus, which can lead to cell death down the road.
"Flow cytometry further confirmed that 65.45% and 77.23% of cells were ROS positive after RF treatment with 1× MIC and 2× MIC, respectively, while only 10.95% of cells were ROS positive in the control group (Fig. 4B). These results suggested that RF markedly promotes the excessive production of ROS (Fig. 4C)."

Then they looked at the effects of riboflavin on candida infected, immuno-suppressed mice. And there as well: b2 inhibited cell count and growth of candida, not as much as fluconazole, but still significantly. Which can be seen when you compare the tongues of mice in the different study groups (Ca= Candida).

Why was B2 less effective in vivo and more effective in vitro than fluconazole?
As far as I understand the concentration used in-vivo was fairly high (0.4 /0.8mg/ml) and the dosage used in vivo was fairly low, only translating to a human equvialent dose of a couple mgs of B2.
I don't know why the researchers used such a low dose of B2 in-vivo, since the dosages used previously and successfully for other infections were much higher. But, interestingly B2 was still effective even in relatively low dosages.

Would B2 be more effective in vivo at higher doses ?
I guess yes. Because in-vitro there is a clear dose-dependant effect, meaning higher dose of b2 --> higher inhibition of pathogen, causing a stronger inhibition than fluconazole. So by taking higher doses in vivo one might be able to increase the effectiveness and restore the superiority of b2 over fluconazole in terms of candida inhbtion.

- Riboflavin Targets the Cellular Metabolic and Ribosomal Pathways of Candida albicans In Vitro and Exhibits Efficacy against Oropharyngeal Candidiasis

 

[youngsinatra]

I love riboflavin

 

[Mauritio]

Another angel through which we can target fungal infections are the Heat shock proteins (HSPs).

"Moreover, it has been demonstrated that Hsps confer drug resistance to C. albicans. Many studies have shown that disrupting the normal functions of C. albicans Hsps inhibits fungal growth or reverses the tolerance of C. albicans to traditional antifungal drugs."

HSP90 seems to be the main actor at confering drug resistance to Candida.
"Hsp90 augments virulence factors and confers antifungal drug resistance to common pathogenic fungi, C. albicans, Aspergillus fumigatus, and Cryptococcus neoformans (Cordeiro Rde et al., 2016; Lamoth et al., 2016; Chatterjee and Tatu, 2017)."

The study also mentions that HDAC-inhibotrs can inhibit HSP90's effect on candida, meaning they can remove the resistance to anti-fungals.
"Furthermore, HDAC inhibitors, such as trichostatin A (TSA) and MGCD290, a Hos2 HDAC inhibitor, have been shown to abrogate Hsp90-dependent azole resistance in C. albicans."
- Candida albicans Heat Shock Proteins and Hsps-Associated Signaling Pathways as Potential Antifungal Targets
- Histone deacetylase inhibitors enhance Candida albicans sensitivity to azoles and related antifungals: correlation with reduction in CDR and ERG upregulation - PubMed

As we have discussed in another thread fungal infections seem to be very good at adapting to new anti-fungals and combining an anti-fungal with a HDAC inhibitor, you might be able to kill the fungus witout it becoming resistant.

Commom HDAC inhibitors: "...Other dietary agents such as butyrate, biotin, lipoic acid, garlic organosulfur compounds, and metabolites of vitamin E have structural features compatible with HDAC inhibition."
- Dietary HDAC Inhibitors

So if you take B2 as an anti-fungal you could simply take another B-vitamin like biotin, niacinamide or butyrate as HDAC inhibitors with it.

So combingin B2 and Biotin (and niacinamide) might be a cheap, safe and effective OTC option for treating candida infections, without them becoming resistant.

Does anybody know the dosages for HDAC-inhibition for Biotin or Niacinamide? I suspect they are on the higher side.

 

[Mauritio]

Another angel through which we can target fungal infections are the Heat shock proteins (HSPs).

"Moreover, it has been demonstrated that Hsps confer drug resistance to C. albicans. Many studies have shown that disrupting the normal functions of C. albicans Hsps inhibits fungal growth or reverses the tolerance of C. albicans to traditional antifungal drugs."

HSP90 seems to be the main actor at confering drug resistance to Candida.
"Hsp90 augments virulence factors and confers antifungal drug resistance to common pathogenic fungi, C. albicans, Aspergillus fumigatus, and Cryptococcus neoformans (Cordeiro Rde et al., 2016; Lamoth et al., 2016; Chatterjee and Tatu, 2017)."

The study also mentions that HDAC-inhibotrs can inhibit HSP90's effect on candida, meaning they can remove the resistance to anti-fungals.
"Furthermore, HDAC inhibitors, such as trichostatin A (TSA) and MGCD290, a Hos2 HDAC inhibitor, have been shown to abrogate Hsp90-dependent azole resistance in C. albicans."
- Candida albicans Heat Shock Proteins and Hsps-Associated Signaling Pathways as Potential Antifungal Targets
- Histone deacetylase inhibitors enhance Candida albicans sensitivity to azoles and related antifungals: correlation with reduction in CDR and ERG upregulation - PubMed

As we have discussed in another thread fungal infections seem to be very good at adapting to new anti-fungals and combining an anti-fungal with a HDAC inhibitor, you might be able to kill the fungus witout it becoming resistant.

Commom HDAC inhibitors: "...Other dietary agents such as butyrate, biotin, lipoic acid, garlic organosulfur compounds, and metabolites of vitamin E have structural features compatible with HDAC inhibition."
- Dietary HDAC Inhibitors

So if you take B2 as an anti-fungal you could simply take another B-vitamin like biotin, niacinamide or butyrate as HDAC inhibitors with it.

So combingin B2 and Biotin (and niacinamide) might be a cheap, safe and effective OTC option for treating candida infections, without them becoming resistant.

Does anybody know the dosages for HDAC-inhibition for Biotin or Niacinamide? I suspect they are on the higher side.

Might have to remove B2 from the anti fungal + HDAC-inhibitor combo, since they only work when the anti-fungal's effect is related to ergosterol ,which to my knowledge is the one mechanism B2 does NOT affect. So we'll have to see which of the common anti-fungals targets ergosterol. B2 stills works as a stand alone candida-treatment ,it just doesnt seem to have any synergy with HDAC inhibitors.

"Similar effects were observed with other HDA inhibitors and with the antifungals terbinafine and fenpropimorph, which target, as do azoles, enzymes in the ergosterol biosynthetic pathway. In contrast, HDA inhibitors had minimal effect on the activities of amphotericin B, flucytosine, and echinocandin, which have unrelated targets."
- Histone deacetylase inhibitors enhance Candida albicans sensitivity to azoles and related antifungals: correlation with reduction in CDR and ERG upregulation - PubMed

 

[Mauritio]

I found some anti-fungal things that work through ergosterol pathway! Heptanoic acid (7:0), nonanoic acid (9:0) and lauric acid (12:0) seem to fit the part.
So combining one or more of those fatty acids with biotin or another of the above mentioned HDAC inhibotors migth make sense.

"Treatment with 7:0, 9:0 or 12:0 at 10 μg ml−1 markedly inhibited sterol production,..."
"The current result indicates that the antifungal effects of medium‐chain fatty acids at high concentrations (Table S1) are probably due to the inhibition of sterol production (Fig. 3B), which could impede cell wall synthesis."
" Interestingly, the inhibition of sterol production by medium‐chain fatty acids was reminiscent of the action mechanism of commercial antifungal azoles that inhibit 4‐α‐sterol demethylase (encoded by the ERG11 gene), which is required for the biosynthesis of ergosterol. "


Interestingly caprylic acid wasnt effective for that purpose.
Nonanoic acid was very effective against candida via different mechanisms.

"Notably, four key biofilm‐ and hypha‐related genes, that is, ALS3 (agglutinin‐like protein 3), ECE1 (hypha‐specific protein, also known as HWP2), HWP1 (hyphal cell wall protein, also known as ECE2), and UME6 (filament‐specific regulator), were repressed by 7:0, 9:0 and by farnesol. For example, 9:0 (the most active) down‐regulated ALS3 by 25.5‐fold, ECE1 by 93‐fold, HWP1 by 17.2‐fold and UME6 by 7.9‐fold, while farnesol down‐regulated these genes by 3.5, 11.4, 3.0 and 2.7‐fold, respectively."


Consuming PUFA (linoleic acid) might be pro-fungal!
"Interestingly, (R)‐3‐hydroxytetradecanoic acid, a metabolite of linoleic acid, was able to reverse the farnesol‐induced inhibition of biofilm formation in C. albicans (Nigam et al., 2011), which suggests fatty acids and fatty acid‐like compounds structurally similar to farnesol might interfere with farnesol‐induced QS signalling (Fig. 4C)."

- Antibiofilm and antifungal activities of medium‐chain fatty acids against Candida albicans via mimicking of the quorum‐sensing molecule farnesol

 

[Mauritio]

Here's another study showing B2's anti-microbial effects:

"stand-alone riboflavin solution successfully inhibited Staphylococcus aureus, Enterococcus faecalis, Salmonella typhi, and Pseudomonas aeruginosa with an inhibition range of (18.7 ± 0.6) mm; (17.7 ± 0.6) mm; (17.3 ± 0.6) mm, and (15.7 ± 0.6) mm, respectively. Intermediate zones of inhibition were observed for Escherichia coli and Candida albicans with a range of (11.7 ± 0.6) mm and (11.7 ± 0.6) mm, respectively. Klebsiella pneumoniae was the only resistant pathogen at (7.7 ± 0.6) mm for the riboflavin concentration used in this work. These results may indicate the exciting prospects of the applications of riboflavin as a complementary approach toward inactivation of pathogenic infections."
- https://www.tandfonline.com/doi/full/10.1080/10942912.2015.1076459

 

[Lejeboca]

Thanks for the studies. Amazing results with B2. I wonder whether it will work well topically on the skin or membrane as anti-microbial/anti-fungal?

 

[Mauritio]

Thanks for the studies. Amazing results with B2. I wonder whether it will work well topically on the skin or membrane as anti-microbial/anti-fungal?

It should, and the concentration will be higher because its applied locally . I would be carefull with sunshine though.

 

[frannybananny]

I love riboflavin

Why?

 

[Mathgirl]

If I remember correctly, Ribloflavin also cured Christ Masterjohn of lifelong allergies overnight...

 

[A.R]

Great thread Thankyou for your efforts @Mauritio

 

[webbt]

Just so people are aware of the kind of doses that are possible, I've taken is 800 mg a few times. Can't say definitively if there were positive or negative effects. It's possible it helped digestion and sleep a bit.

 

[Mauritio]

Great thread Thankyou for your efforts @Mauritio

Youre welcome

Just so people are aware of the kind of doses that are possible, I've taken is 800 mg a few times. Can't say definitively if there were positive or negative effects. It's possible it helped digestion and sleep a bit.

B2 is incredibly non toxic. I suspect that over 300-400mg per dose you dont get any higher absorption.

 

[Mauritio]

I gradually increased my dosage of B2 to 200mg with only positive effects as far as I can tell. Energy is definetly up, gut seems more quiet and calm just as my mind , which is able to focus longer. I also feel more calm and social generally. It seems to have an anti-endotoxin effect as well, when I eat something bad and I swallow a tablet of B2 I can feel the whole stress response calming down 30 minutes later, something that only magnesium glycinate was able to give me until now.
The dry lips are gone ,but I still have a little bit of red eyes so I want to see when that disappears.

 

[frannybananny]

I gradually increased my dosage of B2 to 200mg with only positive effects as far as I can tell. Energy is definetly up, gut seems more quiet and calm just as my mind , which is able to focus longer. I also feel more calm and social generally. It seems to have an anti-endotoxin effect as well, when I eat something bad and I swallow a tablet of B2 I can feel the whole stress response calming down 30 minutes later, something that only magnesium glycinate was able to give me until now.
The dry lips are gone ,but I still have a little bit of red eyes so I want to see when that disappears.

Thanks for the update Mauritio! I have been having some anxiety issues lately so I want to get some B2 and see if that helps.

 

[frannybananny]

I found some anti-fungal things that work through ergosterol pathway! Heptanoic acid (7:0), nonanoic acid (9:0) and lauric acid (12:0) seem to fit the part.
So combining one or more of those fatty acids with biotin or another of the above mentioned HDAC inhibotors migth make sense.

"Treatment with 7:0, 9:0 or 12:0 at 10 μg ml−1 markedly inhibited sterol production,..."
"The current result indicates that the antifungal effects of medium‐chain fatty acids at high concentrations (Table S1) are probably due to the inhibition of sterol production (Fig. 3B), which could impede cell wall synthesis."
" Interestingly, the inhibition of sterol production by medium‐chain fatty acids was reminiscent of the action mechanism of commercial antifungal azoles that inhibit 4‐α‐sterol demethylase (encoded by the ERG11 gene), which is required for the biosynthesis of ergosterol. "


Interestingly caprylic acid wasnt effective for that purpose.
Nonanoic acid was very effective against candida via different mechanisms.

"Notably, four key biofilm‐ and hypha‐related genes, that is, ALS3 (agglutinin‐like protein 3), ECE1 (hypha‐specific protein, also known as HWP2), HWP1 (hyphal cell wall protein, also known as ECE2), and UME6 (filament‐specific regulator), were repressed by 7:0, 9:0 and by farnesol. For example, 9:0 (the most active) down‐regulated ALS3 by 25.5‐fold, ECE1 by 93‐fold, HWP1 by 17.2‐fold and UME6 by 7.9‐fold, while farnesol down‐regulated these genes by 3.5, 11.4, 3.0 and 2.7‐fold, respectively."


Consuming PUFA (linoleic acid) might be pro-fungal!
"Interestingly, (R)‐3‐hydroxytetradecanoic acid, a metabolite of linoleic acid, was able to reverse the farnesol‐induced inhibition of biofilm formation in C. albicans (Nigam et al., 2011), which suggests fatty acids and fatty acid‐like compounds structurally similar to farnesol might interfere with farnesol‐induced QS signalling (Fig. 4C)."

- Antibiofilm and antifungal activities of medium‐chain fatty acids against Candida albicans via mimicking of the quorum‐sensing molecule farnesol

So in your linked article I read this: << We hypothesized that fatty acids mimicking farnesol might influence hyphal and biofilm formation by C. albicans. Among 31 saturated and unsaturated fatty acids, six medium‐chain saturated fatty acids, that is, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and lauric acid, effectively inhibited C. albicans biofilm formation by more than 75% at 2 µg ml−1 with MICs in the range 100–200 µg ml−1. These six fatty acids at 2 µg ml−1 and farnesol at 100 µg ml−1 inhibited hyphal growth and cell aggregation. The addition of fatty acids to C. albicans cultures decreased the productions of farnesol and sterols. Furthermore, down‐regulation of several hyphal and biofilm‐related genes caused by heptanoic or nonanoic acid closely resembled the changes caused by farnesol. In addition, nonanoic acid, the most effective compound diminished C. albicans virulence in a Caenorhabditis elegans model. Our results suggest that medium‐chain fatty acids inhibit more effectively hyphal growth and biofilm formation than farnesol.>>

@Mauritio do you happen to know what food or elements contain these 6 fatty acids? Where are they?

 

[Lejeboca]

B2 is incredibly non toxic. I suspect that over 300-400mg per dose you dont get any higher absorption.

yet, for the anti-bacterial action of B2, the doses were quite low, and even lower in vivo. From the OP paper:

"Interestingly, the effective concentration of RF (0.125 or 1 mg/kg) in vivo is much lower than that of FCZ (13.6 mg/kg). RF treatment showed a 2-order-of-magnitude reduction in kidney fungal burden, which was superior to the 1-order-of-magnitude reduction in the tongue. The metabolism of RF is inextricably linked to the kidney (12), suggesting that understanding the absorption and transport of RF is vital to studying the mechanism of anti-infection activity in vivo."​

Perhaps, it may be explained by the following quote from the paper The promise of endogenous and exogenous riboflavin in anti-infection, found from the references in the OP:

"Exogenous riboflavin directly controls infection in three ways:

1. it inhibits transcription of bacterial LPS (Figure 2b);
2. it reduces the level of NO or expression of iNOS; and
3. it regulates the functions of innate immune cells (such as macrophages and neutrophils) and the levels of immunoreactive materials, including TNF, ILs, and IFN.

The key factors affecting the anti-bacterial or anti-fungal effects of riboflavin are the time of administration and the dose [20,21,94,95]."​

I think that the fact that B2 reduces inducible NO in a gene (not stress related mechanism) is very interesting. It also reduced plasma elevated NO levels by reducing expression of inducible NO synthase (iNOS) gene. I seemed to read that there are many substances that can reduce iNOS once its process started.

 

[Mauritio]

So in your linked article I read this: << We hypothesized that fatty acids mimicking farnesol might influence hyphal and biofilm formation by C. albicans. Among 31 saturated and unsaturated fatty acids, six medium‐chain saturated fatty acids, that is, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and lauric acid, effectively inhibited C. albicans biofilm formation by more than 75% at 2 µg ml−1 with MICs in the range 100–200 µg ml−1. These six fatty acids at 2 µg ml−1 and farnesol at 100 µg ml−1 inhibited hyphal growth and cell aggregation. The addition of fatty acids to C. albicans cultures decreased the productions of farnesol and sterols. Furthermore, down‐regulation of several hyphal and biofilm‐related genes caused by heptanoic or nonanoic acid closely resembled the changes caused by farnesol. In addition, nonanoic acid, the most effective compound diminished C. albicans virulence in a Caenorhabditis elegans model. Our results suggest that medium‐chain fatty acids inhibit more effectively hyphal growth and biofilm formation than farnesol.>>

@Mauritio do you happen to know what food or elements contain these 6 fatty acids? Where are they?

Im not sure if they are all in one food, especially not in qunatities that suffice. Some of them are not even available as commercial supplements. But caprylic acid and lauric acid are and those are two of those mentioned fatty acids.

 

[Mauritio]

yet, for the anti-bacterial action of B2, the doses were quite low, and even lower in vivo. From the OP paper:

"Interestingly, the effective concentration of RF (0.125 or 1 mg/kg) in vivo is much lower than that of FCZ (13.6 mg/kg). RF treatment showed a 2-order-of-magnitude reduction in kidney fungal burden, which was superior to the 1-order-of-magnitude reduction in the tongue. The metabolism of RF is inextricably linked to the kidney (12), suggesting that understanding the absorption and transport of RF is vital to studying the mechanism of anti-infection activity in vivo."​

Perhaps, it may be explained by the following quote from the paper The promise of endogenous and exogenous riboflavin in anti-infection, found from the references in the OP:

"Exogenous riboflavin directly controls infection in three ways:​

1. it inhibits transcription of bacterial LPS (Figure 2b);
2. it reduces the level of NO or expression of iNOS; and
3. it regulates the functions of innate immune cells (such as macrophages and neutrophils) and the levels of immunoreactive materials, including TNF, ILs, and IFN.

The key factors affecting the anti-bacterial or anti-fungal effects of riboflavin are the time of administration and the dose [20,21,94,95]."​

I think that the fact that B2 reduces inducible NO in a gene (not stress related mechanism) is very interesting. It also reduced plasma elevated NO levels by reducing expression of inducible NO synthase (iNOS) gene. I seemed to read that there are many substances that can reduce iNOS once its process started.

Yes , I was wondering why they used such a low dosage in vivo. Im pretty sure higher doses would have been more effective ,since this is what the in vitro study points at. Also other studies that use B2 as an anti-microbial use way higher dosages.
The NO thing is interesting. Im wondering if that is downstream from a reduction in endotoxin.

 

[Dave Clark]

Looks great about B2, but is anyone concerned about higher doses and photosensitivity? I know Ray mentioned it a few times.