The NAC Protocol

S. Peribsen, M. MacLir*

A natural fungal mitigation and

immunomodulatory protocol

Introduction
Fungal infections and their toxins (called mycotoxins) can be a major contributor to inflammation in the body. As a result
of that inflammation, it can cause or exacerbate various conditions, including autoimmune diseases.
Relevant links to science articles will be presented as we go, listed so you can click on the link for more information. In
addition, a more detailed scientific review of the protocol compounds is available at the end of the document for researchers
and clinicians alike.
Fungal infections and their mycotoxins are seriously under-addressed in symptom management and the treatment of
disease. This natural protocol focuses on effective elimination of fungal infections and modulation of immune response to
improve outcomes.

Common Fungal Infections even cold like symptoms. This necessary discomfort
is required to remove detrimental fungal infections.
Common Fungal Infections include Candida, As- Overall, it can take 2 to 4 months to see a significant
pergillus and Cryptococcus species. Until recently, improvement in health and well-being. Periods of
these fungi have been considered commensal, or a detoxification symptoms will come and go as you
common part of the biome and generally considered progress. It is common to notice small improvements
harmless. along the way, and it can be a motivator to complete
More recent research indicates that their byprod- the process.
ucts (or metabolites) can be carcinogenic, inflamma- The process takes longer because depending on
tory and even mutagenic to DNA [1]. the fungi in question, you may have infection in the
These byproducts, or mycotoxins, are commonly lungs, gastrointestinal tract, upper respiratory sys-
measured in the body and regular exposure occurs tem, spinal fluid, synovial fluid in the joints, various
through both food contamination [2] and inhalation tissues and organs and even the brain itself.
of spores [3] in the home and outside environments. Another thing that makes this process take longer
By reducing fungal infection levels and their myco- is biofilms. What you normally call plaque on the
toxins, we can reduce the corresponding inflamma- teeth is an example of biofilms. 80% of the pathogens
tory response in the body and promote homeostasis you are targeting live inside these biofilms. As a
for improved immunity. result they gain protection from the immune system.
Biofilms can take time to break down, but all
3 components of the protocol serve this purpose.
A Natural Anti-Fungal Protocol The pathogens targeted by this protocol are both
pathogenic fungi and bacteria. Both can be the cause
There are 3 components to this natural protocol.
of dysbiosis, or an imbalance in your gut biome,
Those are N-Acetylcysteine (NAC), Oregano Oil (OO)
which can lead to various issues [5].
and Black Seed Oil (BSO).
All 3 components work safely and effectively at
reducing fungal infection and supporting the body’s The NAC Protocol
detoxification process. Part of the process of remov-
ing fungal infections in the body is dealing with the Morning
cell death of pathogens, which release toxins and
byproducts that the body must eliminate through • 1200mg NAC
detoxification. This process occurs in the liver and • Oregano Oil (40mg Carvacrol)
kidneys. During this cleanup process, a Jarisch- • Black Seed Oil (1 teaspoon)
Herxheimer reaction is common [4].
Night
This reaction can include various symptoms like
headaches, tiredness, gastrointestinal symptoms or • 600mg NAC
• Oregano Oil (40mg Carvacrol)
* with contributions from S. C. • Black Seed Oil (1 teaspoon)
The NAC Protocol

Continue daily for a minimum of two months and a more long term solution.
count out 3 weeks with no die off symptoms prior to To approach correction of potential long-term im-
discontinuing. mune dysfunction related to commensal or invasive
fungi, the following page details a maintenance pro-
Fungal die off symptoms may include: Tiredness, tocol for regular long-term use.
exhaustion, muscle soreness, increased chest or nasal dis-
charge, cold or flu like symptoms, cold sores, headaches, The Maintenance Protocol
rash, acne, irritability, change in stool frequency, volume
The Maintenance Protocol focuses on a more gentle
or color; increased urination, bloated stomach, cramps,
antifungal approach combined with immune modu-
increased gas.
lation to prevent overactive response (autoimmune).
Choosing Your Supplements To get technical for a moment, Niacin provides a
Oregano Oil’s bioactive ingredient is called Car- needed boost in the NAD+ pool [6], which works
vacrol. You want to purchase your OO in capsule with Pterostilbene as a SIRT1 activator [7] to promote
form and purchase a product that has around 40mg homeostasis by countering oxidative stress, inflam-
of Carvacrol per dose. Read the bottle instructions to mation and mitochondrial dysfunction.
determine if 1 or 2 capsules will provide the neces- Pomegranate Extract serves to provide additional
sary Carvacrol amount. antifungal, anti-inflammatory and anti-oxidative sup-
NAC can be found easily in 600mg per capsule port.
dosages at various retailers and online shops.
Morning
Black Seed Oil should be purchased in the cold
pressed, unfiltered oil form. A large 16 ounce bottle • 600mg NAC
is available through Horbaach and SVA Organics. • 500mg Slo Niacin (nicotinic acid)
These products have been tested by users to be effec- • 100mg Pterostilbene
tive. • 250mg Pomegranate Extract (40% Ellagic Acid)
The morning and evening dose can be taken with • Black Seed Oil (1 teaspoon)
or without food, but should be taken with food if
you have a sensitive stomach. Increase water intake Night
and fiber while on the protocol to ease detoxification. • 500mg Slo Niacin
#1 Rule: Listen To Your Body • 100mg Pterostilbene
Everybody is different, and various factors can in- • 250mg Pomegranate Extract
fluence how strong your herxheimer reactions are, • Black Seed Oil (1 teaspoon)
including level of infection, age and general health.
After every 3 weeks on the maintenance protocol,
Consult with your doctor before starting.
take 1 week off. Continue to use black seed oil during
When you start this natural protocol, die off symp-
the off cycle.
toms may be immediate or may take up to a month
to begin. When this detox begins, listen to your body.
If at any time the symptoms become too overwhelm- Safety & Adverse Reactions
ing, take a few days off, rest and increase your water
intake. Always consult with your doctor before starting this
You can then continue at lower dosages by reduc- protocol and receive prior approval. They can appro-
ing the Oregano Oil or taking it once daily, reducing priately address interactions with medications or any
NAC to once daily, and gradually increasing amounts current health conditions you have.
as symptoms improve to reach protocol amounts. In addition, the protocol may lower specific vita-
As you advance further, it can be beneficial to scale mins and minerals including zinc, iron and calcium.
up your dosages slowly to continue making progress. A multi-vitamin is recommended to address this.
If you decide to increase amounts, a general guide- The protocol is known to lower blood sugar, blood
line for maximum daily intake based on studies is pressure and can have a blood thinning effect.
400mg for Carvacrol amount, and 1800mg daily for In addition, asthmatics on corticosteroids should
NAC. Black Seed Oil can be maintained at 2 to 4 consider additional caution with NAC due to poten-
teaspoons daily. tial spasmodic activity. Refer to the science section
Once you reach a period where die off stops oc- for more detailed information.
curring for 3 to 4 weeks and you are feeling good, Black seed oil contains thujone derivatives, which
you can take a break for a month and see how you may aggravate certain conditions that are prone to
react. If any symptoms return quickly, you may need seizures.

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Protocol User Feedback pension should prompt genetic testing if dysbiosis

can be ruled out.
We’ve shared this protocol online for over a year and
Candida spp. are associated with a number of spe-
a half as we gathered anecdotal reports from users
cific gene mutations that predispose to fungal in-
on their experience.
fections, impair immune response and improve the
In that time, we’ve received hundreds of positive likelihood of both chronic infection and disseminated
anecdotal reports from users of all ages and back- Candidiasis [9, 10, 11, 12, 13, 14, 15, 16, 17].
grounds. Various inflammatory issues improved, These autosomal dominant traits have been classi-
aches, pains, flexibility issues, mood and well-being fied under Familial Candidiasis [18]. Molecular genet-
increased, various forms of dysbiosis were corrected ics tests are available to confirm [19].
(including irritable bowel disorder) and general
It is important to stress that if a patient shows
health and well-being improvements were reported.
positivity for Familial Candidiasis, they will need a
We hope you experience the wonderful improve- long-term antifungal solution in addition to an im-
ments in health and vitality that many have reported. munomodulatory solution to prevent autoimmune re-
If it helps improve your quality of life, please con- sponse. The NAC Protocol meets these requirements
sider sharing this important information with the without the known hepatotoxicity of Amphotericin B
people you care about. or Fluconazole.
We wish you the best in health and vitality. The NAC Protocol can be considered as a natural
treatment option whenever signs of recurrent fun-
gal infections are presented. The review at the end
Advice For Clinicians of this document covers the protocol’s anti-fungal,
anti-inflammatory, hepatoprotective and restorative
Frequent fungal infections like vaginitis (yeast infec- benefits.
tion), autoimmune skin disorders including sebor-
Additionally, mold exposure at home or in the
rheic dermatitis, oral thrush on the tongue, chronic
workplace should be an additional query. Aspergillus
sinus issues, tooth decay and gingivitis, or recurrent
spp. and their mycotoxins (Gliotoxin, Aflatoxin,
infection of the skin, nails or mucous membranes
Ochratoxin) should be ruled out in cases of chronic in-
should prompt further diagnostic tests to rule out
flammation, frequent headaches, chest tightening or
Candidiasis, mold and mycotoxin exposure and ge-
asthma, hemoptysis, eye symptoms or lung nodules
netic predispositions to fungal infections.
detected during x-ray or CT scan. Both Cryptococcus
Candidiasis generally manifests as frequent yeast and Aspergillus are frequently misdiagnosed as lung
infections, fungal nail infections, white or yellow carcinoma [20, 21].
tongue thrush, advanced tooth decay, autoimmune
Antibody testing with Aspergillus-specific IgG can
skin disorders and infection of the mucous mem-
be used if Pulmonary Aspergillosis is suspected. Com-
branes.
mercially available tests to detect serum galactoman-
Candida infections can vary in severity, with most nan (early detection) and 1, 3 β-D-glucan can be used
testing done for Invasive Candidiasis, which is a more as a non-culture based diagnostic.
serious late stage infection. Candida actively works
Urinalysis detecting the primary mycotoxins (afla-
to penetrate the epithelial barrier of the gut, and if
toxin, gliotoxin, ochratoxin) can give a good baseline
successful can be detected using blood culture tests,
of exposure [22], especially in absence of known en-
however culture tests have been shown to be mostly
vironmental exposure, and can indicate an active
unreliable.
infection. Baselines would be lower from food con-
To rule out Invasive Candidiasis using non-culture tamination [23].
tests, The Fungitell test (Associates of Cape Cod,
East Falmouth, MA), multiplex PCR assays and
T2Candida nanodiagnostic panel can be used, with Science Behind The NAC Protocol
75% to 98% sensitivity. The Fungitell test should be
confirmed by two consecutive tests (80% sensitivity)
N-Acetyl Cysteine
and does not detect Cryptococcal infection [8].
If there is no positivity for Invasive Candidiasis N-Acetyl Cysteine (NAC) is a derivative of
and the patient is not immunosuppressed or com- amino acid L-cysteine, used clinically to treat ac-
promised, a pattern of recurring infection should etaminophen overdose and associated hepatic injury.
prompt further investigation. Frequent vaginitis non- It’s commonly used off label in the treatment of lung
responsive to fluconazole treatment, positive sputum conditions, including COPD and cystic fibrosis [24].
cultures for Candida or persistent oral thrush that is The sulfhydryl grouping confers antioxidant effect,
non-responsive to fluconazole or nystatin oral sus- and NAC acts as a precursor to glutathione (GSH)

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tive DNA damage and apoptosis, starting with glu-

tathione depletion. Animal studies suggest that OTA-
dependent oxidative stress is the precursor to cell
lysing [30]. OTA concentrations were tested on a hu-
man renal proximal tubular epithelial cell line (HK-2),
further confirming the role of oxidative stress in geno-
toxicity [31]. A study of OTA genotoxicity on porcine
ovarian granulosa cells showed similar response to
Aflatoxin, damaging repair related genes PARP1 and
RAD51 [32].
Acetaldehyde is a metabolite of Candida Albicans re-
sulting from glycolysis [32]. Both ROS and Ca2+ path-
ways are involved in Drp1 phosphorylation and mi-
tochondrial fragmentation. Elevation of Drp1 phos-
phorylation was partly dependent on ROSmediated
activation of c-Jun-N-terminal kinase (JNK) and p38
mitogen-activated protein kinase (MAPK) [33]. Ac-
etaldehyde chemically induced DNA adducts follow
a dose-response relationship, with mutagenicity fre-
quently occurring as aldehyde dehydrogenase reduc-
tions become overwhelmed [34, 35].
Figure 1: N-Acetyl Cysteine
Guanine is the most frequently oxidized DNA
base, causing transversions in DNA replication [36].
production [25]. O6-methylguanine (O6mG) is a common mispairing,
causing GC to AT transversion. Repair of 06mG to
Primary Benefit And Methodology
guanine is done by O6-alkylguanine-DNA alkyltrans-
N-Acetyl Cysteine (NAC) features a restorative and
ferase (AGT), which requires cysteine [37]. 8-Oxo-7,8-
protective role in The NAC Protocol, both by amelio-
dihydroguanine (8-oxoG) is also frequently oxidized,
rating the genomic damage caused by fungal toxins
causing GC to TA transversions [36].
and restoring excision and chemical repair of DNA.
A study on mice containing Ataxia telangiectasia,
The specific metabolites studied were Aflatoxin,
which show continuous oxidative stress, showed
Gliotoxin, Ochratoxin and Acetaldehyde.
that Thiol-containing NAC counteracts 8-OH de-
Aflatoxin is a secondary metabolite of Aspergillus,
oxyguanosine, a marker for DNA deletions and
specifically A. flavus and A. parasiticus [26]. Aflatoxin
genome instability [38]. Further, NAC was also
B1 (AFB1) is considered hepatotoxic, teratogenic and
shown to restore 06mG, likely by preventing modifi-
immunotoxic in humans [27].
cation of essential thiol groups [39].
Studies on a human epidermal cell line showed
that concentrations of AFB1 >10 µM are toxic to Ha- By modulating the intracellular redox state, NAC
CaT cells and induce oxidative stress via ROS1 and can directly reduce oxidative-mediated apoptosis and
NO2 generation [27]. DNA damage, acting as a scavenger for ROS and
Substantial damage to IMR32 neuronal cell lines maintaining reduced glutathione (GSH) production
was also observed, upregulating NOX2 and trigger- in the liver [40]. Increased levels of GSH from sup-
ing DNA damage via downregulation of PARP1, plementary NAC intake act as a catalyst with Glu-
BRCA2, and RAD51 [28]. tathione S-transferases (GSTs) to reduce AFB1 by
Gliotoxin is also a toxic metabolite of Aspergillus, metabolizing and excreting it [41].
species A. fumigatus, and works via uptake of the NAC inhibits Gliotoxin-induced apoptosis by
disulfide bridge, which cycles between oxidized and blocking activation of caspase-3-like proteases and
reduced state, in turn generating ROS and destroying also scavenging intracellular ROS [42]. With OTA it
plasmid DNA [22]. Gliotoxin is also responsible for inhibits apoptosis by preventing glutathione deple-
activating ROS-mediated apoptosis, and disrupting tion [30].
the integrity of the epithelial and endothelial barriers Finally, NAC binds to Acetaldehyde acting as a
to enhance systemic fungal invasion [22]. scavenger, attenuating ROS and further carcinogenic
Ochratoxin (OTA) is produced by multiple species or genotoxic effect [43].
of Aspergillus [29]. It is capable of inducing oxida- Anti-Biofilm Activity
1 Reactive Oxygen Species The extracellular matrix of biofilms must be con-
2 Nitric Oxide sidered a target when eliminating fungal infections

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due to antimicrobial drug resistance and persistence through membranes and biofilms due to it’s mu-
of infections. colytic effect, hydrolyzing glycoproteins and lipids
Biofilm formation by fungi and bacteria contribute via disulfide bonds and decreasing viscosity [55].
to various pathogenic processes including gastroin- Additional benefit is provided by correcting the
testinal diseases, systemic autoimmune diseases, and imbalance between reactive oxygen species (ROS)
neurodegenerative diseases [44]. Until more recently and glutathione depletion, which offers a protective
it was assumed that biofilms were formed exclusively effect combined with antifungals. As an inhibitor
by bacteria. Various pathogenic fungi can also form to c-Jun N-terminal kinase (JNK) it can also reduce
biofilms, including Candida Albicans, Cryptococcus ne- endothelial dysfunction, inflammation and invasion
oformans, Cryptococcus gatti, Aspergillus fumigatus and [56].
Saccharomyces cerevisiae. The persistence of fungal The antifungal activity of Carvacrol induces ROS
infections is greatly enhanced by it’s ability to form [57] which is ameliorated by NAC, as it is commonly
biofilms [45, 46]. used in clinical settings to identify and test ROS
NAC is a powerful mucolytic antioxidant that ef- inducers [58].
ficiently inhibits and disrupts biofilms. Pseudomonas NAC plays a supportive role, including ROS scav-
aeruginosa is an encapsulated bacterium that fre- enging, disulfide reduction and glutathione replen-
quently causes infections in humans that are difficult ishment.
to treat due to quick formation of biofilm. A recent study on NAC further investigated the
At a concentration of 0.5 mg/ml NAC can de- method of action and proposed an alternative func-
tach mature P. aeruginosa biofilms, and at 10 mg/ml tion for antioxidant activity, suggesting that NAC
biofilms were completely disrupted [47]. A study uptake and deacetylation decelerate and prolong Cys
on treatment of endodontic multi-species biofilms delivery, releasing hydrogen sulfide(H2S), a product
using NAC showed minimum inhibitory concentra- of Cys catabolism. A further product of H2S, sul-
tion (MIC) of 0.78–3.13 mg/ml. Multi-species culture fate sulfur species, is also proposed to contribute to
consisted of Actinomyces naeslundii, Lactobacillus sali- NAC’s beneficial effects as a cytoprotective [25].
varius, Streptococcus mutans, and Enterococcus faecalis
Safety Studies
[48].
NAC has a well-established safety profile, and its
A study of NAC on Candida Albicans biofilm ad-
toxicity is rare. Elimination of NAC occurs through
hesion and disruption showed that NAC works ef-
the renal system, with approximately 30% excreted
fectively on mature biofilms (50-95% disruption) but
through urine. In oral administration the most re-
less effectively on adhesion (≥ 32.8%). The study
ported adverse effects are gastrointestinal symptoms
also showed increased efficacy when combined with
such as nausea, vomiting or diarrhea [59].
ketoconazole, an antifungal [49].
Intravenous or oral inhalation can cause more se-
Cryptococcus Neoformans requires capsular polysac-
rious adverse effects, including anaphylactoid re-
charide for biofilm formation, which primarily con-
actions of flushing, itching, and angioedema, and
sists of Glucurunoxylomannan (GXM) and is also a
systemic symptoms, such as bronchospasm and hy-
constituent of cryptococcal biofilm. These biofilms
potension [60].
are composed of 80% GXM which provides a unique
Oral dosages of 600mg and 1200mg daily showed
challenge [50]. The trans-cell wall vesicular trans-
no significant increase in adverse effects. Dosages as
port system of Cryptococcus is dependent on laccase
high as 3000mg daily resulted in minor gastrointesti-
[51], which is susceptible to NAC via a superoxide
nal symptoms [61].
reaction to copper, converting it to H2O2 [52]. This
There was 1 fatal case of anaphylactoid reaction
reaction in the laccase containing vesicle and corre-
in a 40 year old woman with chronic asthma who
sponding membrane disruption appear to prevent
received intravenous treatment [62]. A potential his-
further virulence and tissue adhesion.
tamine response with asthmatic patients increases
A study on wound biofilm formation treated with
susceptibility to anaphylactoid reactions, and can
NAC showed interference with bacterial cellular re-
potentially occur via oral administration [63]. Addi-
dox states (NADH) and interference with ECM. Dis-
tionally, review of all available literature found no
ruption of biofilms was primarily due to the molecu-
incidence of sulfa or sulfonamide allergy reactions
lar structure of NAC with acetyl and carboxyl groups
with administration of NAC.
[53].
Reports of NAC preventing apoptosis have been a
Protocol Synergy subject of debate. As an example, NAC can be benefi-
NAC has shown a synergistic effect with many anti- cial to neuronal cells by preventing apoptosis caused
fungals, decreasing the MIC values significantly [54]. by trophic factor deprivation [64] but in other cases
It is believed that this is due to better penetration can promote tumor progression by downregulating

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Figure 3: Thymol

Figure 2: Carvacrol
position, as revealed by an increased abundance of
beneficial bacteria and reduced the proportion of
tumor antigen P53 [65]. Thymoquinone provides detrimental flora [74].
a counter to this with p53-mediated apoptosis [66], A similar study on weaned piglets found that
but more importantly is the action of Origanum Vul- Oregano Oil supplemented in chow (25 mg/kg)
gare (Oregano) as it binds to sterols on the fungal showed a lowered population of Escherichia coli in the
membrane, specifically ergosterols and disrupts the jejunum, ileum, and colon. They found that Oregano
permeability of the membrane leading to apoptosis. Oil promotes intestinal barrier integrity by correcting
The two primary active compounds, Carvacrol [67] dysbiosis and lowering inflammation by measuring
and Thymol [68] both contribute to this process. mitogen-activated protein kinase (MAPK), protein ki-
nase B (Akt), and nuclear factor κB (NF-κB) signaling
Oregano Oil pathways [75].
A study on oregano oil’s effect on the intestinal bar-
Origanum Vulgare (Oregano) contains two active com-
rier integrity of Hyland rabbits revealed that oregano
pounds in abundance, Carvacrol and Thymol. Car-
essential oil increased significantly the gene expres-
vacrol is the primary constituent, a p-menthane
sion of junctional adhesion molecule 2 (JAM2) and
monoterpenoid derived from cymene that provides
JAM3 in jejunum (p < .05), showing a direct improve-
many benefits to the human body [69].
ment in intestinal barrier function [76].
Primary Benefit & Methodology A study on broiler chickens fed dietary oregano in
Oregano serves multiple purposes as part of their feed showed a reduction in Campylobacter spp.
The NAC Protocol, including antifungal, anti- and E. coli, with a significant increase in Lactobacillus
inflammatory and immunomodulatory roles. spp [77]. While another broiler study showed similar
Several additional benefits are obtained by using results, with Lactobacilli raised (P < .001) in ileum and
Oregano over other natural antifungals. One instance cecum of all groups supplemented with Oregano
is dysbiosis, where imbalances in the mycobiota can [78].
influence homeostasis and disease progression [70]. These additional benefits to dysbiosis and intesti-
Carvacrol works effectively against pathogenic bac- nal barrier function were considered when choosing
teria and fungi [67, 71, 72, 73] to ameliorate dysbio- Oregano as the primary antifungal. Altered microbial
sis. In one study of mice with C. difficile infection, composition, termed dysbiosis, has been implicated
Oregano Oil positively altered the microbiome com- in mucosal barrier dysfunction and inflammatory

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responses. Restoring the epithelial barrier can po- Effectiveness against a wide variety of pathogenic
tentially prevent autoimmune response and systemic fungi and bacteria have been observed [88]. Carvacrol
infection [79]. and Thymol are effective antifungal compounds that
directly disrupt membrane integrity and ergosterol
Antibiofilm Activity synthesis against Candida isolates [88].
The two primary compounds of Origanum Vulgare, Inhibitory activity against Candida globosa, Candida
Carvacrol and Thymol [69], show both powerful albicans, Cryptococcus laurentii, Trichosporon asahii, Ko-
inhibitory and disruptive activity against biofilms. damaea ohmeri and Saccharomyces using an Oregano
Pathogenic fungi can make their own biofilms [80] or ethanolic extract showed a MIC value of 1.56 mg/mL
cohabitate in multi-species bacterial biofilms where [89].
they arrange micro colonies with distinct features
A study of Oregano against Aspergillus flavus and
[81].
Penicillium commune as possible alternatives for food
Therefore it is important to address mixed biofilms preservation showed a MIC of 4 mg/mL [90]. Ef-
to effectively treat fungal infections. In a study on fectiveness against Aspergillus niger and Aspergillus
Staphylococcus aureus and Candida albicans in single flavus was compared between oregano (Origanum vul-
and mixed cultures, Carvacrol showed a strong de- gare), thyme (Thymus vulgaris) and clove (Syzygium
crease of cell count, biomass, metabolic activity, and aromaticum), with Oregano showing the highest in-
vitality of established 24- and 48-h biofilms [82]. hibitory levels [91].
A synergy was also shown between Carvacrol and Tests of essential oils against heat resistant molds
Thymol in a similar study on Candida albicans and Aspergillus fumigatus and Paecilomyces variotii using
Staphylococcus epidermidis, where this combination citrus (Citrus sinensis L. Osbeck), laurel (Laurus nobilis
killed highly tolerant persister cells of mono-species L.), myrtle (Myrtus communis L.), oregano (Origanum
and mixed-species biofilms and demonstrated less vulgare L.), and savory (Satureja thymbra L.) showed
risk of resistance development [83]. Effectiveness Oregano as the most effective inhibitor of growth
against Salmonella Enteritidis biofilms also showed [92]. Another study on Aspergillus niger, Aspergillus
Carvacrol and Thymol as effective, showing inhibi- carbonarius, and Aspergillus wentii showed inhibitory
tion of biofilm formation at sub-minimum inhibitory effect of 95.6%, 45.6%, and 100% at 2.5mL/100mL,
concentration and effectiveness against preformed respectively [93].
biofilms [84].
Effectiveness of essential oils tested against Crypto-
Effectiveness was also found against biofilms pro- coccus neoformans and Cryptococcus laurentii showed
duced by pathogenic fungi. In a study on oral can- Carvacrol and Thymol as most effective (16 and 32
didiasis, carvacrol and thymol significantly reduced µg/mL) as planktonic inhibitors, compared to Cinna-
both mature biofilm biomass and metabolic activ- mon oil (Cinnamaldehyde), Lemongrass oil (Citral),
ity [85]. A study on the antibiofilm and antifungal Clove oil (Eugenol), Peppermint oil (Menthol) and
activity against Cryptococcus neoformans and Crypto- Thyme oil (Thymol) [86].
coccus laurentii compared Oregano oil (Carvacrol),
Cinnamon oil (Cinnamaldehyde), Lemongrass oil Protocol Synergy
(Citral), Clove oil (Eugenol), Peppermint oil (Men- There are a number of likely synergies between
thol) and Thyme oil (thymol). The top 2 compounds NAC, Oregano Oil and Black Seed Oil based on avail-
for antibiofilm activity were Thymol and Carvacrol, able studies.
respectively [86]. Thymoquinone (TQ) is the active compound in
Method of inhibitory action on biofilms was eluci- Black Seed Oil (Nigella Sativa). In a study of oral
dated in a Salmonella typhimurium biofilm study. Pro- candidiasis, TQ was tested against Candida albicans,
teomic analysis showed changes in the proteins DsbA Candida tropicalis, Candida glabrata and Candida krusei
(thiol: disulfide interchange protein DsbA), LuxS (S- strains and the synergistic antifungal activity of these
ribosylhomocysteine lyase), DksA (RNA polymerase strains in combination with nystatin. With TQ alone
binding transcription factor DksA), and SODs (su- C. albicans was significantly inhibited at 7.5 µg/mL.
peroxide dismutases) A, B and C showed inhibited Nystatin showed inhibition against C. albicans at 1.875
synthesis [87]. µg/mL, but when combined with TQ it lowered MIC
to 0.234 µg/mL showing a strong synergy [94]. TQ
Antifungal Activity has also shown a synergistic effect against multi-
Origanum Vulgare (Oregano) and it’s primary drug resistant bacteria and fungi when combined
constituents Carvacrol and Thymol have shown with antibiotics [95] or antifungal treatments [96].
anti-oxidant, antiseptic, anticarcinogenic, anti- We believe there will be a similar synergy between
inflammatory, antidiabetec, immunomodulatory, an- Carvacrol, Thymol, and Black Seed Oil compounds,
timicrobial, antispasmodic and antibacterial benefits. decreasing inhibitory concentrations and increasing

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effectiveness against multi-drug resistant fungi. vivo, as well as human clinical trials, approval as a
Determining chemical composition of N. sativa food additive by the FDA, and used extensively as a
shows many potential synergies. Some of the ad- food preservative to prevent spoilage.
ditional active compounds found by GC and GC/MS A Phase I clinical study on the safety of Carvacrol
analysis were trans-anethole, p-cymene and limonene studied 1mg/kg and 2mg/kg groups in a human
[97]. Carvacrol and p-cymene have shown synergy as trial for one month, finding all post-treatment mea-
compounds, reducing the minimum inhibitory con- sured parameters were within normal range. The
centration of Carvacrol [98]. Studies on Limonene- results of this phase I study regarding carvacrol ef-
Carvacrol (Lim-Car) have also shown synergy in in- fects on healthy subjects, showed clinical safety and
hibitory concentrations [99]. tolerability [106].
Synergy between NAC and antifungals has been A Phase II clinical trial on the possible therapeutic
shown previously with Fluconazole and Caspofungin effect of Carvacrol on asthmatic patients also showed
[100]. Data infers that the mucolytic activity of NAC no adverse outcomes [107].
combined with the antifungal activity of Oregano Due to strong interest by the food industry for
provide an effective treatment against eukaryotic and natural options for food preservatives, animal studies
sessile forms of pathogenic fungi. are also plentiful. A study on in vivo genotoxic effects
produced in rats orally exposed to 81, 256 or 810 mg
Immune Modulation
cavacrol/kg body weight (bw) at 0, 24 and 45 h found
Oregano as part of The NAC Protocol acts as an im-
that carvacrol (81-810 mg/kg bw) did not induce in
munomodulatory compound through several mecha-
vivo genotoxicity or oxidative DNA damage in any
nisms. Both Carvacrol and Thymol play a role, with
of the tissues investigated [108].
Thymol suppressing expression of iNOS and COX-2,
blocking the phosphorylation of IκBα, NF-κB p65, Studies on Oregano Oil (OO) and Oregano Essen-
ERK, JNK, and p38 MAPK [101]. Carvacrol showed tial Oil (OEO) showed similar safety profiles. A study
similar effect against pro inflammatory IL-1b, COX- on OEO’s oxidant effect (DPPH and ABTS assays)
1 and COX-2, while upregulating IL-10 [102, 103] and cytotoxicity found OEO to be nontoxic [109]. A
and demonstrating tissue healing ability against gas- similar study on Wistar rats tested for genotoxicity
tric ulcers and remodeling ability in a skin disease over a 90 day trial, using 50, 100 and 200 mg/kg ad-
study. OEO significantly inhibited several inflam- ministered daily. Results obtained in the genotoxicity
matory biomarkers, including monocyte chemoat- assays indicated lack of effect in micronucleus and
tractant protein 1 (MCP-1), vascular cell adhesion standard comet assay under the conditions tested,
molecule 1 (VCAM-1), intracellular cell adhesion showing no genotoxicity or oxidative damage to tis-
molecule 1 (ICAM-1), interferon gamma-induced sue [110].
protein 10 (IP-10), interferon-inducible T-cell alpha The evidence currently suggests that Oregano Oil
chemoattractant (I-TAC), and monokine induced by is safe for more long-term use, showing no indicators
gamma interferon (MIG) [104, 105]. of oxidative damage, genotoxicity, mitochondrial dys-
Oregano oil supports the immune system overall function or morphological changes in healthy cells.
by also reducing mycotoxin burden via fungicidal Oregano Oil and it’s active compounds do show cy-
action against susceptible pathogens like Aspergillus totoxicity against cancerous cells, however.
and Candida, as shown in Oregano Oil Antifungal In a study on Acute myeloid leukemia cell lines
Activity section (AML) carvacrol and thymol showed powerful syn-
Additional immune modulatory benefit was ergy, inducing tumor cell death with low toxicity on
demonstrated in Oregano Oil Primary Benefit & normal cells. Cell death induced by the carvacrol
Methodology section, showing how Oregano can and thymol combination is caspase-dependent in
ameliorate dysbiosis issues which disrupt homeosta- the HL60 cell line and caspase-independent in the
sis and promote disease progression. other cell lines tested [111]. Furthermore, a study on
Finally, the repair of the intestinal epithelial barrier F1 DBA C57 Black hybrid mice studied OEO effect
by reducing inflammation and stabilizing dysbiosis on Lewis carcinoma tumor engraftment. Mice were
promotes further immune enhancing effect. In all, fed a low uptake dose of oregano essential oil with
Oregano is a powerful tool against pathogen-derived drinking water for three months, showing a tumor en-
disruption of homeostasis and acute inflammatory graftment decreased by 1.8 times, its size decreased
response. by 1.5 times, and the development of tumor was
significantly suppressed. Interestingly, activity of an-
Safety Studies tioxidant enzymes was found to increase after three
Oregano is one of the most widely studied natural months of essential oil uptake (by 1.5–3 times) as
antimicrobials, with animal studies in vitro and in compared to the control group [112].

8 Document version 1.0.4

 The NAC Protocol

ide dismutase against superoxide [119]. A reduc-

tion of TQ in the liver to dihydrothymoquinone
is part of this antioxidant mechanism, and com-
bined they appear to mediate this protective action
[120] and also act as effective OH radical scaveng-
ing agents [121]. TQ is known as a scavenger for
hydroxyl and carbon centered radicals. It also short-
ens ROS-facilitated stress by yieling glutathionylated-
dihydrothymoquinone via non-enzymatic reaction
[122].
Antioxidant and Anti-inflammatory actions of TQ
are the primary mechanisms that protect hepatocytes
from injury. Myeloperoxidase activity in the liver
tissue is an aggravating factor by increasing lipid
peroxidation and free radical formation [123].
The hepatoprotective role of BSO is crucial as part
of The NAC Protocol.

Antibiofilm Activity
Figure 4: Thymoquinone As part of The NAC Protocol BSO acts primarily as
hepatoprotective, immune modulatory and an anti-
fungal potentiator. Anti-Biofilm activity is also robust
Oregano oil as part of The NAC Protocol is rec-
due to the abundant monoterpines and sesquiter-
ommended at 40mg Carvacrol twice daily, or 80mg
pines [114]. Minimum biofilm inhibitory concentra-
total intake per day. Safe levels were tested up to
tion (MBIC) for Thymoquinone ranges from 25-100
600mg per day in the above referenced Phase I trial
µg/mL, with Candida Albicans being highly suscep-
(2mg/kg), and up to 800mg/kg daily in animal trials
tible using in vitro assays [124].Staphylococcus aureus
showed no cytotoxic effects.
and Staphylococcus epidermidis minimum biofilm in-
hibition concentration (BIC50) was reached with 22
Black Seed Oil and 60 µg/ml, respectively. TQ also prevented cell
adhesion [125]. N. sativa oil (BSO) showed highest
Thymoquinone, derived naturally from Nigella Sativa,
microbial activity when compared to aqueous and
is a natural compound with widespread protective
methanolic extracts. BSO was also shown to reduce
effects, including anti-oxidative, anti-inflammatory,
preformed biofilms of multi-drug resistsnt MRSA
immunomodulatory, anti-cancer, and anti-microbial
1294, MRSA 1295 and MRSE 1297 effectively [126].
[113].
The complexity of bioactive ingredients plays a
Primary Benefit & Methodology
major role. In a study testing BSO against Liste-
Black seed oil is typically produced using a cold
ria monocytogenes, a common food contaminant,
press process, extracting the active compounds from
30 ligands were tested. α-longipinene was selected
the Nigella Sativa seed. A GC-MS analysis re-
based on in silico docking studies. Further in vitro
vealed more than 30 active compounds, including
studies demonstrated the anti-biofilm activity of α-
thymoquinone, fenchone, p-cymene, trans-anethole,
longipinene [127]. The complexity of terpines in the
limonene, carvone, carvacrol, longifolene and many
volatile oil likely contributes to it’s broad spectrum
additional active compounds [114].
effectiveness. This complexity leads to many po-
The effect of Black seed oil (BSO) as part of The
tential synergies. p-cymene, a major constituent of
NAC Protocol is multi-purpose, serving as hepato-
BSO based on GC-MS analysis [114] has shown to
protective, a potentiator for antifungal activity, a
have a synergistic effect with y-terpinene, carvacrol
biofilm disruptor, immune modulator, and a restora-
and other active compounds in BSO to increase anti-
tive which can increase t-cell count and differentia-
biofilm activity [128].
tion [115, 98, 116, 117, 118].
A study of doxorubicin-induced cardiotoxicity in Studies on the individual active compounds in BSO
rats using 10mg/kg daily TQ in drinking water show several unique anti-biofilm qualities. Limonene
showed amelioration of induced cardiotoxicity. TQ interferes with C. albicans biofilm adhesion, while
proved to be a potent superoxide radical scavenger, trans-Anethole shows synergy with biofilm inhibition
with scavenging power being as effective as superox- against S. aureus [129, 130].

Document version 1.0.4 9

The NAC Protocol

Antifungal Activity rect synergy with Carvacrol, including p-cymene and

Nigella sativa has been studied extensively for its limonene [98, 99]. Carvacrol and Thymol, the pri-
pharmacological benefits, but antifungal research is mary active compounds in Oregano are also featured
limited. In a study of N. sativa in a methanolic extract, in BSO [114]. BSO has been found effective against
it was found effective against 20 different strains of multi-drug resistant S. aureus, P. aeroginosa and C.
Candida [131]. albicans, and Carvacrol performs similarly [140, 141].
A further study on candidiasis of mice using an Carvacrol and Thymol concentrations in BSO are in
aqueous extract of N. sativa (6.6 mL/kg) showed sig- lower concentrations [114] but when adding Oregano,
nificant inhibitory effect, only 24 hours after inocula- which contains higher levels of Carvacrol and Thy-
tion. A 5-fold decrease in Candida in kidneys, 8-fold mol, two distinct methods of action are present [69].
in liver and 11-fold in spleen was observed [132]. In- Thymoquinone has been observed to disrupt C. albi-
hibitory effect on Aspergillus parasiticus (CBS 921.7) cans cell wall synthesis, disintegrate the cytoplasm
and Aspergillus flavus (SQU 21) was also demon- and act as a pro oxidant inducing oxidative stress via
strated (1-3mg/100ml) using N. sativa oil (BSO) with ROS generation [142, 143]. Differentially, Oregano
potential metabolic effects on biosynthesis pathways disrupts the cell membrane by interrupting ergosterol
for aflatoxin [133]. synthesis [88].
Investigating the composition of N. sativa volatile N-Acetylcysteine (NAC) was studied on chronic
oil yields several active compounds, including thy- wound biofilms using mice with a 20 day maturation
moquinone, p-cymene, a-thujene, limonene, trans- period. Pseudomonas, Staphylococcus, Acinetobacter,
anethole, fenchone and carvacrol [114]. and Enterobacter were identified in the wound biofilm.
Thymol, thymoquinone (TQ) and thymohydro- NAC demonstrated effectiveness in disrupting the
quinone (THQ), all constituents of N. sativa, were extracellular matrix of the biofilm, penetrating the
tested against 30 pathogens acquired from patients bacterial cell membrane, inducing oxidative stress
at a concentration of 1mg/mL. 100% inhibition was and disrupting protein synthesis [53].
demonstrated against eight dermatophyte, five yeast We believe the mechanism of NAC combined with
and five mold isolates. TQ was found to be the the antifungal compounds in N. sativa and Oregano
strongest antifungal compound against dermato- provides a specific advantage when treating fungal
phytes and yeasts. Thymol was the most effective and bacterial infections. This combination is crucial
against molds [134]. with up to 80% of the targeted pathogens residing in
A study on human infection by Fusarium solani, a biofilms [144].
filamental fungi from the Nectriaceae family, was per-
Immune Modulation
formed comparing Thymoquinone to Amphotericin
Nigella Sativa has been used in Middle Eastern folk
B. A 10 day inhibition test using 1mg/mL was per-
medicine since biblical times, with modern research
formed. TQ demonstrated 100% inhibition by day
showing N. sativa has effect on respiratory problems,
10, however Amphotericin B only inhibited 72.4% of
dyspepsia, metabolic syndrome, diabetes mellitus, in-
growth in the same time range [135].
flammatory diseases, and various types of cancer
P-cymene has shown to be effective against drug-
[145, 146].
resistant forms of Candida, showing synergy when
The primary purpose of Black Seed Oil (BSO) as
combined with Thymol [136].
part of The NAC Protocol is both hepatoprotective
Trans-anethole also has strong antifungal proper-
and immunomodulatory. BSO has potent antioxi-
ties. Fennel is known as a strong antifungal, which
dant effect over several pathways, modulating NF-Kβ,
is composed primarily of trans-anethole [137]. Trans-
inhibiting iron-dependent lipid peroxidation, eleva-
anethole has demonstrated effect with other drugs
tion in total thiol content and (GSH) level, radical
as it exhibits synergistic activity against several fungi
scavenging, increasing the activity of quinone reduc-
[136].
tase, catalase, superoxide dismutase (SOD) and glu-
Fenchone was also shown to inhibit fungal growth
tathione transferase (GST) and inhibiting COX/LOX
(32-64 µg/mL) testing against Candida albicans ATCC-
[147, 148].
76645 and LM-05, Candida tropicalis ATCC-13803
As an anti-inflammatory, Thymoquinone (TQ)
and LM-20, and Candida Krusei ATCC-6258 [138].
inhibits JNK, ERK and P38 phosphorylation and
Limonene was also shown effective against C. tropi-
PI3K/mTOR signaling activation. In addition, BSO
calis (20-40 µL/mL) using potato dextrose broth [139].
was shown to decrease lipid profiles (TG, TC, LDL,
Protocol Synergy VLDL), liver enzymes (AST and ALT), hs-CRP in-
N. sativa oil (BSO) has shown synergy with both flammatory marker, IL-6 and TNF-α [149, 150, 151].
antifungals and antibacterials as a potentiator [95, As an immunomodulator, BSO can directly im-
96]. Active compounds in BSO have also shown di- prove immune response to fighting infection. A study

10 Document version 1.0.4

 The NAC Protocol

of immunostimulation on a murine macrophage cell (TG). Renal function markers (creatinine) were also
line showed that N. sativa ethanolic extract directly tested. Recruited participants did not exhibit any
increased macrophage count in a cell proliferation clinical signs of toxicity or adverse effects. Liver
assay, showing up to an 138% increase (25 µg/ml) toxicity and kidney function markers showed no
[152]. An additional study using ethanolic extract on change, however, lipid profiles showed significant
blood derived, splenic and peritoneal macrophages decrease but were within safe limits. Change in
showed a remarkable increase in phagocytic activity TC, TG, LDL, VLDL and HDL were 12.1%, 19.66%,
[153]. 16.33%, 12.76%, 8.21% and 15.27%, respectively [167].
Immunostimulatory effect has also been demon-
strated with peripheral blood mononuclear cells
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