Licorice

Glycyrrhiza is a plant family that is commonly known as Liquorice. It is divided into several species, where Uralensis and Glabra are the two most commonly used. Other species that do exist and belong to the licorice family are inflata and echinata.^[8] Glycyrrhiza derives its botanical name from the Greek words Glycos (sweet) and rhiza (root), and is literally called SweetRoot^[9] in reference to its sweetness; mediated by Glycyrrhizinic Acid that is said to be 50 times as sweet as sucrose on a molecular weight basis^[10] and is sometimes added to cigarettes and chewing tobacco to enhance sweetness as a natural sweetener.^[11]

When used in traditional medicines, it is sometimes referred to as Yashtimadhu (Ayurveda^[12]) or Gan cao (Traditional Chinese Medicine in reference to the root extract^[13]) or Kanzoto (aqueous extract^[14]). In Traditional Chinese Medicine, it is usually recommended to consume 8-25g of Licorice daily (as a decoction) and up to 100g during periods of disease^[15][16] and tends to be catered to respiratory, cardiovascular, endocrine, and digestive system diseases.^[16] In greek medicine, it has the standard usages of treating pectoral and respiratory diseases but appears to have been a treatment for Addison’s Disease^[9] while in Chinese Medicine licorice is said to be useful for (according to Shen Nong Ben Cao Jing) life-enhancing properties, improving health, cure of injury or swelling, and for its detoxification effects.^[17]

Licorice is a plant where the roots are both used for medicinal properties and also sometimes used to make confectionaries. The root is highly sweet, from which it derives its name

Another form of “licorice” exists and is referred to as Brazilian Licorice (with the official name of Periandra Mediterranea) and is unrelated to the Glycyrrhiza family of plants. The phrasing of “Chinese Licorice” tends to refer to the species of Glycyrrhiza Uralensis,^[18] although sometimes pallidiflora is used in replacement of Uralensis for confectionary purposes.^[9]

Other common species of Glycyrrhiza that are not commonly used for medicinal purposes are typica (Central Europe and England), glandulifera (Southern Russia), violacea (Midde East), and lepidota (North America); there are 13 species known in total.^[9]

(Potentially) bioactive components of Glycyrrhiza include:

• Glycyrrhetinic Acid and its diglycoside of Glycyrrhizin. The latter said to be between 2-15% dry weight of the plant^[19] but measured appears to be in the range of 1.212-40.7mg/g (0.1-4%; most values around 1.5%).^[20] Up to 9.1% can be found in a hydroalcoholic extract,^[21] and when identifying both the 18α and 18β isomers of Glycyrrhetinic Acid, their ranges in mg/g dry weight are 0.062-0.475 and 0.015-0.13 respectively^[20]

• Glycyrrhyzic Acid (inactive analogue of Glycyrrhetic Acids)^[22]

• Glabridin at 0.92mg/g licorice by dry weight,^[23] although another study suggests a range of 0.08 to 0.35% (0.92mg/g being 0.09% and within this range)^[24] and one study reporting up to 11.9%;^[25] 100g of Licorice gives 80–350mg Glabridin within the normal range

• Liquiritigenin (flavanoid; 0.108-2.174mg/g) and when diprenylated, Glabrol (2.3mg/g dry weight).^[26] Also Isoliquiritigenin (0.073-0.489mg/g),^[14] and Liquiritigenin’s glycoside Liquirtin (0.451-30.7mg/g; up to 3% Licorice by dry weight). Liquirtigenin is unique to Licorice and Alfalfa^[27]

• Coumarins (Licopyranocoumarin, Licoarylcoumarin, Glycycoumarin)^[28][29]

• Formononectin^[30]

• Glisoflavone^[29]

• Various prenylated flavanoids (Prenylicoflavone A, Glysasperin A, Licoricidin, Hispaglabridin A, Isoagnusone A, Kanzonol K, and Glycyrrhisoflavone)^[17]

• Various volatiles such as β-caryophyllene oxide, decadienol, 1α, 10α-epoxyamorpha-4-ene, β-dihydroionone, thymol, and carvacrol (mostly related to flavor and scent)^[8]

• Isoangustone A (Glycyrrhiza Uralensis)^[31]

• Licochalcone A-E (mostly from the inflata species^[32][33]) with at least A^[34] and C^[35] isolated form Glycyrrhiza Glabra and limited amounts in Uralensis^[36]

The main active component of Liquorice is known to be Glycyrrhizin and its metabolite 18-β glycyrrhetinic acid, flavanoids which focus mostly on Liquirtigenin and Isoliquirtigenin, and the subset of polyphenolics centering on Glabridin. Although there are many different bioactives (and an entire class of prenylated flavanoids with promise), those three categories seem to be most researched

And polysaccharides found in Licorice (bioactive compounds that belong to the carbohydrate class; found in caloric vessels of Licorice but not isolated calorie-free extracts):^[17]

• Glycyrrhizin UC (69,000kDa; L-arabinose, D-galactose, D-glucose, and L-rhamnose at 10:30:27:1 in arabino-3, 6- galactoglucan-type structure)^[37][38] found in Uralensis

• Glycyrrhizin UA^[39] found in Uralensis

• A collection of lesser known Uralensis polysaccharides (n=10)^[40]

• Glycyrrhizin GA (85,000kDa; L-arabinose: D-galactose: L-rhamnose: D-galacturonic acid: D-glucuronic acid at 22:10:1:2:1)^[41][42] found in Glabra

• Unidentified pro-immunity polysaccharide^[43]

Like many medicinal herbs, licorice has a collection of bioactive polysaccharides

About 20% of the bioactives can be removed from the water extract, which consist of the sweetened fragment (mostly Glycyrrhizin up to 7-25% of the water extract) and flavanoids related to Liquirtigenin (1-1.5% total weight, 5-7.5% of the water extract).^[9]

Glabridin may become unstable during heat and light exposure.^[44]

One study assessing confectionaries noted that the amount of Glycyrrhizin in treats ranged from 2.96-5.37% of product weight (assessing chips and cylinders), and the content was not significantly different with commerical quality roots;^[19] due to this, it has been estimated that the American public has an average consumption of 0.027-3.6mg/kg daily consumption of GLycyrrhizin secondary to confectionary consumption and cigarettes.^[9]

One study noted that, in a small uncontrolled trial of 4 persons, that consuming 200g of Licorice (containing 100mg Glycyrrhisin and 1.6mg Glycyrrhetic Acid) as food product in 45 minutes resulted in a (range) of values from 250-434ng/mL after 250 minutes that persisted until the experiment ended at 500 minutes; one of the four subjects had a significantly lesser (150ng/mL) peak at 350 minutes.^[45] This is despite Glycyrrhizin having apparently reduced absorption when consumed via licorice products (relative to isolated supplements).^[46]

Food products may have the same or comparable levels of bioactives as basic licorice root and unconcentrated supplements, and licorice should be viewed as a functional food due to this

At least in the US, regulations are in place for maximal Glycyrrhizin content in food products:^[9][47]

• Baked goods at 0.05%

• Alcoholic beverages and “unclassified” at 0.1%

• Plant protein products, seasonings, and non-alcoholic beverages at 0.15%

• Dietary supplements (for the purpose of flavor enhancement) at 0.5%

• Chewing gum at 1.1%

• Soft candy at 3.1%

• Hard candy at 16.0%

Licorice as a flavoring agent has a Generally Recognized as Safe (GRAS) classification.^[48]

Glavonoid is a brand name used in some trials as “Licorice Flavanoid Oil” (LFO) patented by Kaneka Nutrients and sourced from Glycyrrhiza Glabra, concentrated for the flavanoid Glabridin. It is standardized to 30% Polyphenolics with 3% Glabridin according to one study^[49] while another measuring the oil found that 90% of LFO by weight consisted of Medium Chain Triglycerides, 10% of Licorice extract, and 1% by total weight was Glabridin; other components included glabrene (0.2%), glabrol (0.2%), and 0.1% 4′-O-methylglabridin with under 0.005% glycyrrhizin (diglycoside of Glycyrrhetic Acids).^[6] These claims of undetectable levels of Glycyrrhizinic Acid have been confirmed by a European Food Safety Panel.^[10]

Glavanoid is a concentrated (patented) formula of flavanoids standardized for its Glabridin content with little to no Glycyrrhizin content

Deglycyrrhizination is a pseudo-term to refer to the removal of Glycyrrhizin and the active 18β-hydroxyglycyrrhizic acid from Licorice supplements. These two compounds are the reason behind some properties of Licorice that are seen as undesirable, such as increases in cortisol and decreases in testosterone.

Extracts that are wholly water (such as tea) or a mixture of water ethanol (70:30 or 50:50) are seen as optimal to preserve contents of Glycyrrhizin,^[23] and as such extractions with fat-soluble solvents are preferable for deglycyrrhizination; a 90:10 hexane:ethanol extract is able to reduce Glycyrrhizin contents to undetectable levels.^[50][31]

The aforementioned Glavanoid product (which is a Medium-Chain Triglyceride extraction) claims to have under 0.005% Glycyrrhizic Acid by weight.^[10]

In Caco-2 cells, Glabridin appears to be absorbed in vitro and reaches serum when orally absorbed either in isolation of as a component of Licorice root Oil.^[51] Absorption appears to be relatively higher when consumed via Licorice root oil, judging by serum levels of Glabridin.^[51] As assessed by serum, Glabridin has an oral bioavailability of approximately 7.5% and is constant for various doses^[52][15] and this low absorption rate is due to excessive efflux by P-Glycoprotein transporters (drug efflux transporters).^[15]

Glabridin does appear to be an inhibitor of P-Glycoprotein itself when incubated in vitro, as it can accumulate Digoxin, but this does not appear to biologically relevant as mice lacking this receptor have enhanced absorption of Glabridin (relative to normal mice) and a stronger P-Glycoprotein inhibitor (Vermapril) can enhance absorption rates.^[15]

P-Glycoprotein inhibitors may increase intestinal absorption of Glabridin

In regards to Glycyrrhizin, it appears to also have relatively low absorption with 50mg/kg being the lowest dose that can be detectable in rat plasma after oral administration;^[53][54] despite this correlating to 8mg/kg in humans (545mg for a 150lb person), Glycyrrhizin can be found circulating in blood of humans at lower doses of 100mg increasing relatively lineraily up to 1600mg.^[55][56][57] Absorption of Glycyrrhizin and its metabolites are dependent on metabolism by intestinal bacteria prior to absorption, as germ-free rats do not get spikes in serum after oral ingestion.^[58][59]

The diammonium salt of Glycyrrhizin results in an apparent bioavailability of 98.88±12.98%, near complete absorption over 24 hours.^[60]

Glycyrrhizin and its aglycone Glycyrrhetic Acid can both be absorbed after oral ingestion of either licorice or isolated supplements (with higher apparent bioavailability with supplements), and hydrolysis by intestinal bacteria appears to be critical for absorption. Glycyrrhetic Acid has near perfect absorption but is prolonged

After oral ingestion of Glabridin, it appears in serum unconjugated in humans,^[61] and this lack of conjugation appears to hold true for rats as well in serum,^[51] but incubating Glabridin in hepatic rat microsomes can produce glucuronide derivatives (via UDPGA), with a lower conversion rate in intestinal cells.^[15]

After ingestion of 10mg/kg Glabridin in rats either in isolation or as a component of Licorice Flavanoid Oil (LFO), the relative pharmacokinetic values are a C_max of 87nmol (isolated Glabridin) or 145nmol (as LFO) at a T_max of one hour, an AUC_inf of 825nM/h (isolated Glabridin) or 1,301nM/h (as LFO) with a half-life of 8.2-8.5 hours.^[51] When replicated in humans following oral consumption of at doses of 300, 600, or 1200mg LFO, there appears to be a dose-dependent increase in serum concentrations with the three respective C_max values being 1.12±0.29ng/mL, 1.27±0.25ng/mL, and 2.65±0.46ng/mL with 300-600mg having a T_max of 3.2-3.6 hours and 1200mg having a T_max of 6 hours.^[6] Half-life fluctuated with no relation to dose (8.9-13.9 hours) and AUC_inf increased dose dependently with 12.27±3.31, 21.78±5.40, and 40.28±3.72 for 300, 600, and 1200mg respectively.^[6]

During multiple dosing testing, the increase in serum Glabridin after consumption of LFO appears to be enhanced after repeated doses (4-55% higher, with higher doses resulting in more serum levels).^[6]

Glabridin appears to have a favorable pharmacokinetic profile and remains bioavailable (unconjugated) in serum

In serum, Licochalcone A appears to bind to serum albumin.^[62][63]

In response to 150mg Glycyrrhizin (as diammonium glycyrrhizinate; metabolized to Glycyrrhetic Acid in a similar manner as Glycyrrhizin^[59][64]) in otherwise healthy volunteers in a fasted state noted that a C_max of 95.57±43.06 resulted at T_max 10.95±1.32 hours, giving a half-life of 9.65±3.54 hours and an AUC to infinity of 1367.74±563.27ng/h/mL.^[60]

Glyyrrhizin (Diglycoside of Glycyrrhetic Acid) is metabolized to its aglycone form (Glyccyrhetic Acid) by intestinal bacteria, either completely or via an intermediate known as Monoglucuronyl-glycyrrhetic acid; the former “complete” metabolism occurs when both sugar molecules are removed at once, and the intermediate exists when they are removed in succession of each other.^[11][9]

After absorption, the aglycone terpenoid (Glycyrrhetic Acid) can be metabolized to 18β-glycyrrhetyl-3-O-hydrogen sulfate, 18β-glycyrrhetyl-3-O-monoglucuronide, or 18β-glycyrrhetyl-30-monoglucuronide via hepatic biotransformation.^[65][66] Another metabolite known as 3-ketoglycyrrhetic acid has been reported in animals, but no human data exists for this particular metabolite.

Some metabolism of Glycyrrhizin metabolites further in the liver

Liquirtigenin appears to be hydroxylated by the Aromatase enzyme (specifically CYP1A2, 1A1 not tested) into 4′′,5,7-trihydroxyflavanone by C5 hydroxylation and is prevented with aromatase inhibitors,^[67] Liquirtigenin can also be rapidly metabolized (6.2 minutes after incubation) into 7,4′′-dihydroxyflavone and is mediated by CYP3A4.^[67][68] 

Beyond those two metabolites linked to an enzyme, other metabolites that have been noted in rat liver microsomes are 7,3′′,4′′-trihydroxyflavone, a hydroxyl quinine metabolite, two A-ring dihydroxymetabolites, and 7-hydroxychromone.^[69]

Liquirtigenin goes through metabolism via Phase I enzymes of P450

In a comparative study between the two isomers of Glycyrrhetic Acid (18α-Glycyrrhetic Acid and 18β-Glycyrrhetic Acid), it was found that both were rapidly distributed to tissues when injected into rats at high doses (21mg/kg) with β having a more general distribution and α appearing to favor the liver.^[70] For both isomers, distrubution was relatively lower in the muscles and the brain, and appeared to be relatively higher in organs of drug metabolism (kidney, liver) as well as both the lungs and cardiac tissue.^[70]

An oral dose of Glabridin can be detected in the liver (23-53nmol; 0.37-0.41% oral dose), kidney (0.02% oral dose; 1.14-2.21nmol), and mesenteric body fat (minimally).^[61]

After injection of the two isomers of Glycyrrhetic Acid, both are rapidly depleted from tissues within 180 minutes.^[70] Elimination of Glycyrrhetic Acid and metabolites appear to be mostly mediated by bile acids and excretion via the feces, and less than 2% of an oral dose is detectable in the urine.^[66] Some enterohepatic circulation appears to be present.^[66][71]

One study on cAMP phosphodiesterases (PDE, subunit not specified) noted that a variety of flavanoids and courmarins had inhibitory potential; mostly Isoliquiritigenin (IC₅₀ 18uM), Glabridin (8.2uM), Licoricidin (4.9uM), Licoarylcoumarin (1uM), Glycycoumarin (0.7uM) and Licoricone (2.3uM).^[28] Liquiritin and Liquiritigenin were less potent.^[28]

Appears to have inhibitory potential towards Phosphodiesterases in vitro; practical relevance of these inhibitory potentials is unknown

Glycyrrhizin and Licorice extract itself are both able to induce P-Glycoprotein expression and CYP3A4 activity, which have been shown to reduce Cyclosporin AUC after both are given to rats.^[72] P-Glycoprotein inhibition has been noted elsewhere with both Glycyrrhetic Acid and Glabridin, while Glycyrrhetic Acid also inhibited MRP1.^[73]

Glycyrrhizin induces (increases) activity of P-Glycoprotein and CYP3A4 activity, while other compounds in Licorice may inhibit P-Glycoprotein

When looking at Glabridin (one of the main bioactives in Licorice supplements without Glycyrrhizin or its derivatives), Gliabrin also appears to inhibit P-Glycoprotein weakly^[73] and inhibited the P450 enzymes of CYP2B6, CYP2C9, and CYP3A4.^[74] When licorice extract is incubated with CYP3A4, activity was reduced at concentrations of 1.4, 6.9, 14, and 69ug/mL to 73%, 45%, 25%, and 12% of control levels after 15 minutes and was completely inhibited with 50uM of pure Glabridin and was not reversible.^[74] With CYP2B6 inhibition was weaker but followed a similar irreversible trend, and it appeared Glabridin destroyed the heme fragment of these P450 enzymes and thought to be due to its anti-oxidant abilities (as the derivative 2,4-Dimethylglabridin, with no anti-oxidant capabilities, failed to inhibit CYP3A4).^[74] Inhibition of CYP2C9 appeared to be reversible and weaker (50% inhibition at 100uM), CYP2D6 was weakly inhibited (15% at 100uM), and CYP2E1 was unaffected.^[74]

Glabridin appears to be a potent CYP3A4 inhibitor and possible relevant CYP2B6 inhibitor, and thus has high potential for adverse drug-nutrient interactions

Deglycosylation of Liquirtin (the glycone of Liquirtigenin) appears to enhance inhibitory potential towards UDP-Glucuronosyltransferase enzymes, with the inhibitory K_i of Liquirtigenin towards UGT1A1 and UGT1A9 appearing to be 9.1μM and 3.2μM respectively.^[75]

Absorption of Glabridin into the brain appears to be 2-3-fold higher than efflux rates, and both are increased when the concentration of Glabridin increases.^[52] The accumulation of Glabridin is increased (nearly doubled in vitro) when the efflux proteins of P-Glycoprotein and MRP1/2 were inhibited but not MRP4.^[52] The efflux kinetics suggest multiple methods of efflux from neural tissue (assessed by RBMVECs), and are ATP-dependent.^[52]

Oral ingestion of Glabridin at 5mg/kg to rats notes that the brain accumulates less Glabridin than any other organ in the body and only reached 2.8ng/g wet weight in the brain with a half-life of 3.56±1.08 hours.^[52] Ingestion of Vermapril (P-Glycoprotein inhibitor) at 25 or 100mg/kg rested in brain concentrations of 4.5±1.3 and 9.5±2.8ng/mL (60% and 240% increase) while increasing the brain 24-hour AUC by 200%, and similar increases were seen with Quinidine (another P-Glycoprotein inhibitor).^[52]

Any neural effect of Glabridin may be enhanced with concomitant ingestion of P-Glycoprotein inhibiting compounds

Glycyrrhiza uralensis root (20µg/mL) is able to prevent glutamate induced cell death to a level comparable with 20µg/mL Uncaria rhynchophylla (both components of Yokukansan, preserving viability to control levels);^[76] this was noted with isolated isoliquirtigenin and glycycoumarin conferring near full protection at 10µM, both of which had binding affinity to NMDA receptors although only isoliquirtigenin showed concentration-dependent inhibition reaching around 15-50% inhibition at 1-100µM.^[76] The protection is mostly due to NMDA antagonism, since elsewhere in PC12 cells (not expressing the major NR2A and NR2B subunits of NMDA receptors, and NMDA receptors not mediating excitotoxicity) the protective effects of licorice were found to be lesser than uncaria rhynchopylline.^[77]

Isoliquirtigenin appears to have the ability to suppress dopamine release induced by Cocaine as well as the subsequent locomotion (reflective of dopaminergic release)^[78] which is also mimicked by Glycyrrhiza Glabra methanolic extracts and acts in a dose-dependent manner via GABA(B) receptor agonism.^[79] These results were later replicated with Liquirtigenin (an isomer of Isoliquirtigenin) at 5mg/kg oral ingestion where this dose appeared to halve the locomotion induction of 20mg/kg cocaine and almost normalized CREB and c-Fos phosphorylation induced by Cocaine in the Nuclear Accumbens.^[80]

Liquirtigenin isomers appear to attenuate the dopaminergic effects of Cocaine at oral doses which can be achieved via Licorice supplementations, showing promise for reducing addiction to dopaminergic compounds

At least one study has noted that Glabridin at 150mg/kg in mice has suppressed food intake in mice, which resulted in the researchers making a pair fed group to assess the effects of Glabridin on fat loss independent of food intake.^[81]

Liquorice is thought to be sedative due to its inclusion in suanzaorentang, a Traditional Chinese Medicine for insomnia that also includes Ziziphus jujuba, Poria cocos, Ligusticum wallichii, and Anemarrhena asphodeloides.^[26][82]

Glycyrrhiza is able to bind to the benzodiazepine binding site of GABA_A receptors, and was thought to act as a positive modulatory due to its pro-sedative effects.^[26] These effects were attributed to the flavonoids in Glycyrrhiza, and thought to be mostly due to Glabrol as 50mg/kg Glabrol as as effective as 10mg/kg zolpidem.^[26]

The ethanolic extract of Glycyrrhiza is able to decrease sleep latency (time required to fall asleep) and increase sleep duration in rats in a dose dependent manner from 50-500mg/kg, being statistically significant at 250-500mg/kg and its potency was not significantly different than Diazepam at 2mg/kg.^[26] These changes correlated with how both Diazepam and the higher doses of Glycyrrhiza were able to increase the amount of non-REM sleep, but not REM sleep.^[26]

Glabrol and related flavanoids may be able to act as a sedative; these effects may not be achived by a whole licorice extract (due to Glycyrrhetic Acids increasing cortisol acutely, which is not conducive to sleep)

In microglia cells (BV-2) incubated with Glabridin prior to LPS-induced inflammation noted that Glabridine was able to concentration-dependently reduce inflammation from LPS as measured by TNF-α (41% reduction) and IL-1β (58% reduction); this was thought to be related to inhibition of AP-1 and NF-kB DNA transcription.^[83]

In an assessment of Traditional Chinese Medicine that holds potential to treat disease of cognitive decline, Licorice was one of the top five promising herbs (alongside Radix rehmanniae, Radix angelica sinensis, Radix polygalae, and Poria cocos) and three bioactives were said to contribute; Glabridin, Liquirtigenin, and Isoliquirtigenin.^[84]

Some studies that use Chinese Licorice per se note a protective effect against β-Amyloid 25-35 proteins (associated with Alzheimer’s Disease) with 0.5-1% Glycyrrhiza Uralensis,^[85] and general protective effects with roasted licorice after Ischemic injury;^[86] the former study correlates with the known bioactivities of Liquirtigenin, while the latter hypothesized that the effects were related to Glycyrrhizin derivatives.

The decrease in latency (indicative of cognitive decline in rats) induced by both scopolamine and diazepam both appear to be wholly abolished with pretreatment of 150mg/kg of a Glycyrrhiza Glabra extract (5.19g dry plant extract) when preloaded;^[87] this study is duplicated in Medline.^[88]

Licorice itself appears to be neuroprotective and, by protecting cognitive during periods of exogenous toxins or organic cognitive decline, may appear cognitive enhancing

250-500mg/kg of the aqueous root extract of Glycyrrhiza (glabra) for 14 days was able to reduce the negative effects of hypoxia (induced by sodium nitrite in drinking water), with the higher dose of 500mg/kg effectively normalizing the impairment to locomotion and memory that occurred with hypoxia.^[89] This study also noted that the increase in glutamate and acetylcholinesterase were normalized with the higher dose, and that the levels of dopamine that were increased in hypoxia were not only normalized but reduced by 7% relative to control;^[89] a reduction in injury-induced acetylcholinesterase activity has been noted elsewhere with isolated Glabridin at 2-4mg/kg (in response to scopolamine (toxin) administration) with similar efficacy as 400mg/kg Piracetam.^[90] These protective effects have been tested in an animal model of stroke (MCAO stroke induction) where one week of injections of Glabridin at 25mg/kg was able to attenuate the stroke-induced neurological deficit (with 5mg/kg being ineffective).^[91]

Glabridin has been tested in diabetic rats at 5-50mg/kg was able to prevent the reduction in cognitive (assessed by passive avoidance learning over 30 days) that occurred in Type II Diabetic control.^[92]

Glabridin appears to attenuate deficits in learning induced by exogenous toxins or states of cognitive decline (limited evidence for the latter claim)

Liquirtigenin (flavonoid) at 3, 10, or 30mg/kg oral ingestion in mice genetically predisposed to Alzheimer’s Disease-like symptoms and overproduction of Beta-amyloid pigmentation (Tg2576 mouse line) for 90 days noted that subsequent learning was less hindered with the two higher doses of Liquirtigenin (10-30mg/kg) with 30mg/kg almost normalizing the rate of learning (compared to control).^[93] This was related to a significant reduction in oligomeric Aβ proteins by 77.5±6.1% and 65.3±5.3% with 10 and 30mg/kg, respectively, and a decrease in astrocytosis in the hippocampus which correlated in magnitude with decreased Notch-2^IC expression.^[93] These benefits have been replicated in a model where Aβ proteins were injected into the hippocampus, where Liquirtigenin confered a protective effect thought to be secondary to its ability to act as a Selective Estrogen Receptor Modulator (SERM) on ERβ.^[94]

Liquirtigenin appears to have some anti-Alzhiemer’s properties, of unknown practical relevance (no comparison to other known compounds)

The powder of Glycyrrhiza Glabra (aqueous) was compared to Myristica Fragrans (Nutmeg; hexane extract) and Metrifonate (known acetylcholinesterase inhibitor) at 150mg/kg, 5mg/kg, and 50mg/kg respectively in an oral study in rats, and it appeared that the aqueous extract of Glycyrrhiza Glabra inhibited 25.4% of brain acetylcholinesterase which outperformed Nutmeg (14.5%) and Metrifonate (20%), as well as 60mg/kg injections of Vitamin C (17.13%).^[95]

One study assessing the neuroprotective effects of isolated Glabridin in diabetic mice that had healthy mice used as a control noted that 25-50mg/kg Glabridin given to healthy mice enhanced learning and memory more than mice given either low doses or no Glabridin.^[92]

Improvements in cognitiion have been noted with 75, 150, and 300mg/kg concentrated licorice extract (Glycyrrhiza Glabra) when control groups were given these doses and compared to toxin groups; these groups themselves (when compared to true control) noted improvements in memory (as assessed by transfer latency) with 150mg/kg yet impairment with 300mg/kg.^[87] This optimal dose of 150mg/kg was said to be equivalent to 5.19g of dried plant material.^[87]

Limited evidence, but it appears to support the notion that Licorice extracts can improve cognition in otherwise healthy rodents; at least one study has noted higher doses than optimal are detrimental to cognition

When looking at component of licorice that could inhibit the serotonin receptor (and act as SSRI-like compounds), it was found that the compounds that were able to inhibit serotonin uptake at 50uM were Glabridin (59.5±5.8%), Glabrene (45.7±0.3%), and 4’-O-methylglabridin (53.3±10.3%). All compounds were weaker than the active control of 5uM Imipramine (74.5±11.3%).^[96]

Glabridin at 5, 10, or 20mg/kg given to mice for 28 days orally prior to a swimming test was able to enhance swimming time in a dose-dependent manner by 22.58%, 43.55% and 50.04% (5, 10, 20mg/kg respectively) which was associated with less exercise-induced lactate and reduced serum BUN (both indicative of less glycolysis).^[97]

When bioactives from licorice were screened for their ability to reduce lipid absorption in the intestines, four molecules showed inhibition against Pancreatic Lipase with IC₅₀ valuse of 7.3μM (Isoliquiritigenin), 35.5μM (3,3′,4,4′-tetrahydroxy-2-methoxychalcone), 14.9μM (Liquiritigenin) and 37.6μM (Licuroside)^[98] with all compounds showing more than 90% inhibition at 250mcg/mL and Liquirtigenin recording 99±1.9% inhibition.^[98]

Licorice flavanoids may reduce lipid absorption from the intestines (may also underlie anti-obesogenic actions)

Cardiomyocytes (heart muscle cells) are connected to one another via what are known as Gap Junctions or Nexus’, where protein channels called connexins adhere one cell to another; coupling of cells during periods of Ischemia/Reperfusion (reduction of oxygen, then resurgence of oxygen that causes damage) enhances damage to cells from one to another^[99] and uncoupling these junctions by 18α-glycyrrhetinic can be a novel mechanism of cardioprotection.^[100] Actual clinical relevance of licorice to cardioprotection is not known.

Consumption of 100g Licorice (150mg Glycyrrhetic Acid) over four weeks appears to increase blood pressure, by 3.5mmHg systolic in persons with otherwise normal blood pressure but more drastically by 15.3mmHg in persons with hypertension at baseline (laboratory measurements) and to a lesser but still statistically significant degree in 24-hour ambulatory measurements.^[101] A later study using 500mg Glycyrrhetic Acid noted that cortisol increased in both normotensive and hypertensive subjects to the same degree^[102] and systemic activity of 11βHSD2 did not differ between groups.^[102] Cortisol doesn’t appear to mediate blood flow acutely (assessed by forearm vascular resistance) regardless of 11βHSD2 inhibition,^[103]

Isolated Glabridin at 20mcg or 200mg of an ethanolic extract of licorice to ApoE^-/- mice is able to reduce LDL oxidation after 6 weeks of consumption,^[104] and when giving a small sample (n=10) of human subjects 100mg of the ethanolic licorice extract daily for 2 weeks noted that, when LDL was extracted from their serum and then tested in vitro after the 2 weeks, that the subject’s LDL was significantly protected from Copper-induced oxidation^[104] and 6 months of consumption of Licorice root (60mg of LicoLife™ conferring 5mg Glabridin) is associated with up to 20% reduced LDL oxidation in humans.^[105][1] Glabridin appears to be the most potent polyphenolic protecting LDL from oxidation that comes from licorice (despite structurally related compounds and Isoliquirtigenin having protective effects^[25]) and having similar potency to Quercetin.^[106]

It appears that Glabridin has high affinity for LDL, with 75-85% of Glabridin binding to LDL ex vivo,^[106][104] and appears to protect LDL from oxidation via binding to LDL-C and preventing oxidation to that site;^[107] with an IC₅₀ of 6-22uM.^[107]

Appears to reduce oxidation of Low density Lipoprotein (LDL), and appears to be active in humans at reasonably low oral doses. Appears to be a relevant mechanism of action

Another possible mechanism to explain the reduced LDL oxidation, rather than direct anti-oxidative effects, would be the ability of Glabridin to accumulate in macrophages and drastically reduced Macropage-mediated LDL oxidation (a prooxidative event due to immune cell activation) due to possible being a PKC inhibitor.^[108] Inhibition of PKC has been noted previously with Licorice, but due to Glycyrrhetic acid (direct inhibitor with 1mM reducing activity of PKC by 90%).^[109]

Glabridin also appears to, secondary to anti-inflammatory effects, reduce secretion of cellular adhesion factors (ICAM-1, VCAM-1, and E-Selectin)^[110] which can possibly reduce adhesion of immune cells to the endothelium, as in vitro 3uM and 10uM of Glabridin inhibited 46 and 65% binding of Monocytes.^[110]

Some anti-artherogenic effects of Glabridin appear to be mediated by anti-inflammatory effects and the immune system

The dose range of 5-10mg Glabridin can feasibly be consumed via foods (at 0.92mg/g Chinese Licorice (Glabra); lower levels in the Uralensis species)

Licochalcone A and Licochalcone E appear to inhibit the PTP1B enzyme with IC₅₀ values of 19.1±0.1 and 20.7±1.8uM, both of which were weaker than the active control of Ursolic Acid at 3.9±0.3uM.^[111]

An apparent phenomena exists where women are more protected from Cardiovascular disease than men when diabetes is not present, but this protective effect associated with gender is lost during Type II Diabetes.^[112][113] This may be related to high blood glucose antagonizing the effects of estrogens on the endothelium (seen as protective).^[114]

One study assessing monocytes (immune cells that contribute to artherosclerosis) noted that Glabridin was able to reverse the glucose-induced decrease in PON2 (as well as estradiol, possibly mediated by the estrogen receptor) and preserved levels of Mn-Superoxide Dismutase and Catalase, possibly via anti-oxidant properties.^[115]

May reduce the adverse effects of hyperglycemia on artherosclerosis

In regards to aldose reductase (an enzyme that, when inhibited, confers protection to neurons and eyes from high blood glucose) a few compounds form licorice appear to have inhibitory potential; semilicoisoflavone B (exclusive to Glycyrrhiza Uralensis) inhibits with an IC₅₀ of 1.8-10.6uM (rat and human aldose reductase) and both liquirtigenin and isoliquirtigenin had IC₅₀ values of 2.0uM and 3.4uM, comparable to the active control of Quercetin of 2.5uM.^[116]

In vitro with adipocytes, Glabridin (flavanoid from Glycyrrhiza) appears to interfere with the transcription of PPARγ and CCAAT binding protein and reduce lipogenesis in differentiated adipocytes,^[117] while similar anti-adipogenic effects have been seen in preadipocytes with 18β-Glycyrrhetinic acid (5-40uM) secondary to downregulation of PPARγ and inhibition of Akt.^[118] Finally, a third study isolated four molecules in the nonaqueous fragment (could potentially be in Licorice Flavanoid Oil; glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D) where binding of 30mg/L of the overall oil had as much affinity as 0.44mg/L troglitazone (active control) and while the former two compounds were equivalent at 5mg/L the latter two were equivalent to troglitazone at 1-2mg/L.^[119] This study also claimed to have tested 70 plants for PPARγ activity with Glycyrrhiza Uralensis having most affinity; plants tested not reported.^[119] Glycyrrhetinic acid and Glycyrrhizin exert no activity on PPARγ at 2-10mg/L.^[119]

Other studies that look at anti-obese effects (attenuating weight gain over time) with 2% Licorice Flavanoid Oil (LFO; 1% Glabridin content) have noted that the reduction of fat mass was accompanied by a reduction in liver triglycerides, which were thought to be due to decreased mRNA of ACCα, FAS, and SREBP-1c (79.9%, 52.2%, and 64.8%) with an increase in PPARα mRNA (134.8%) over 21 days.^[120] Reductions in adipogenesis genes of the liver have been noted elsewhere with 2% LFO in rats.^[121]

Possible anti-obese mechanisms of unknown relevance, but seem to be related to interference with PPARγ (a pro-adipogenic gene) or otherwise attenuating an adipogenic gene shift in the liver in response to caloric intake

One study has mentioned that Glycyrrhetic Acid is, in vitro, a potent oxidative uncoupler (claimed to be nearing 2,4-Dinitrophenol in potency)^[122] and is dependent on the 3-hydroxy, 11-oxo, and 30-carboxyl groups in its structure and was more potent than two other triterpenoids measured, polyporenic acid A and fusidic acid.^[122] The potency of the two has been compared at a concentration of 0.05mM (in cartilage cells as the experiment was arthritic in goalset) and the uptake of inorganic phosphorus by 35% with Glycyrrhetic acid and 65% with 2,4-Dinitrophenol, making Glycyrrhetic acid 54% as potent as DNP at the same concentration.^[123] Another study noted an increase in liver ATPase of 23.1% (no effect on brain ATPase) in normal rats given an injection of 20mg/kg Glycyrrhetic acid for 10 days,^[124] which was said to be a biomarker of mitochondrial uncoupling; neither weight nor body heat were measured.^[124]

Another novel mechanism of Glycyrrhetic Acid in adipocytes is enhancement of β2-Adrenergic signalling secondary to lipid rafts.^[125] This study noted that Glycyrrhetic acid competed with ATGA for deposition in cellular membranes (thought to be due to its cholesterol-like structure) and decreased cholesterol content of lipid rafts (which has previously been connected to enhanced β-adrenergic signalling^[126][127]) and increased membrane fluidity in hydrophobic regions of the plasma membrane;^[125] G-protein coupling to the β2-Adrenergic receptor increased while internalization of the receptor decreased, and the final biomarker of isoproterenol-induced cAMP induction was synergistically increased with preincubation of Glycyrrhetic Acid and abolished with a β2-Adrenergic receptor antagonist.^[125]

Other studies on Glycyrrhizin metabolites note that 18β-Glycyrrhetic Acid can activate Hormone Sensitive Lipase (HSL) and increase lipolysis.^[118]

Glycyrrhetic Acid may also inhibit the 11βHSD enzymes in adipocytes, which would have varying results based on which enzyme is inhibited (as cortisol positively mediates lipolysis). Although inhibition of 11βHSD1 results in less lipolysis secondary to less cortisol,^[128] a study using the pharmaceutical derivative of Glycyrrhetic Acid (Carbenoxolone) noted that oral administration failed to alter 11βHSD1 activity in adipocytes.^[129] The relevance of 11βHSD interactions in adipocytes is not known.

A variety of mechanisms that insinuate that Glycyrrhetic Acid is a potent fat burning compound (mitochondrial uncoupling; augmenting adrenergic signalling), but these mechanisms have not been demonstrated in interventions

Additionally, Glabridin appears to activate AMPK (Adenosine Monophosphate Kinase) in a variety of tested tissues (adipocytes, muscle tissue, liver cells, kidney cells) in a dose and time dependent manner.^[81] Secondary to activation of AMPK, a trend towards more fatty acid oxidative and less lipogenic genes result.^[81] The activation of AMPK appears to be secondary to the mitochondria, where Glabridin interfered with Mitochondrial Complexes I and II and (by reducing ATP production about 15%) increased the AMP/ATP ratio;^[81] increases in this ratio are known to signal energy deficit to AMPK and activate its activity.^[130] When compared to an active control of Berberine, although they were not significantly different at increasing cAMP Glabridin trended to underperform relative to Berberine (with Berberine outperfoming significantly on the ratio of AMP/ATP).^[81]

Appears to activate AMPK which may underlie potential fat burning properties of Licorice. On a molecular basis, Glabridin is almost as effective as Berberine (but with less reaching systemic circulation)

One study noted that some compounds, including Isoliquirtigenin and Licuroside, were able to attenuate weight gain from a high-fat diet when consumsed at 30mg/kg secondary to attenuating absorption of fatty acids; both were less potent than the active control of Orlistat.^[98]

Overall, there are a wide variety of mechanisms that lead to the assumption that Licorice can be useful as a fat burning compound; these have not been thoroughly tested in vivo so their practical relevance is currently unknown

One intervention using 100g of Licorice (150mg Glycyrrhetic Acid) for 4 weeks noted increases in weight and BMI (1.4%), which was attributed to an increase of water weight (edema);^[101] caloric intake was not controlled in this study, but another study using 3.5g Licorice noted that an increase in water weight that occured obscured the reductions in body fat as a result of licorice ingestion over 2 months.^[131]

Two animal studies have noted that the addition of Glycyrrhiza flavanoids concentrated for Glabridin was able to suppress the gain in body fat at 2% of food intake,^[132] with similar potency to 0.5% Conjugated Linoleic Acid in rats.^[133]

One study noted that isolated Glabridin (150mg/kg) to mice was associated with a reduction in body weight that was highly significant when it suppressed food intake, and remained significant even when food intake was controlled for.^[81] 20mg/kg of Glabridin for 28 days is not associated with body weight changes in lean mice.^[97]

In mice, studies note lacklustre fat burning effects with feasible doses of Licorice extract but may have minor anti-obese effects. Fat burning effects can be forced with high dose Glabridin supplementation

In a trial on recreationally active obese men given Licorice Flavanoid Oil (LFO) at 300mg (brand name Glavonoid) for 8 weeks failed to provide any benefit to fat loss or body weight.^[49] This study also had a subset of athletic men undergoing slight overfeeding conditions, and the addition of LFO at 300mg did not alter body composition any different than placebo (although trended towards better body composition).^[49] One other study using 300mg LFO for 12 weeks in obese men noted that the intervention group did not increase body fat when placebo gained weight (resulting in significant differences at the end of the trial).^[134]

One study has concluded reductions in body fat in normal weight persons when assessed by Bioelectrical Impedence (BIA), but changes in weight did not occur due to water retention.^[131] This study noted that men lost 10% of their body fat (12.4% increase in extracellular water) and women lost 11.3% of their body fat (2.4% increase in extracellular water) over in response to 3.5g oral licorice for 2 months.^[131]

One study has been conducted in humans with Glycyrrhetic acid applied topically to 18 non-obese females, where a cream containing 2.5% Glycyrrhetic acid (80mg total) was compared against a placebo cream for one month and noted that while body weight did not change between groups there was a reduction in the size of superficial fat on the thigh as assessed by Ultrasound.^[135]

Limited evidence for the efficacy of Licorice and body fat reductions, with lower doses showing some promise for actual fat burning associated with Glycyrrhetinic acid while a Glycyrrhetinic acid free suppleent (Licorice Flavanoid Oil; mostly Liquirtigenin and Glabridin) exert anti-obesity effects

Oral administration of 50mg/kg Glycyrrhizic acid for one week to otherwise healthy rats was associated with an increase of Lipoprotein Lipase expression in the quadricep muscles by 102% over control, where other muscles trended to increase nonsignificantly in the abdominal muscle (87%), kidney (43%), liver (29%) and visceral adipose tissue (8%) and both subcutaneous adipose and the heart had about a 4% increase.^[136] This study noted that the observed effects could not be accounted for the the classical mechanism of 11β-HSD1 inhibition, as the inhibitory effects of glucocorticoids on LPL protein content^[137] only appeared to influence adipocytes.^[136] Increases in LPL expressionin muscle cells have been noted with 100mg/kg Glycyrrhizin Acid in rats with metabolic syndrome where muscular expression of LPL in the quadricep and abdominal reached 278% and 271% of metabolic syndrome control.^[138] This study noted an increase in insulin sensitivity possible secondary to this.^[138]

The mechanisms underlying the above are not known, but have been hypothesized to be involved with PPARα in skeletal muscle.^[136]

Liquirtigenin (flavanoid) appears to induce Nrf2 transcription and activity at 10-30mg/kg oral ingestion in rats.^[139]

Glabridin appears to possess anti-inflammatory actions via inhibiting NF-kB/Rel activation (which does not appear to be cell type specific)^[110] secondary to preventing TNF-α induced Akt and ERK activation (both of which induce NF-kB activity) with weak influence on p38 MAPK and SAPK/JNK; these effects are known to be mediated by Sphingosine-1-Phosphate (S1P)^[140] which is produced from the enzyme Sphingosine Kinase (SK) in response to TNF-α.^[141] Glabridin inhibits this enzyme weakly at basal levels and strongly prevents the increase of activity of SK in response to TNF-α.^[110] This theory was strengthed as exogenous addition of S1P bypassed SK inhibition and overcame the effects of Glabridin.^[110]

Anti-inflammatory effects secondary to preventing induction of a pro-inflammatory mediator, appears to be a relatively novel mechanism of action for nutraceuticals

Licochalcone A (a chalcone found mostly in Glycyrrhiza Inflata, but to a degree in Glabra) is able to suppress secretion of pro-inflammatory cytokines (IL-1β, IL-6, iNOS) and the enzyme COX-2 secondary to inhibiting activation of AP-1 and NF-kB both in vitro and when given mice undergoing septic shock.^[142] These results were replicated in macrophages indubated with 5-20uM Licochalcone A with minimal suppression of IL-6 (more dose-dependent suppression of TNF-α and IL-1β),^[143] and when 20, 40, or 80mg/kg of Licochalcone A given an hour (injected) before LPS injection was able to attenuate inflammatory changes to lung tissue.^[143]

The inhibition of NF-kB from Licochalcone appears to be mediated via preventing IKKβ activation via interfering with activation of the IkB complex,^[144] which is required for NF-kB activation. The double bond on the ketone group appears to be vital to this inhibition,^[145] thought to be possibly related to releasing a rigid conformation and allowing Licochalcone to rotate freely.^[145] The importance of this area of the Licochalcone A molecule may explain why NF-kB inhibition is preserved among several Licochalcone analogues (A, B, and D), but not Licochalcone C.^[146] Inhibition of NF-kB via Licochalcone A is mediated via inhibition of Serine 276.^[146][147]

Licochalone A is also a specific STAT3 inhibitor.^[148]

Licochalcone A shows anti-inflammatory effects, which may be biologically relevant but are otherwise not too remarkable (in their efficacy in suppressing cytokines as well as the active concentration)

Similar to other nutrients, Liquirtigenin is also able to reduce cytokine release from activated macrophages.^[149] 3-30uM Liquirtigenin was able to attenuate iNOS protein and mRNA levels and almost abolished iNOS genomic activity; nitrite accumulation in the macrophage and subsequent release of nitric oxide were also attenuated in response to LPS.^[149] These effects were thought to underlie the anti-inflammatory effects of Liquirtigenin when rats were fed 50mg/kg (or injected with 15mg/kg) where paw edema was reduced, but with less potency than 1mg/kg dexamethasone.^[149]

Macrophages appear to accumulate Glabridin in vitro, with 20uM of incubated Glabridin increasing intracellular Glabridin to 1.8±0.2ug/mg and this accumulation is associated with Glabridin inhibiting NADPH oxidase activity, which appears to be downstream of inhibiting P47 translocation in macrophages possible due to 70% less Protein Kinase C activity.^[108] The overall effect is less pro-oxidative macrophage activation,^[108] and similar results (less superoxide production associated with less P47 translocation) have been observed with other PKC inhibitors.^[150][151]

Ingestion of 7.5mL of Liquorice extract twice daily (total 0.87g dry herb equivalent) for a week in otherwise healthy volunteers noted that expression of CD25 on T cells occured within 24 hours (at a higher potency than Echinacea, lesser than Astragalus Membranaceus) but was not present after a weak of ingestion (although it was with Echinacea).^[152] CD69 appears to be induced with all three herbs, and possibly in an additive manner that was not observed with CD25.^[153]

One study investigating Liquirtigenin and Isoliquirtigenin noted that these compounds were able to inhibit IL-4 and IL-5 secretion from Memory T-cells (Th2),^[154] and studies using ASHMI (Licorice, Sophora Flavescens, Ganoderma Lucidum) note reductions in Th2 cytokine levels that may be due to licorice; of weaker potency than Prednisone.^[155]

Angiogenesis is the process of constructing new blood vessels, and tends to be a process that is abnormal during tumorogenesis (production of tumors) as growth factors facilitate vessel formation and nutrient delivery to tumors.

Licochalcone A appears to potentially have anti-angiogenic properties, as 20uM or less concentration of Licochalcone in rat HUVECs was able to suppress proliferation, migration, and tube formation.^[156] Similar antiangiogenic potential exists with Liquirtigenin by inhibiting HIFα-mediated VEGF expression, exerting complete inhibition at 75nmol/mL and abolishing HIFα protein content at 100nmol/mL which preceded an inhibition of angiogenesis in vitro in HUVECs; these effects were mediated via Liquirtigenin greatly attenuating activation of Akt/mTOR.^[157] Inhibition of angiogenesis by Liquirtigenin has been replicated elsewhere in vivo when tumors were implanted, where dose-dependent benefits were noted with oral intakes of 10-40mg/kg Liquirtigenin with the highest tested dose inhibiting half of tumor size over 28 days.^[158]

Compounds in licorice appears to have anti-angiogenic potential, and Liquirtigenin appears to have animal evidence that it can be effective

A minor constituent of Glycyrrhiza, dibenzoylmethane, appears to downregulate the androgen receptor in prostatic cancer cells.^[159]

Isoangustone A (ethanolic extract of Uralensis species) is able to dose-dependently decrease DNA synthesis rates and induce apoptosis at G1 in prostatic cancer cells (DU145; androgen insensitive prostate cells).^[31]

The 11βHSD2 enzyme is able to attenuate the antiproliferative effects of corticosteroids on breast cancer cells by metabolizing active corticosteroids into inactive derivatives; inhibiting this enzyme with glycyrrhetic acid is able to preserve the anti-proliferative effects of corticosteroids.^[160] Abnormal expression of 11βHSD2 has been noted in up to 66% of a sample of breast cancer cells,^[161] and inhibiting it can normalize levels of apoptosis (which is seen as a increase of 40% relative to preserved (11βHSD2) without inhernetly causing apoptosis secondary to cortisol;^[161] cortisol, in breast cancer cells, are anti-proliferative.

A possible direct role of Glycyrrhetic Acid in preventing proliferation can be to inhibit gap junctions between cells, as assessed by highly invasive MDA-MB-231 cells.^[162]

Glycyrrhetic Acid itself is able to exert anti-proliferative effects via apoptosis in MCF-7 cells (thought to be selective apoptosis^[163]) secondary to a reduction in mitochondrial membrane potential; the apoptotic effects appeared to be associated with a reduction of Akt signalling and an increase in FOXO3 nuclear accumulation (Activation of Akt excludes FOXO3 from the nucleus^[164]).^[165] The degree of proliferation inhibition measured 91.1% after 48 hours incubation with 100uM Glycyrrhetic Acid with an IC₅₀ of 32.6µM, and this dose was not associated with any toxicity in MCF-10A cells (noncancerous breast cells) and the level of apoptosis after 24 hours at 100uM (20.5%) was greater than the active control of Paclitaxel at 10nM (14.5%).^[165]

The bioactive Glycyrrhetic Acid (derivative of Glycyrrhizin) appears to have anti-proliferative effects which may be secondary to preserving the anti-proliferative effects of cortisol, and may have direct cytotoxic effects on breast cancer cells

Isoangustone A (ethanolic extract of Uralensis species) is able to dose-dependently decrease DNA synthesis rates and induce apoptosis at G1 in 4T1 murine mammary cancer cells.^[31]

The Hexane/ethanolic extract of Glycyrrhiza Uralensis (Isoangustone A as main active ingredient, no active Glycyrrhizin) is able to attenuate cardiotoxicity induced by the chemotherapeutic agent Doxorubicin.^[50] This study was in vitro in nature, and the extract dose-dependently reduced cardiotoxicity with complete cell preservation at 10ug/mL, and 15ug/mL of this extract actually increased the count of cardiomyocytes above control.^[50]

This same study noted that the same extraction was able to induce apoptosis (regulated cell death) in three cancer cell lines (prostatic, breast, and HT-29) and in the cell line that was least responsive to the extract (prostatic DU145) it showed additive cytotoxicity with Doxorubicin.^[50]

Interconversion of some hormones (of most pertinent concern is glucocorticoids) can be mediated by two enzymes, 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2); Type 1 converts inactive hormones such as cortisone to active hormones such as cortisol, and the type 2 enzyme reverses this process and converts active hormones such as cortisol to inactive cortisone.

In this sense, inhibition of 11βHSD1 increases cortisone relative to cortisol (reducing cortisol levels) while inhibition of 11βHSD2 increases cortisol relative to cortisone (increasing cortisol). Glycyrrhetic Acid from Licorice is able to inhibit both enzymes with the IC₅₀ of inhibiting 11βHSD2 at 0.36uM and the IC₅₀ of 11βHSD1 being 0.09uM.^[166] Inhibition of 11βHSD2 is well noted with Licorice^[167] as is the subsequent increase of bioavailable cortisol.^[168] 

Glycyrrhetinic Acid inhibits both 11β-hydroxysteroid dehydrogenase enzymes (Type 1 and Type 2), with slightly more affinity for Type I (Anti-cortisol) than Type II (Pro-cortisol)

The degree of cortisol increase has been noted to be 39-50% after 500mg glycyrrhetinic acid (salivary cortisol)^[169] and has been noted with serum measurements following the same dose.^{[170][171][172][173][102]} Cortisone has been demonstrated to be reduced as well by around 45%, which further implicates 11βHSD2 inhibition as the root cause.^{[174][173][172]}

At least one study using 3.5g Licorice (half the oral dose of the aforementioned studies) suggested that the observed effects in this study were secondary to 11βHSD1 activation with no apparent increases in cortisol.^[131]

Furthermore, compounds in Licorice appear to have a direct agonistic effect on mineralocorticoid receptors.^[171][175] The affinity is lesser than that of normal circulating corticosteroids and aldosterone,^[176] which may explain why an oral dose of 500mg Glycyrrhetic acid is required to decrease testosterone (see below).

Via inhibition of 11βHSD2, an increase of cortisol results alongside a concomitant decrease in cortisone (as 11βHSD2 converts cortisol to cortisone). This occurs at around 500mg oral intake of Glycyrrhetic Acid, and although dose-dependent lower oral doses of Glycyrrhetic Acid may not increase cortisol to a significant degree

When looking at cortisol synthesis, glycyrrhetinic acid does not appear to influence either ACTH-stimulant of forskolin-stimulated cortisol synthesis.^[174]

Independent of the enzymes mediating the balance of cortisol and inactive corticosteroids, Glycyrrhetinic acid does not appear to otherwise induce synthesis of Cortisol

Glycyrrhetinic and Glycyrrhyzic acid are thought to inhibit 17,20-lysase and 17-HSD type 5; two enzymes in the testosterone synthesis pathway.^[177][178] This latter enzyme (17βHSD5) catalyzes the conversion of androstenedione to testosterone and catalyzes the degradation of DHT androstanedione, and Glycyrrhetic Acid inhibits this enzyme with an IC₅₀ value of 20-30uM;^[179] which is weaker inhibition than Biochanin A and Quercetin (IC₅₀ values of 8-14uM and 5-9uM respectively).^[179]

In otherwise healthy men given 7g of liquorice containing 0.5g glycyrrhizic acid for one week, testosterone was reduced to 55% of baseline by day 4 and maintained this reduction to day 7, and was normalized 4 days after cessation.^[180] As a relative increase of 17-hydroxyprogesterone and no significant influence was seen on androstenedione, this effect was thought to be mediated via 17-HSD inhibition.^[180] This study was later attempted to be replicated by another research group but a reduction in testosterone was not achieved when men were given 5.6g liquorice with 0.5g glycyrrhizic acid and noted a nonsignificant decrease in testosterone of 9.5%; these authors mentioned the aforementioned 55% could have been overstated due to inclusion of outliers in the data.^[169] The first research group replicated their results with the same methodology and a larger sample and found a 26% (statistically significant) reduction in testosterone after one week concomitant with increases in 17-hydroxyprogesterase and luteinizing hormone,^[181] and this reduction in testosterone is similar to the maximally observed reduction in testosterone when rats are given 2,000mg/kg for 9 weeks (28.6%).^[182]

One other study giving 100g of candy containing 3% liquorice to partipants failed to find any influence on total testosterone in either gender, but noted a slight but statistically significant increase in unconjugated (free) testosterone by 25.6%.^[174] Exact dose of glycerrhetinic acid not disclosed, but another study using 100g of licorice (150mg Glycyrrhetic Acid) also noted a failure of licorice to reduce testosterone levels.^[183]

500mg of Glycyrrhizic Acid (5-7g Licorice extract) appears to be able to reduce testosterone in otherwise healthy men, although the degree of reduction seems to vary between individuals and the sample sizes of human studies are not the best to assess this variability. The reduction in testosterone appears to be benign and transient

As the reduction in testosterone appears to be dose dependent, small intakes of liquorice (100g of licorice as food product, ot 150mg Glycyrrhizic Acid) does not appear to significantly reduce testosteron levels as the dose is too low

Reductions in testosterone synthesis have been noted in ovarian slices in response to glycerhetinic acid and an increase in the estrogen:androgen ratio,^[184] and a herbal (Kampo) mixture known as Shakuyaku-Kanzo-To with the active ingredients of Liquorice and Paeonia lactiflora has shown some efficacy in women with Polycystic Ovarian Syndrome (PCOS), a state characterized by abnormal testosterone synthesis in women.^[185] Similar to men, testosterone reductions can occur in otherwise healthy women given Licorice which is normalized upon cessation of Licorice,^[178] and has been measured at 32-37%.^[186]

One study assessing the potential for licorice to be used as combination therapy alongside Spironolactone (androgen receptor and glucocorticoid receptor antagonist) found that the normally adverse effects of Licorice (increase in blood pressure and edema) were effective in reducing the side-effects of Spironolactone related to reduced blood volume.^[7]

Limited evidence suggesting benefit to females with PCOS in reducing androgen status (preliminary evidence suggests it works well with Spironolactone), but also appears to work in otherwise healthy women as well. The testosterone reducing effects of Glycyrrhiza appear to influence both genders

Glycerrhizin (Glycoside of Glycerrhetinic Acid) has been found to interact with the androgen receptor with similar affinity to paeoniflorin and DHT itself;^[187] this study did not note whether this was an agonistic or antagonistic property.^[187] A study in rats where the prostate weight was measured after testosterone injections (as a in vivo measurement of androgenicity) did note reductions of androgenic effects possibly related to interfering with the receptor, but the magnitude was minimal (150-300mg/kg alcoholic extract reducing the growth in prostate rate around 10% compared to testosterone-only control).^[188]

Might have direct anti-androgenic effects, but the evidence is limited and direct androgenic effects (weaker than testosterone, thus being an androgen modulator) have not been ruled out

At least one rat study noted that, after injections of testosterone into castrated rats, that those consuming 150-300mg/kg alcoholic extract had less circulating testosterone than the active control group;^[188] this study used castrated rats (excluding reductions in testosterone synthesis) implicating metabolism, and the authors mentioned aromatase could be an explanation (although did not demonstrate this).^[188] When assessing Glycyrrhiza Uralensis for possible bioactives that interact with aromatase, 7 compounds appeared to have good affinity for the aromatase enzyme and thus interacted while four of these compounds appeared to inhibit aromatase by 64.48%, 63.25%, 57.2% (Dihydrolicoisoflavone^[189]), and 82.78% (Glyasperin F^[190]) at 20ug/mL, with the former two unnamed compounds sharing structural similarity to Glabrene.^[191] One other study using MCF-7 cells noted that isoliquiritigenin was able to inhibit aromatase with an IC₅₀ of approximately 2.5-3.8uM as a mixed inhibitor, and appeared to be active in vivo due to reducing the rate of testosterone-induced breast tumor growth in mice (mediated after conversion to estrogen) at 0.05-0.5% of the diet.^[192]

Possible interactions at the level of aromatase (CYP1A), the enzyme that converts androgens to estrogens. Evidence of induction has been seen yet inhibitors have been noted, so the practical relevance of orally ingested licorice is not clear

Isoliquirtigenin appears to be most likely to be manipulated as an AI due to not being inherently estrogenic

When looking at the level of the receptor not in the presence of estrogen, the ethanolic extract of licorice root appears to induce signalling via the estrogen receptor (alpha) in HEK293 cells as well as a Yeast assay with 50-60% the potency of 10nM estradiol when the ethanolic extract was at 1/2000^th of solution (with descending activity thereafter, similar to a bell curve).^[193] These estrogenic actions appear to be mediated via the prenylated isoflavonoids Glabrene^[194] and Glabridin,^[195] and both compounds appear to be agonists of the estrogen receptor alpha (ERα) with EC₅₀ values of 5-50uM.^[196][197] There are most likely other active components, as water extracts have some activity.^[193] As Genistein (one of the soy isoflavones) has an EC₅₀ of 1.73uM for ERα,^[198] Licorice root phytoestrogens have comparatively less estrogenicity.

Glabridin, despite having affinity for the ERα receptor,^[195] appears to act as a receptor antagonist when incubated with estrogen by reducing estrogenic signalling via ERα by 80% at 10uM; establishing Glabridin as a Seletive Estrogen Receptor Modulator (SERM) or the ERα with no antagonism of ERβ.^[198]

Liquiritigenin (flavanoid) appears to be a selective ERβ agonist, activating the receptor between concentrations of 1-500nM with an EC₅₀ of 36.5nM and appeared to act in multiple tested cells lines.^[199] It binded to both receptors, although the affinity for the ERβ receptor was only 20-fold that of ERα (while activation of ERβ was much more selective), it appeared that Liquirtigenin activated cofactors of ERβ signaling only.^[199] Isoliquirtigenin does not appear to be estrogenic (via ERα) due to not increasing uterine weight of female mice.^[192]

One study coincubating licorice root flavonoids with the estrogen receptor noted that they were capable of superinduction of the estrogen receptor;^[200] and were found to prolong signalling in vitro for up to 24 hours and eventually exceeded 100% of signalling (assuming control was 100%).^[198] It was not established which compound mediated these effects or how.^[198]

Licorice Root contains phytoestrogenic compounds. Glabrene appears to just activate the estrogen receptor while the other related compound Glabridin appears to be a moderately selective SERM and modulate the estrogen receptor

Liquiritigenin appears to be a highly selective ERβ agonist, active at nanomolar concentrations; a promising Selective Estrogen Receptor Modulator (SERM)

For studies that measure serum estrogens, no significant effects are seen after 2 months of using 3.5g Licorice extract (7.9% Glycyrrhetic Acid)^[186] or 100g of Licorice (150mg Glycyrrhetic acid) for 4 weeks.^[183]

No significant influences on circulating estrogens

When comparing the estrogenic effects of Licorice (95% ethanolic extract of Uralensis root) against other medicinal herbs in a yeast assay (transfected with ERα), Licorice appears to be more estrogenic than Sophora Flavescens, similar potency to Rheum undulatum rhizome, Eriobotrya japonica leaf, and Ginger Rhizome, and despite being much more effective than the root of Pueraria lobata it was greatly outperfomed by the leaves (with an EC₅₀ of 9.39ug/mL relative to Licorice’s 76.4ug/mL).^[201]

Sex Hormone Binding Globulin (SHBG) is a protein that sequesters steroid hormones such as estradiol and DHT from acting in a cell. 18β-Glycyrrhetic Acid appears to have stronger affinity for SHBG than does estradiol, and may displace it (freeing up steroid hormones in the cytoplasm).^[202] This competitive inhibition has also been seen with the diglucoside Glycyrrhizin as well as the bioactive paeoniflorin (from Paeonia lactiflora) and also displaces DHT from SHBG.^[187]

When measuring the amount of SHBG in serum, there does not appear to be a significant reduction or increase in SHBG levels of either gender following 100g Licorice (150mg Glycyrrhetic Acid) for 4 weeks.^[183]

Dehydroepiandrosterone can be elevated relative to its sulfated component, due to inhibition of SULT2A1 with an apparent IC₅₀ of 7uM;^[174] this mechanism was thought to be causative due to an increase in hormones that get sulfated by SULF2A1 such as deoxycorticosterone.^[174] A reduction in sulfated DHEA has been noted elsewhere to the degree of 12%, but only appeared to affect men.^[183]

One study in women given 3.5g Licorice extract (7.9% Glycyrrhetic acid) for 2 months noted an increase of Parathyroid hormone to 20.2% higher than baseline after 2 months of usage (with a nonsignificant increase of 13.3% after one month), which normalized after one month of cessation.^[186]

A patch containing Licorice extract that was applied to canker sores in the mouth (Recurrent aphthous ulcers) was able to increase the amount of participants reporting no pain from 40% in no treatment (and 61% in placebo patch) to 81% which reducing the size of the ulcer over one week of application.^[203]

Licorice is sometimes used in cough drops alongside Ephedra (Ma Huang; source of ephedrine),^[204] which may be related to the inherent benefit to vasodilation and breathing associated with beta-2-adrenergic agonists such as ephedrine and the ability of Glycyrrhetic Acid to augment beta-2-adrenegic signalling in vitro.^[125]

Additionally, Liquirtigenin and its isomer Isoliquirtigenin appear to inhibit IL-4 and IL-5 release from memory T-cells, which may provide relief to allergic asthmatic responses^[154] which has been noted in interventions with ASHMI;^[205][206] an anti-asthmatic formulation consisting of a 2:2:3 ratio of Licorice (as Glycyrrhiza Uralensis), Ganoderma Lucidum, and Sophora Flavescens root.

By itself, Glycyrrhiza Uralensis appears to prevent LPS-induced upregulation of ICAM-1 and IL-8 in cultured A549 epithelial cells in a methanolic solution (containing Liquirtigenin and Glycyrrhetic Acid) with an IC₅₀ of 78ug/mL being slightly more potent than Morus alba;^[207] these results were observe in vivo when Xia-bai-san (mixture of four herbs of which include Licorice) was fed at 0.5-4mg/kg 30 minutes before LPS injections.^[207]

Licorice supplementation appears to be effective as an anti-asthmatic supplement, with most potency derived from nutrient-nutrient interactions (increasing the efficacy of other components, with limited evidence to suggest licorice is effective in isolation)

9 weeks of a water extract of Liquorice was able to slightly (but significantly) reduced the weight of the prostate and seminal production, but was not associated with any signs of seminal or testicular toxicity up to 2,000mg/kg.^[182]

More moderate doses of licorice extract appear to have protective effects on toxin-induced testicular damage induced by Carbendazim^[208] and ochratoxin A,^[209] where the latter study compared 100mg/kg Glycyrrhiza Glabra extract against 15mg/kg Melatonin (relatively strong anti-oxidant) and Glycyrrhiza Glabra appeared to slightly outperform Melatonin when both were administrated for 28 days prior to the toxin.^[209]

Despite potential testosterone reducing effects, Licorice appears to be protective of testicular function at low to moderate doses and not significantly toxic to the testicles at a dose commonly considered slightly toxic

Liquirtigenin can attenuate LXRα-induced SREBP-1c activation due to its ability to induce Nrf2 in a dose-dependent manner, which was also observed at 30uM for sulforaphane (another Nrf2 inducer); no comparison of potency was conducted.^[139] Activation of SREBP-1c mediates a variety of pro-lipogenesis genes in the liver and is a major therapeutic target of fatty liver,^[210] and inhibiting this with 10-30mg/kg Liquirtigenin in rats greatly attenuated an increase in liver fat due to a high-fat diet, attenuated weight gain, and abolished the increase in lipid peroxidaton (assessed by TBARS).^[139] Isoliquitigenin may also suppress LXRα-dependent fatty liver induction, although by another mechanism (JNK1 inhibition).^[211]

Inhibition os SREBP-1c has also been noted with Carbenoxolone (synthetic derivative of Glycyrrhetic Acid)^[212] which is downstream of 11βHSD Type 1 inhibition and seen as therapeutic for conditions with a fatty liver.^[213][214] It is possible that Glycyrrhetic acid acts via these means as 11βHSD1 inhibition per se mediates these effects, but this has not yet been demonstrated.

Several components of licorice appear to reduce fatty liver build-up in mice, all by different mechanisms (meaning the root itself could be potentially useful therapy). However, no human studies have been conducted to assess potency of licorice in this regard

The gap junctions of intestinal cells (space between each cell) are able to facilitate muscle contraction along the intestines when the cells have tigher gap junctions,^[215] and the ability of Glycyrrhetic Acid (10uM) to uncouple these junctions can reduce Ca²⁺ currents alongside the intestines with an IC₅₀ of 1.9uM.^[216] Glycyrrhetic Acid isomers (both 18α and 18β) are reversible Tight Junction uncouplers that act in intestinal cells.^[217][218]

Glycyrrhizin derivatives may have anti-motility actions through a mechanism called tight junction uncoupling

The flavonoid isoliquiritigenin appears to have spasmolytic properties in the lower intestines and exerted concentration-dependent inhibition of contraction with IC₅₀ values of 4.96±1.97uM (against CCh), 4.03±1.34uM (against KCl) and 3.70±0.58uM (against BaCl₂) which were similar to Papavarine as active control; pretreatmented inhibited acetylcholine-induced contraction with similar potency as well.^[14] Oddly, the mechanism of action does not appear to be related to PDE inhibition (similar to papaverine), and appeared to have more efficacy in the rectum than in the jujenum or ileum.^[14]

Other compounds found in Glycyrrhiza do appear to have PDE inhibitory properties related to intestinal relaxation, such as Glycycoumarin^[219] and Licochalcone A.^[220]

May have spasmolytic (muscle relaxant) effects on the lower intestines; which is possibly related to both laxative effects and cramp alleviation

Glabridin appears to inhibit tyrosinase activity in concentrations of 0.1-1ug/mL (17.5-39.1% inhibition), and appears to be mediated via the anti-oxidant properties of Glabridin (as 2′,4′-O-diethylglabridin did not have inhibitory effects nor anti-oxidant properties despite otherwise having the same structure).^[221] Maximal inhibition is at 10ug/mL reaching 49% over control.^[221]

Glabridin appears to slightly reduce erythema from UVB exposure when topically applied as a 0.5% cream to rodents, thought to be mediated via cyclooxygenase (COX) inhibition; weaker than the active control of indomethacin.^[221]

ASHMI is a chinese decoction consisting of Ganoderma Lucidum (Reishi), Sophora Flavescens root, and Licorice at a 2:2:3 ratio; ASHMI being a simplification of a traditional chinse medicine for Asthma sufferers consisting of 14 herbs. ASHMI appears to be safe and well tolerated as a combination therapy for asthma suffers at 1200-3600mg daily for 2 weeks (divided into two doses),^[222] it appears to be slightly less potent than prednisone.^[155]

3.5g of licorice (7.6% glycyrrhizic acid) daily for 2 months appeared to increase circulating levels of pro-vitamin D (25-hydroxycalciferol) from 27.3±11.3ng/mL to 36.3±12.3ng/mL (33% increase) without significantly influencing the circulating levels of hormonally active Vitamin D known as 1,25-dihyroxyvitamin D.^[186] Levels did not normalize one month after treatment cessation, and since this study was conducted in spring sun exposure could be a confounding factor.^[186]

Increase in Vitamin D may be a false positive due to seasonal changes

One study comparing Lycopene to a nutraceutical mixture (6% Lycopene, 0.1% beta-carotene, 1% Vitamin E, various polyphenolics including carnosic acid and Glabridin) noted that Lycopene’s 22% inhibition of LDL oxidation prevention was increased to 97% when combined with other nutraceuticals.^[223] Further tested noted that while Glabridin was able to inhibit LDL oxidation in a concentration-dependent manner with an IC₅₀ of 1.8umol/L (more effective than rosmarinic acid or Carnosic acid), that the addiction of 5umol/L lycopene had a degree of synergism of 1.95 (1.95-fold higher actual value relative to additive value); this degree of synergism was similar for Rosmarinic acid and Carnosic acid^[223]

Appears to work synergistically with Lycopene in preventing LDL oxidation

Building off one study assessing the combination of Panax Ginseng and powdered oriental bezoar,^[224] Glycyrrhiza Glabra was added to the pair as it has usage in Kampo medicine as a “harmonizing” agent (nonlegitimate term) in one study (with two publications in Medline^[5][4]). This study noted that 250mg of Panax, 50mg powdered bezoar, and 50mg Glycyrrhiza taken one hour prior to mental arithmatic stress noted a reduction in chromogranin A (salivary stress biomarker^[225] selective for mental stress^[226]) relative to placebo and attenuated stress-induced changes in other biomarkers such as heart rate and SNS activity.^[5]

Proanthocyanidins from cranberries (6.25-25ug/mL) and Licochalcone A (2.5-5ug/mL; from licorice) are able to reduce the growth of Gingivitis in a synergistic manner, and while cranberries themselves inhibited adherence of the bacteria to oral epithelial cells by 25% (Licochalcone by itself inactive), the combination increased this inhibition to 65-85%.^[227]

When testing the macrophage’s response to an inflammatory antigen, Cranberry proanthocyanidins and Licochalcone A synergistically suppress the release of IL-1b, TNF-a, IL-6, and IL-8 with concentrations of 50ug/mL Cranberry proanthocyanidins and 5ug/mL Licochalcone A almost wholly abolishing the increase in the latter three cytokines.^[227]

Cranberry polyphenolics and a compound form Licorice known as Licochalcone A appear to be synergistic in anti-inflammatory and oral anti-bacterial effects; possibly good candy combination

Eating 200g of Licorice within 45 minutes has been shown to make 75% of persons feel queasy and tired, which correlates with serum Glycyrrhizin levels (with one subject having less serum Glycyrrhizin, and no queasiness).^[45]

Dosing of up to 1200mg for 7 days^[6] or 1800mg for 4 weeks^[134] of Licorice Flavanoid Oil (LFO) without Glycyrrhizin appears to not be associated with any serum indicators of toxicity. Up to 800mg/kg bodyweight for 90 days in male mice (400mg/kg in females) has been tested in rats with no apparent toxicity, with the next highest tested doses (1600mg/kg in males, 800mg/kg in females) associated with excessive anticoagulative effects..^[228]

Licorice supplements with Glycyrrhetininc Acid removed do not appear to have significant toxicity associated with them in moderate doses

Pseudohyperaldosteronism is a condition in which inhibition of the 11-βHSD2 enzyme, which elevates cortisol and is a mechanism of Glycyrrhisa, and mimicks the symptoms of legitimate hyperaldosteronism (high blood pressure and metabolic alkalosis); inappropriate usage of licorice (overdosing) can produce Pseudohyperaldosternosim.^[229] This appears to be highly related to a metabolite of Glycyrrhizin known as 3β-D-(monoglucuronyl)18β-glycyrrhetinic acid (3MGA), which is the result of if intestinal bacteria hydrolyzes one but not both of the sugars bound to the glycone, Glycyrrhizin (removing both sugars resulting in 18β-glycyrrhetinic acid).^[230][65] 3MGA inhibits the 11βHSD2 enzyme with pretty much the same potency as Glycyrrhetic Acid (0.32 and 0.26uM), both of which are slightly more effective than Glycyrrhizin (2.2uM) but may have much more biological relevant as it is a substrate for organic anion transporters (OAT1, OAT3, and 4C1) allowing transport into renal tubules.^[231]

Death has been associated with Licorice in one women who only ate Licorice for an undisclosed time, and appeared to have died from a hyperglycemic coma; mostly through interactions with the carbohydrate content of licorice and bioactives (and lack of other nutrients).^[45] Rhabdomyolysis has been associated with licorice intoxication associated with hypokalemia, which is mostly reversed upon cessation.^[232][233]

Licorice intoxication or overdose is possible