Licorice Root

Sources

Glycyrrhiza is a plant family that is commonly known as Licorice. 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]

Composition

(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 Licorice 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]

Structure and Stability

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

Food products

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%

Glavanoid

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

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]

Absorption

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

Serum

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]

Metabolism

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

Distribution

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]

Elimination

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]

Enzymatic Interactions

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]