In these cells, lipid formation was inhibited by theobromine doses greater than 25 μM. Theobromine has no effect on the viability of cells. Adipogenesis gene expression, C/EBPα, and PPARγ protein expression were all suppressed by theobromine at dosages greater than 25 μM. These genes' mRNA levels are likewise decreased by theobromine [1].

Compared to the excipient group, the theobromine group had a lower body weight. Moreover, theobromine prevented the growth of perirenal and epididymal adipose tissue weight. Compared to the vehicle group, the theobromine group's average adipocyte area was lower [1]. The theobromine group had lower counts than the other groups (p=0.021 and p=0.055 compared to the reference group (RF) and the cocoa group (CC), respectively) when calculating the number of bacteria per unit fecal weight. pH levels were greater following the theobromine diet than following the RF and CC diets. The lactic acid content in feces (RF group, 4.26±1.54 mM; CC group, 1.96±0.41 mM; theobromine group, 2.69±0.73 mM) was not significantly affected by the experimental diet [2].

Absorption, Distribution and Excretion
The ratio of theobromine concentration in rat brain tissue to blood decreased continuously from 0.96 at birth to 0.60 in 30-day-old rats. No organ accumulation of theobromine or its metabolites was observed in adult animals 24 hours later. Following oral administration of theobromine to rats, theobromine was rapidly absorbed and distributed, freely balancing between plasma and testicular fluid. Similar kinetic parameters were observed in male and female rabbits upon intravenous or oral administration of theobromine at doses of 1 and 5 mg/kg body weight, with complete gastrointestinal absorption. The absorption rate constant in rabbits decreased as the dose increased from 10 mg/kg body weight to 100 mg/kg body weight. Despite delayed gastrointestinal absorption at high doses (likely due to the compound's low solubility), the absolute bioavailability of theobromine approached 100%. Labeled theobromine was almost completely absorbed after oral administration (1–6 mg/kg); the higher the dose, the later the peak plasma concentration appeared.
When male dogs were given a single oral dose of theobromine at 15–50 mg/kg body weight, peak plasma concentrations occurred within 3 hours, but individual variability was significant. At higher doses (150 mg/kg body weight), peak plasma concentrations were reached after 14–16 hours, indicating delayed intestinal absorption. In rats, plasma protein binding was very low (8–17%) after oral administration of theobromine at 1–100 mg/kg body weight.
For more complete data on the absorption, distribution, and excretion of 3,7-dimethylxanthines (17 in total), please visit the HSDB records page.
Metabolism/Metabolites
Studies have shown that pregnancy and increased theobromine doses alter theobromine metabolism. At a dose of 50 mg/kg body weight, pregnant rabbits excreted a higher proportion of unmetabolized theobromine in their urine (51% vs. 35%). Pregnant rats excreted a higher proportion of unmetabolized theobromine at a 5 mg/kg dose (53%) than non-pregnant rats (39%); this difference disappeared at the saturation dose (100 mg/kg), where the unmetabolized theobromine content in the urine of both pregnant and non-pregnant animals was approximately 60% of the administered dose. Rats given a 100 mg/kg dose excreted more unmetabolized theobromine than rats given a 1 mg/kg dose (73% vs. 51%), and their excretion of the uracil metabolite 6-amino-5-(N-methylformamide)-1-methyluracil was correspondingly reduced (16% vs. 28%). Compounds identified in the bile of phenobarbital-treated rats included 3,7-dimethyluric acid (64-76% of the bile radioactivity), dimethylallantoin (5-8%), 6-amino-5-(N-methylformamide)-1-methyluracil (10-17%), and theobromine (8-10%). In rats treated with 3-methylcholanthrene, the excretion of unmetabolized theobromine in urine decreased from 23-27% to only 2%, while the excretion of 6-amino-5-(N-methylformamide)-1-methyluracil significantly increased. In control rats, hepatic microsomal incubation produced only 3,7-dimethyluric acid, while pretreatment with phenobarbital and 3-methylcholanthrene enhanced biotransformation, leading to the formation of all metabolites and an unknown polar compound. 6-Amino-5-(N-methylformamide)-1-methyluracil is the most important theobromine metabolite in rats, accounting for 20-35% of urinary metabolites. Most theobromine-derived radioactivity in rat feces is attributable to 3,7-dimethyluric acid. Theobromine metabolism was most extensive in rabbits and mice; male mice converted theobromine to this metabolite more efficiently than female mice. Conversely, the oxidation of theobromine to 3,7-dimethyluric acid was significantly higher in female rats than in male rats. Rabbits and dogs primarily metabolize theobromine to 7-methylxanthine and 3-methylxanthine, respectively, while dogs also excrete small amounts of unidentified metabolites. As a metabolite of caffeine, theobromine has been detected in the plasma and urine of humans and various animal species, but in varying amounts. For more complete data on the metabolism/metabolites of 3,7-dimethylxanthine (11 in total), please visit the HSDB record page. Known human metabolites of theobromine include 3,7-dimethyluric acid, 7-methylxanthine, and 3-methylxanthine. Theobromine is a known human metabolite of caffeine. The mean half-life of theobromine in human serum is 6.1 to 10 hours. The mean disposal half-life of theobromine is 7.1 ± 2.1 hours… In dogs, the mean plasma half-life after a single oral dose of theobromine ranging from 15 to 150 mg/kg body weight is 17.5 hours. In rabbits, the mean elimination half-life ranges from 4.3 to 5.6 hours at doses ranging from 1 to 100 mg/kg body weight.

Toxicity Summary
Identification and Uses: Theobromine is a white powder and an alkaloid found in cocoa and chocolate products. It is primarily used in the production of caffeine. Historically, theobromine and its derivatives have been used as diuretics, myocardial stimulants, vasodilators, and smooth muscle relaxants. Theobromine salts (calcium salicylate, sodium salicylate, and sodium acetate) were used to dilate coronary arteries at doses of 300 to 600 mg daily. Currently, theobromine has no therapeutic use. Human Exposure and Toxicity: Studies have shown that high doses of theobromine may cause nausea and anorexia, while daily intake of 50-100 grams of cocoa (containing 0.8-1.5 grams of theobromine) is associated with sweating, tremors, and severe headaches. Reactions to theobromine vary by dose; subjective effects are limited at a dose of 250 mg, while higher doses produce negative mood effects. Furthermore, theobromine can increase heart rate in a dose-dependent manner. Other studies have shown that theobromine increases high-density lipoprotein cholesterol (HDL-C) concentration by 0.16 mmol/L. Furthermore, theobromine significantly increases apolipoprotein AI concentration and decreases apolipoprotein B and low-density lipoprotein cholesterol (LDL-C) concentrations. In human lymphocyte cultures, theobromine did not significantly increase the number of sister chromatid exchanges per cell; however, in another experiment using higher doses, the number of sister chromatid exchanges per cell increased in the absence of an exogenous metabolic system. Contrary to results in rodent cells, theobromine induced chromosome breakage in cultured human lymphocytes. Animal studies: High doses (250–300 mg/kg body weight, adult animals) and 500 mg/kg body weight showed complete thymic atrophy in both male and female rats. This effect was observed in hamsters only at doses up to 850 mg/kg body weight, and in mice at doses up to 1840–1880 mg/kg body weight. In rabbits, theobromine administration caused significant changes in the thymus and testes, and the severity of the lesions appeared to be related to the amount of methylxanthine ingested. Other rat studies have also found theobromine toxicity to the testes. No maternal toxicity was observed in rats fed a theobromine-containing diet (daily dose of 53 or 99 mg/kg body weight) during days 6–19 of gestation. Although no malformations were observed, a slight decrease in fetal weight and a significantly increased incidence of skeletal variations were observed in the high-dose groups. In rabbits, decreased fetal weight and malformations were observed at doses of 125 or 200 mg/kg; the incidence of skeletal variations increased at doses of 75 mg/kg and above. Theobromine was mutagenic to Escherichia coli but not to Salmonella typhimurium under conditions of constant growth rate and cell density. Theobromine induced mutations in the lower eukaryote Euglena. Theobromine did not induce chromosomal aberrations in plants (broad beans). Abstract: No chromosomal aberrations were observed after treatment with 0.45% theobromine in a fruit fly (Drosophila melanogaster) feeding experiment. Theobromine also did not induce chromosomal aberrations in Chinese hamster cells.
Interactions
Rats were co-administered with different concentrations of theobromine (TB) and (-)-epicatechin (EC), and the concentrations of EC and its metabolites in plasma were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed that TB increased EC absorption in a dose-dependent manner. Cocoa powder had a similar effect, and its mechanism of action was thought to be independent of tight junctions.
The experiment aimed to determine the effects of adding 0.5% of methylxanthine compounds caffeine, theobromine, or theophylline to the diet of 4- to 6-week-old male rats for 14 to 75 weeks. In the first two experiments, Osborne-Mendel rats were fed the test substances alone or in combination with sodium nitrite to verify whether these amine compounds could undergo nitrosation in vivo to generate toxic nitrosamine compounds. These compounds failed to induce tumors or precancerous lesions, but a significant positive finding was that 85-100% of rats fed caffeine or theobromine developed severe bilateral testicular atrophy, accompanied by azoospermia or oligospermia. Under repeated dosing conditions, we investigated the effects of allopurinol on plasma theobromine clearance and metabolism. Allopurinol had no effect on theobromine clearance, suggesting that the elimination of this compound depends on an enzyme system other than xanthine oxidase, presumably a mixed-function hepatic oxidase. Allopurinol treatment also had no effect on the excretion of 3-methylxanthine, 6-amino-5-(N-methylformamide)-1-methyluracil, and unmetabolized theobromine. Although allopurinol inhibited the production of 7-methyluric acid (7MU) and increased the excretion of 7-methylxanthine (7MX), the metabolic clearance of (7MX + 7MU) was not significantly different in the presence or absence of allopurinol. This study suggests that the secondary bioconversion of 7MX to 7MU is mediated by xanthine oxidase. Compounds identified in the bile of phenobarbital-treated rats included 3,7-dimethyluric acid (64-76% of the bile's radioactivity), dimethylallantoin (5-8%), 6-amino-5-(N-methylformamide)-1-methyluracil (10-17%), and theobromine (8-10%). In rats treated with 3-methylcholanthrene, the excretion of unmetabolized theobromine in urine decreased from 23-27% to only 2%, while the excretion of 6-amino-5-(N-methylformamide)-1-methyluracil significantly increased. Incubation of liver microsomes in control rats produced only 3,7-dimethyluric acid, while pretreatment with phenobarbital and 3-methylcholanthrene enhanced biotransformation, leading to the production of all metabolites and unknown polar compounds in vivo.

---

Non-human toxicity values
Dog oral LD50: 300 mg/kg body weight
Rat (acute) oral LD50: 950 mg/kg body weight

[1]. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.

[2]. Effect of cocoa's theobromine on intestinal microbiota of rats. Mol Nutr Food Res. 2017 Oct;61(10).

Theobromine is an odorless white crystalline powder with a bitter taste. pH (saturated aqueous solution): 5.5-7. (NTP, 1992)
Theobromine is a dimethylxanthine with its two methyl groups located at positions 3 and 7, respectively. It is a purine alkaloid derived from the cacao plant and found in chocolate and many other foods. It has vasodilatory, diuretic, and cardiac stimulant effects. It functions as an adenosine receptor antagonist, a food component, a plant metabolite, a human serum metabolite, a mouse metabolite, a vasodilator, and a bronchodilator.
Theobromine (3,7-dimethylxanthine) is the main alkaloid in the cacao tree (Theobroma cacao) and other plants. A xanthine alkaloid, it is used as a bronchodilator and vasodilator. Its diuretic effect is weaker than that of theophylline, and its stimulating effect on smooth muscle is also weaker. It has almost no excitatory effect on the central nervous system. It has been used in the past as a diuretic and to treat angina pectoris and hypertension. (Excerpt from Martindale Pharmacopoeia, 30th edition, pp. 1318-1319)
Theobromine has been reported in tea trees (Camellia sinensis), holly (Ilex perado), and several other organisms with relevant data.
3,7-Dimethylxanthine. The main alkaloid in cacao (Theobroma cacao, cacao bean) and other plants. A xanthine alkaloid used as a bronchodilator and vasodilator. Its diuretic effect is weaker than theophylline, and its excitatory effect on smooth muscle is also weaker. It has almost no excitatory effect on the central nervous system. It has been used as a diuretic and to treat angina pectoris and hypertension. (Excerpt from Martindale Pharmacopoeia, 30th edition, pp. 1318-1319)
See also: Guarana seeds (partial).

---

Drug Indications
Theobromine is used as a vasodilator, diuretic, and cardiac stimulant. Similar to caffeine, it may help relieve fatigue and orthostatic hypotension.

---

Mechanism of Action
Theobromine stimulates the medulla oblongata, vagus nerve, vasomotor center, and respiratory center, promoting bradycardia, vasoconstriction, and increased respiratory rate. Previously, this effect was thought to be mainly due to increased intracellular cyclic adenosine monophosphate (cAMP) levels after inhibition of phosphodiesterase (an enzyme that degrades cyclic adenosine monophosphate). It is now believed that xanthine compounds (such as caffeine and theobromine) act as antagonists of adenosine receptors on the plasma membrane of almost all cells. Since adenosine, as an endogenous substance, inhibits the release of neurotransmitters at the presynaptic site but enhances the effects of norepinephrine or angiotensin, the antagonism of adenosine receptors promotes neurotransmitter release. This explains the stimulatory effect of xanthine derivatives (such as theobromine and caffeine). After caffeine intake, blockade of cardiac adenosine A1 receptors leads to a significant increase in heart rate.

---

Therapeutic Uses
Diuretic, bronchodilator, cardiotonic. /Previous Uses/
In the past, theobromine and its derivatives were used as diuretics, myocardial stimulants, vasodilators, and smooth muscle relaxants. Theobromine salts (calcium salicylate, sodium salicylate, and sodium acetate) were used to dilate coronary arteries at doses of 300 to 600 mg daily. Currently, theobromine has no therapeutic use. /Previous Uses/
Veterinary Use: Diuretic, myocardial stimulant, vasodilator. /Previous Uses/
/Exploratory Treatment/ Coughing is a common protective reflex, but persistent coughing can severely impact a patient's quality of life. The use of opioids for cough suppression is limited by their unacceptable side effects, thus a more effective treatment method is urgently needed. This study shows that theobromine (a methylxanthine derivative found in cocoa) effectively inhibits citric acid-induced cough in guinea pigs. Furthermore, in a randomized, double-blind, placebo-controlled human study, theobromine inhibited capsaicin-induced cough without adverse effects. We also demonstrated that theobromine directly inhibited capsaicin-induced sensory depolarization of the vagus nerve in guinea pigs and humans, suggesting an inhibitory effect on afferent nerve activation. These data indicate that theobromine's effects appear to be peripherally mediated. We conclude that theobromine is a novel and promising treatment that may form the basis for a new class of antitussives.

---

Pharmacodynamics
Theobromine is a xanthine derivative, similar to caffeine and the bronchodilator theophylline, and can be used as a central nervous system stimulant, a mild diuretic, and a respiratory stimulant (used to treat apnea in preterm infants).

C7H8N4O2

180.167

180.064

83-67-0

Theobromine-d6;117490-40-1;Theobromine-d3;65566-69-0

5429

White to off-white solid powder

1.6±0.1 g/cm3

495.5±37.0 °C at 760 mmHg

345-350 °C

253.5±26.5 °C

0.0±1.3 mmHg at 25°C

1.737

-2.08

1

3

0

13

267

0

YAPQBXQYLJRXSA-UHFFFAOYSA-N

InChI=1S/C7H8N4O2/c1-10-3-8-5-4(10)6(12)9-7(13)11(5)2/h3H,1-2H3,(H,9,12,13)

3,7-dimethylpurine-2,6-dione

Santheose; Teobromin; Diurobromine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~1.1 mg/mL (~6.11 mM)
H2O : ~1.1 mg/mL (~6.11 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)