Higenamine acts as a beta2-adrenergic receptor (β2-AR) agonist, and its anti-apoptotic and cardiac protective effects are mediated by the β2-AR/PI3K/AKT signaling pathway [1].
Higenamine functions as a β2-AR agonist to antagonize bronchoconstriction [1].
The compound is also considered to be a β2-AR agonist that may stimulate lipolysis and thermogenesis [2].

In neonatal rat ventricular myocytes (NRVMs), Higenamine dose-dependently attenuated H2O2-stimulated early and late apoptosis as analyzed by annexin V FITC/propidium iodide (PI) assay with flow cytometry. Higenamine inhibited H2O2-induced increase of cleaved-caspase-9 and cleaved-caspase-3 in a dose-dependent manner (concentration tested: 100 μM) [1].
In NRVMs, the inhibitory effects of Higenamine (100 μM) on H2O2 (250 μM for 24 h)-induced cleaved-caspase-9 and cleaved-caspase-3 were abolished in the presence of the β2-AR antagonist ICI118551 (0.5 μM) but not by the β1-AR antagonist CGP20712a (1 μM) [1].
In adult mouse ventricular myocytes (AMVMs), H2O2 (20 μM for 24 h)-induced cell death (assessed by trypan blue staining, round/rod cell ratio) was significantly attenuated by Higenamine (100 μM). This protective effect was blocked by the β2-AR antagonist ICI118551 (0.5 μM) and the PI3K inhibitor wortmannin (10 μM), but not by the β1-AR antagonist CGP20712a (1 μM) [1].
In AMVMs, TUNEL staining showed that the anti-apoptotic effect of Higenamine (100 μM) on H2O2 (20 μM for 24 h)-induced apoptosis was blocked by the β2-AR specific inhibitor ICI118551 (0.5 μM) [1].
Higenamine stimulated AKT phosphorylation at Ser473 (activation) in AMVMs, which was blocked by the β2-AR antagonist or PI3K inhibitor but not changed by the β1-AR antagonist [1].
The β2-AR specific antagonist butoxamine blocked the effect of Higenamine on H2O2-stimulated caspase-3 activity and AKT activation. The β2-AR specific agonist formoterol hemifumarate reduced caspase-3 activity and increased AKT activation similar to Higenamine, and addition of formoterol hemifumarate to Higenamine did not elicit an additive effect [1].
In H2O2-treated NRVMs, Higenamine dose-dependently decreased cleaved-caspase-9 and cleaved-caspase-3 proteins (doses tested: 0, 10, 50, 100 μM). The effects of 100 μM Higenamine were significantly blocked by 0.5 μM ICI118551 but not by 1 μM CGP20712a [1].

In C57BL/6J mice subjected to I/R injury (30 min ischemia by LAD ligation followed by 24 h reperfusion), intraperitoneal injection of Higenamine (10 mg/kg body weight, 2 h prior to surgery) significantly reduced myocardial infarct area (MI) compared to saline-treated mice (19% vs. 51%, with similar areas at risk). TUNEL staining of heart sections showed that apoptotic cells were drastically reduced in Higenamine-treated I/R hearts compared to vehicle-treated I/R hearts [1].
In ex vivo perfused mouse hearts (Langendorff system, 30 min no-flow global ischemia followed by 30 min reperfusion), perfusion with Higenamine (100 μM added to working buffer) significantly decreased myocardial infarction area compared to vehicle (11.6% vs. 42.7%). The cleaved-caspase-3 level was reduced by Higenamine treatment, and this reduction was abolished in the presence of the β2-AR antagonist ICI118551 (0.5 μM). AKT phosphorylation was increased by Higenamine, and this increase was abolished by β2-AR antagonism [1].
In healthy human subjects (8 men, 8 women; ages: men 26.1±2.5 yrs, women 22.4±3.1 yrs), acute oral ingestion of a dietary supplement containing Higenamine (combined with caffeine 270 mg and yohimbe bark extract) in a double-blind, randomized, cross-over design (two separate occasions separated by 6-8 days, following a 10-hour overnight fast) resulted in significantly higher plasma free fatty acids (FFA) compared to placebo at 60 min (p=0.0004), 120 min (p=0.0004), and 180 min (p=0.004) post-ingestion. Kilocalorie expenditure was significantly higher for supplement compared to placebo at 60 min (p=0.03) and 120 min (p=0.02) post-ingestion, with a trend at 180 min (p=0.07). No statistically significant effects were noted for glycerol or respiratory exchange ratio (RER). Heart rate (condition effect p=0.03) and systolic blood pressure (condition effect p<0.0001) were higher for supplement compared to placebo (overall heart rate increase ~3 bpm, systolic blood pressure increase ~12 mmHg) [2].

Western blot analysis was performed to detect protein levels. Cell lysates were prepared using ice-cold modified RIPA buffer containing Tris-HCl pH 7.4, NP-40, NaCl, EDTA, PMSF, sodium orthovanadate, NaF, aprotinin, leupeptin, and pepstatin. Lysates were loaded on SDS-polyacrylamide gel electrophoresis, electrophoresed, and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 5% nonfat dry milk in 0.01% Tween/PBS, incubated in primary antibody overnight at 4°C, then incubated in horseradish peroxidase conjugated secondary antibodies and developed using enhanced chemiluminescence detection reagent. Primary antibodies used: cleaved and total caspase 3 and 9; p-AKT and total AKT; and GAPDH [1].
Caspase-3 activity assay: After different treatments of adult mouse ventricular myocytes, measurement of caspase-3 activity was carried out according to the manufacturer's instruction. Briefly, 30 μl of cell lysates was mixed with 80 μl of reaction solution containing caspase-3 substrate (Ac-DEVD-pNA) and incubated for 120 min at 37°C. Absorbance was measured by a microplate reader at 405 nm. Enzyme activity was expressed as absorbance normalized by protein concentration in each group, and results were shown as fold compared to the control group [1].
Free fatty acids determination: Plasma samples were assayed using a fatty acid detection kit following the manufacturer's instructions. Glycerol was determined using a free glycerol determination kit and glycerol standard following the manufacturer's instructions [2].
Indirect calorimetry measurement: Kilocalorie expenditure and respiratory exchange ratio (RER) were measured using indirect calorimetry. Total oxygen consumption (L·min⁻¹) was determined from gas collection and used to estimate total kilocalorie expenditure. RER was determined from gas collection data (VCO₂/VO₂) as a measure of substrate utilization [2].

Isolation and culture of neonatal rat ventricular myocytes (NRVMs): Hearts were excised from 2-3 day old rat pups and ventricles separated and rinsed in Hank's balanced salt solution prior to digestion with multiple rounds of collagenase type II containing HBSS. Cells were collected by centrifugation, resuspended in DMEM containing 5% fetal bovine serum, 5% horse serum, penicillin, and streptomycin. Non-myocytes were removed by preplating the cells at 37°C for 1 hour. Cells were then cultured in DMEM containing 10 μM cytosine arabinoside for 24 hours on gelatinized plates before switching to serum-free DMEM medium containing 0.2× insulin-transferrin-selenium [1].
Isolation and culture of adult mouse ventricular myocytes (AMVMs): Mouse hearts were enzymatically dissociated using collagenase type II in a Langendorff perfusion system. Heparinized and anesthetized mouse hearts were rapidly excised, cannulated, and perfused with Ca²⁺-free isolation buffer for 3 min followed by collagenase type II mixed isolation buffer until digestion was complete. Myocytes were exposed to stopping buffer for increasing Ca²⁺ concentration to 1.4 mM, plated cells in laminin-precoated dishes with plating buffer, and after 2 hours, cells were cultured with prewarmed culture medium containing BSA, HEPES, NaHCO₃, penicillin/streptomycin, blebbistatin, and insulin-selenium-transferrin in MEM + Earl's salts + L-glutamine medium [1].
Trypan blue viability assay: AMVMs were seeded in 6-well plates. After treatments, cells were incubated with trypan blue solution (final concentration 0.04%) for 5 minutes and then counted under a phase-contrast inverted microscope. Thirty fields in every dish were randomly selected for quantification. Dead cells were expressed as percentage of total counted cells [1].
TUNEL staining in AMVMs: Adhered cells were fixed with 4% paraformaldehyde for 1 hour at room temperature, then permeabilized with 0.5% Triton X-100 at room temperature for another 1 hour. TUNEL reaction mixture (50 μL) was added to each dish and incubated at 37°C for 1 hour. DAPI in PBS was incubated for 5 min at room temperature for nuclear staining. The ratio of cell apoptosis was expressed as the percentage of TUNEL staining positive cells versus DAPI staining positive cells [1].
Flow cytometry analysis for Annexin V-FITC/propidium iodide (PI) staining: NRVMs were collected, washed with PBS. Cells were stained with Annexin V/PI detection kit according to manufacturer's instructions. Annexin V-FITC (5 μl) and PI (10 μl) were added to 2×10⁵ suspended cells and incubated for 30 min at 37°C in the dark prior to analysis using flow cytometry. Cells that were Annexin V-positive only and both Annexin V- and PI-positive were considered early and late apoptosis cells, respectively [1].

In vivo ischemia/reperfusion injury in mice: C57BL/6J male mice were subjected to ischemia by ligating the left anterior descending artery (LAD) below the tip of the left auricle. Occlusion of LAD was confirmed by change of color and elevation of ST segment on electrocardiogram. After 30 minutes of occlusion, the suture was untied for reperfusion, and the chest cavity and skin incision were closed. After 24 hours of reperfusion, LAD was re-occluded at the same position. Hearts were perfused with 2% Evans Blue to delineate the area at risk, and heart tissue slices were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) for infarct areas. Higenamine (10 mg/kg body weight) or vehicle (saline) was administered via intraperitoneal injection 2 hours prior to surgery [1].
Ex vivo ischemia/reperfusion injury (Langendorff perfused mouse hearts): C57BL/6J male mouse hearts were perfused in Langendorff mode with oxygenated Krebs-Henseleit buffer. Hearts were stabilized at a flow rate of 4 ml/min for 20 minutes before 30 minutes of no-flow global ischemia, then 30 minutes of reperfusion. In the I/R + Higenamine group, 100 μM Higenamine was added to the working buffer. In the I/R + Higenamine + ICI group, 0.5 μM ICI118551 and 100 μM Higenamine were added to the working buffer. Myocardial necrosis was evaluated by 1% TTC staining of viable tissue [1].
Human study protocol: Healthy exercise-trained men (n=8) and women (n=8) participated in a double-blind, randomized, cross-over design. After a 10-hour overnight fast and without caffeine for 24 hours, subjects reported to the lab. Testing was conducted on two separate occasions separated by 6-8 days at the same time of day. Subjects ingested a dietary supplement (two caplets containing a proprietary combination of Higenamine, yohimbe bark extract, and caffeine 270 mg) or placebo (microcrystalline cellulose caplets). No food was allowed during the 3-hour post-ingestion period, but water was allowed ad libitum and matched for both test days. Subjects remained inactive in the laboratory during the entire 3-hour test period. Heart rate (via 60-second radial artery palpation) and blood pressure (via auscultation) were measured, blood samples were obtained, and 5-minute breath samples were collected at pre-ingestion, 30, 60, 120, and 180 minutes post-ingestion. Subjects were asked not to exercise or perform strenuous physical activity for 48 hours prior to each test day, and to duplicate food and beverage intake during the 24-hour periods prior to both test days [2].

In human subjects, acute oral ingestion of a dietary supplement containing Higenamine (combined with caffeine and yohimbe bark extract) resulted in a moderate increase in heart rate (~3 bpm) and systolic blood pressure (~12 mmHg). No adverse events were experienced by subjects, only moderate increases in heart rate and blood pressure were noted. The supplement was well tolerated [2].
The lethal dose (LD₅₀) of Higenamine following injection in animals is 50 mg/kg, suggesting less concern for acute toxicity with oral consumption [2].
Higenamine administration has been reported to actually decrease blood pressure in animals and was concluded to be safe for human intake following acute intravenous administration [2].

[1]. Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of β2-AR/PI3K/AKT signaling pathway. Pharmacol Res. 2016 Feb;104:115-23.

[2]. Acute oral intake of a higenamine-based dietary supplement increases circulating free fatty acidsand energy expenditure in human subjects. Lipids Health Dis. 2013 Oct 21;12:148.

(RS)-norcocaine is a type of norcocaine, and it is the conjugate base of (RS)-norcocaine. Norcocaine is currently being investigated in the clinical trial NCT01451229 (Pharmacokinetics and Pharmacodynamics of Norcocaine in Healthy Chinese Subjects). Norcocaine has been reported to be found in Gnetum montanum, Gnetum parvifolium, and several other organisms with relevant data.

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Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Long-term activation of β-AR signaling regulates myocyte hypertrophy and survival. β1-AR activation is pro-apoptotic while β2-AR activation is anti-apoptotic to cardiac myocytes. Stimulation of β2-AR can also activate pertussis toxin-sensitive Gi signaling, which activates the PI3K/AKT signaling cascade that promotes myocyte survival [1].
Higenamine has been shown to have anti-apoptotic effects in several cell types including cardiomyocytes. It can attenuate doxorubicin-induced neonatal rat cardiomyocyte apoptosis and protect against I/R-induced cardiac injury through upregulating heme oxygenase-1 (HO-1). It also stimulates PI3K/AKT/Nrf2 signaling and subsequently induces HO-1 expression, which is critical for antagonizing C6 apoptosis and hypoxia-induced brain injury [1].
Higenamine exerts positive chronotropic and inotropic effects likely through activating β-AR signaling. It functions as a β2-AR agonist to antagonize bronchoconstriction. The compound also has vasodilatory effects [1].
Higenamine is being used as an ingredient in dietary supplements for weight loss. β-AR agonists enhance lipolysis and thermogenesis, and Higenamine as a β-AR agonist may be considered an anti-obesity agent. Caffeine and yohimbe bark extract are often combined with Higenamine in supplements to enhance lipolytic and thermic effects [2].

C16H17NO3

271.3111

271.12

5843-65-2

Higenamine hydrochloride;11041-94-4;(S)-Higenamine hydrobromide;105990-27-0;(S)-Higenamine;22672-77-1

114840

White to off-white solid powder

1.3±0.1 g/cm3

522.4±50.0 °C at 760 mmHg

208-210℃

209.6±20.7 °C

0.0±1.4 mmHg at 25°C

1.666

1.41

4

4

2

20

317

0

WZRCQWQRFZITDX-UHFFFAOYSA-N

InChI=1S/C16H17NO3/c18-12-3-1-10(2-4-12)7-14-13-9-16(20)15(19)8-11(13)5-6-17-14/h1-4,8-9,14,17-20H,5-7H2

1-[(4-hydroxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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