In silico docking studies highlighted Lotusine's affinity for proteins involved in diabetes pathophysiology including: Glucose fructose 6 phosphate amidotransferase (PDB: 2zj4), Glycogen Synthase Kinase (PDB: 3f7z), aldose reductase (PDB: 3g5e), Glucokinase (PDB: 4ixc), Hydroxysteroid dehydrogenase (PDB: 4k11), Proliferator-activated receptor gamma (PPAR gamma, PDB: 3dzy), and Pyruvate dehydrogenase kinase (PDB: 4mp2). [2]

In HepG2 cells, treatment with 25 mM and 50 mM D-glucose and Lotusine (with or without D-glucose) did not significantly affect cell viability over 48 and 72 hours, maintaining 85-90% cell viability. Lotusine treatment combined with 25 mM D-glucose significantly ameliorated the reduction in SOD activity induced by D-glucose [2].
Lotusine treatment mitigated the reduction in CAT activity induced by 25 mM D-glucose at 48 and 72 hours intervals. Lotusine treatment for 48 hours significantly increased CAT activity compared to the control [2].
Lotusine treatment combined with 25 mM D-glucose increased GPx activity relative to the control group, with a considerable increase noted at 50 µM after both 48 and 72 hours [2].
Lotusine dramatically reduced MDA levels in cells exposed to 25 mM D-glucose. In HepG2 cells fed with 50 mM D-glucose for 72 hours, MDA levels increased by 41% but decreased GSH by 50%; however, the Lotusine-supplemented diet-treated rats expressed considerable protection by increasing the GSH levels [2].
Lotusine (200 μg/mL) inhibited alpha-amylase activity with an inhibition rate of 80.36% and an IC50 of 30.60 μg/mL [2].
Lotusine (200 μg/mL) inhibited alpha-glucosidase activity with an inhibition rate of 82.6% and an IC50 of 36.15 μg/mL [2].
Lotusine treatment significantly altered mRNA expression levels, with IRS-1 showing a noteworthy increase, comparable to the effect of Metformin. AKT-2 and GLUT-4 mRNA levels also exhibited significant changes following Lotusine treatment [2].
In H9c2 cells, pretreatment with 10 and 50 µM Lotusine for 24 hours followed by exposure to 1 µM doxorubicin (DOX) showed cell viability near to control (10 µM) and cell proliferation (118.53±9.05 and 110.36±1.35% respectively in SRB and MTT assays). The concentration of 100 µM Lotusine resulted in significant reduction of cell viability (84.41±6.07 and 79.84±11.20% in SRB and MTT assays) [3].
In H9c2 cells, pretreatment with 10 and 50 µM Lotusine significantly protected cardiomyocytes by increasing the antioxidant levels of SOD and CAT, while only the 50 µM concentration increased GSH content after DOX exposure. Lotusine-pretreated cells significantly reduced lipid peroxidation (MDA content) compared to DOX-alone-treated cells [3].
In H9c2 cells, Lotusine-pretreated (10 and 50 µM) cells did not show significant morphological changes (shrinkage, blebbing, reduced size, detachment) and exhibited well-arranged intact nuclei (Giemsa staining) compared to DOX-alone-treated cells which showed darkly stained nuclei indicative of chromatin condensation [3].
In H9c2 cells, Lotusine-pretreated (10 and 50 µM) cells showed no green fluorescence (DCFDA staining) as similar to control cells, whereas intense green fluorescence (ROS) was observed in DOX-alone-treated cells [3].
In H9c2 cells, Lotusine-pretreated cells showed downregulation of pro-apoptotic gene Bax and apoptotic executor caspase-3, and upregulation of anti-apoptotic gene Bcl-2 in qPCR analysis, compared to DOX-alone-exposed cells which showed downregulation of Bcl-2 (0.61±0.11 fold change) and upregulation of Cas-3 (2.5±0.52 fold change) and Bax (2.08±0.29 fold change) [3].
In H9c2 cells, the Caspase 3/7 activity was found to decline in Lotusine-pretreated cells even after exposure with DOX. The 50 µM Lotusine treatment showed similar luminescence values near to untreated control, whereas 10 µM Lotusine-pretreated cells had significantly increased caspase activity [3].

Diabetic rats (STZ-induced, 50 mg/kg) receiving a Lotusine-supplemented diet (50 mg/kg) for four weeks showed enhanced growth parameters including higher body weight gain (BWG) compared to diabetic controls. Lotusine-treated rats (G3) recorded the highest BWG (77.8±0.5g); the combined treatment STZ+Lotusine (G4) enhanced BWG by 13% and 45% compared to control and G2 (diabetic untreated), respectively [2].
Dietary Lotusine significantly restored reduced serum, liver, and pancreatic vitamin C and E levels in diabetic rats to near normal values [2].
Lotusine significantly increased liver cell protein content to 11.6 mg/g, with a relative increase of 21% compared to glibenclamide-treated rats [2].

Alpha-Amylase Inhibition (AAI) assay: A reaction mixture containing 5 µL of porcine pancreatic enzyme solution and 15 µL of Lotusine diluted in phosphate buffer (1.56 - 200 μg/mL) was incubated at room temperature for 10 minutes. Then, 20 µL of starch solution was added and the mixture was incubated for 30 minutes at 37°C. The reaction was stopped by adding 10 µL of 1M HCl and 75 µL of iodine reagent. Absorbance was measured at 540 nm. Acarbose was used as a positive control and PBS as a negative control. The percentage inhibition was calculated using the formula: Inhibition (%) = [Abs(control) - Abs(sample)] / Abs(control) 100 [2].
Alpha-glucosidase inhibition assay: A reaction mixture containing 5 µL of Lotusine (1.56 - 200 μg/mL) and 20 µL of alpha-glucosidase solution (50 μg/mL) was incubated in a 96-well plate at 37°C for 20 minutes after adding 10 µL of p-nitrophenyl-α-D-glucopyranoside (PNP-GLUC, 10 mM) and 60 µL of 67 mM KH2PO4 buffer (pH 6.8). The reaction was halted with 25 µL of 100 mM sodium carbonate (Na2CO3), and absorbance was measured at 405 nm. Epigallocatechin gallate (EGCG) was used as a positive control [2].

Cell viability assay (MTT): HepG2 cells (12 x 10^3 cells/well) in 96-well plates were incubated for 48 and 72 hours at 37°C with 25 μM and 50 μM D-glucose and/or 25 μM Lotusine. Following treatment, cells were incubated for 3.5 hours in a serum-free media containing 0.5 mg/ml of MTT. After dissolving formazan crystals in 100 μL DMSO, absorbance was measured at 570/650 nm. Cell vitality was determined as the ratio of treated cells' absorbance to that of untreated cells [2].
Lipid peroxidation inhibitory activity (MDA assay): HepG2 cell lysates (100 μL) treated with D-glucose and/or Lotusine were mixed with 0.25 mol/L HCl, 15% trichloroacetic acid, and 400 μL TBA reagent containing 0.375% TBA. After 30 minutes of heating at 95°C and a quick cooling period, the mixture was centrifuged for 15 minutes at 4°C at 8000 x g. The supernatant's pink absorbance was measured at 532 nm [2].
Antioxidant enzyme assays: SOD activity was recorded using the Nitro Blue Tetrazolium (NBT) technique. GPx and CAT activities were assessed as previously described. A total protein test assay (Bio-Rad BSA) was performed to test protein levels [2].
GSH concentration measurement: GSH concentration was measured in the serum and supernatants of liver and pancreatic tissue homogenates. The thiol reagent (DTNB, Ellman's Reagent) reacts with free sulphydryl groups to form a yellow complex measured at 412 nm [2].
Real-time PCR: Total RNA was extracted using TRIzol reagent. qRT-PCR reactions were performed using RT^2 SYBR Green mix. The PCR protocol: initial denaturation at 95°C for 4 minutes, followed by 35 cycles of denaturation at 95°C for 90 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 90 seconds, with a final extension at 72°C for 10 min. Primers for IRS-1, Akt-2, GLUT-4, and GAPDH (internal control) were used. Relative gene expression changes were calculated using the 2-ΔΔCT method [2].
Cytotoxicity assay for DOX and Lotusine (MTT) in H9c2 cells: About 6000 cells were seeded in each well of a 96-well plate. After 12h, cells were treated with various concentrations of DOX (0.10, 0.50, 1.00, 2.50, 5.0, and 10 μM) and Lotusine (10, 50, 100, 250, 500, and 1000 μM) and incubated at 37°C for 1 day. Then, 90 μL of DMEM medium without FBS and 10 μL of MTT reagent (5 mg/mL) were added and incubated in dark for 4h. Formazan crystals were dissolved in 0.1 mL of DMSO and readings were recorded at 570 nm [3].
In vitro cardioprotective activity of Lotusine in H9c2 cells: Cells were pretreated with 10, 50, and 100 μM of Lotusine for 24h. After incubation, the media was drained and 1 μM of DOX solution was added and incubated for 24h. Cell viability was assessed with MTT and SRB (Sulforhodamine-B) assays [3].
Estimation of total protein and endogenous antioxidant level in H9c2 cells: After pretreatment with Lotusine (10, 50, and 100 μM) and exposure with DOX (1 μM), cells were lysed with prechilled buffer (1% Triton-X 100, 50 mM sodium chloride, 10 mM tris-HCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 0.1% beta-mercaptoethanol, pH 7.5). The homogenized cell suspension was centrifuged (5000 rpm for 10 min at 4°C). Total protein content was estimated from the supernatant with BSA as a standard [3].
Enzymatic antioxidants (CAT, SOD) and GSH estimation in H9c2 cells: CAT activity was measured by the reduction of H2O2 into water and oxygen; the reaction mixture consisted of 2 mM H2O2 in 100 mM phosphate buffer (pH 7.4) mixed with the cell homogenate, and absorbance was recorded at 240 nm. SOD activity was measured by the enzyme's inhibitory activity of NBT reduction. GSH was estimated by the reaction of NADPH in a reaction mixture containing DTNB, and OD was recorded at 412 nm [3].
LPO assay in H9c2 cells: Cells (6.0 x 10^-3) were pretreated with Lotusine (10, 50, and 100 μM) and exposed to 1 μM DOX. Cells were lysed with prechilled cell lysis buffer and sonicated for 30s under 4°C. The homogenate was mixed with 1 mL of TCA (10%), centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was collected, 1 mL of TBA (0.67%) solution was added and incubated in boiling water bath for 20 min. A mixture of n-butanol and pyridine (15:1 ratio) was added and centrifuged. OD at 532 nm was measured with the butanol layer [3].
Morphological examination of nuclear abnormality in H9c2 cells: Cells were seeded on a glass coverslip. Lotusine pretreatment (10 and 50 μM) and DOX (1 μM) exposure were done for 24h. Nuclear abnormalities were detected using Giemsa staining and observed under a bright field microscope [3].
Intracellular ROS detection in H9c2 cells: Cells (-6 x 10^-3) were seeded on a glass coverslip, pretreated with Lotusine (10 and 50 μM), and exposed to DOX (1 μM). Treated cells were incubated with 20 μM DCFDA under dark for 30 min at 37°C, washed with PBS, and observed under a fluorescent microscope [3].
Gene expression analysis by qPCR in H9c2 cells: Total RNA was extracted using TRIzol reagent. cDNA synthesis was done using SuperScript™ III First-Strand Synthesis System. qPCR analysis was performed with Power SYBR™ Green PCR Master Mix. Primers for Bcl-2, Bax, Cas-3, and beta-actin (housekeeping gene) were used [3].
Caspase 3/7 activity assay in H9c2 cells: About 6000 H9c2 cells were seeded on 96-well plates and pretreated with Lotusine (10 and 50 μM) for 24 h, then exposed to DOX (1 μM) for 24 h. Caspase-Glo 3/7 assay was performed using a commercial kit. Relative light units (RLU) or luminance was measured in a Luminometer at different time intervals (0, 15, 30, and 45 min) [3].

Two hundred male Wistar (Sprague Dawley) rats weighing 160 ± 20 g were used. Rats were housed under a 12 h light/dark cycle at 23°C and relative humidity of 20-50% [2].
Diabetes induction was carried out by a single intraperitoneal injection of streptozotocin (STZ) prepared at 50 mg/kg bw in 0.1 M citrate buffer at pH 4.5 and administered to rats fasted overnight. The STZ solution was prepared in ice-cold citrate buffer and maintained on ice. 5% glucose solution was provided for the rats after six hours of STZ administration for the next 12 hours post-STZ injection. The hyperglycemic state was confirmed after 3 days when fasting blood glucose (FBG) was ≥ 250 mg/dL and postprandial glucose (PPG) was ≥ 350 mg/dL [2].
200 rats were divided into four groups of ten each, with five replicates. Group 1 (control) received normal saline supplements plus a basal diet for four weeks. Group 2 (diabetic control) received STZ 50 mg/kg bw for 4 weeks. Group 3 (glibenclamide-treated diabetic) received glibenclamide at a dose of 50 mg/kg bw, fed the NC diet for four weeks. Group 4 received Lotusine 50 mg/kg. All groups were fed a normal control diet for four weeks [2].
At the end of the experiment, rats were sacrificed by cervical dislocation. Blood samples were collected by cardiac puncture, allowed to clot, and centrifuged at 3,000 x g for 10 minutes. The serum was decanted and stored at -80°C. The liver and pancreas were removed, washed in ice-cold sucrose (250 mM), and homogenized in PB (50 mM, pH 7.4) to obtain a final 10% w/v homogenates solution. Homogenates were centrifuged at 10,000 x g for 30 min at 4°C, and the supernatants were used for assays [2].

In HepG2 cells, treatment with Lotusine (25 μM and 50 μM with or without D-glucose) did not significantly affect cell viability over 48 and 72 hours, maintaining 85-90% cell viability [2].
In H9c2 cells, the IC50 value for Lotusine exposure for 24h was 701 μM. A significant decrease in cell viability was observed only at higher concentrations from 250 to 1000 μM. The lesser concentrations (10, 50, and 100 μM) showed cell viability near to control (above 95%) [3].

[1]. Anti-lung cancer activity of lotusine in non-small cell lung cancer HCC827 via reducing proliferation, oxidative stress, induction of apoptosis, and G0/G1 cell cycle arrest via suppressing EGFR-Akt-ERK signalling. In Vitro Cell Dev Biol Anim. 2025;61(4):450-458.

[2]. Prophylactic Impacts of Lotusine Against Hyperglycaemia-Induced Oxidative Stress in Hepatic Cells Isolated from Diabetic Rats Via Irs-1/Pi3 K/Akt Pathway. Pak Vet J, 45(1): 124-137.

[3]. Lotusine, an alkaloid from Nelumbo nucifera (Gaertn.), attenuates doxorubicin-induced toxicity in embryonically derived H9c2 cells. In Vitro Cell Dev Biol Anim. 2020;56(5):367-377.

[4]. Lotusine ameliorates propionic acid-induced autism spectrum disorder-like behavior in mice by activating D1 dopamine receptor in medial prefrontal cortex. Phytother Res. 2024;38(2):1089-1103.

[5]. Lotusine G: a new cyclopeptide alkaloid from Zizyphus lotus. Fitoterapia. 2002 Feb;73(1):63-8.

Lotusine belong to the isoquinoline class of compounds. It has been reported that Lotusine are found in magnolia (Magnolia officinalis), small magnolia (Xylopia parviflora), and lotus (Nelumbo nucifera), and relevant data are available for reference.

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The study concluded that Lotusine exhibits tolerability and hypoglycemic effects, suggesting its potential as an effective antidiabetic agent for managing type 2 diabetes mellitus (T2DM) [2].
It is the first report to use Lotusine for cardioprotection against doxorubicin-induced oxidative stress using an in vitro model (H9c2 cells) [3].
The biologically active compounds discovered in Lotusine may be invented into prospective therapeutic compounds against enzymes/proteins that contribute to the emergence and progression of chronic inflammation and for monitoring hyperglycemia [2].

C19H24NO3

314.176

6871-67-6

Lotusine hydroxide;3721-76-4

5274587

Typically exists as solid at room temperature

2.981

2

3

3

23

392

1

O(C([H])([H])[H])C1=C(C([H])=C2C([H])([H])C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])(C([H])([H])C3C([H])=C([H])C(=C([H])C=3[H])O[H])C2=C1[H])O[H]

ZKTMLINFIQCERN-QGZVFWFLSA-O

InChI=1S/C19H23NO3/c1-20(2)9-8-14-11-18(22)19(23-3)12-16(14)17(20)10-13-4-6-15(21)7-5-13/h4-7,11-12,17H,8-10H2,1-3H3,(H-,21,22)/p+1/t17-/m1/s1

(1R)-1-[(4-hydroxyphenyl)methyl]-7-methoxy-2,2-dimethyl-3,4-dihydro-1H-isoquinolin-2-ium-6-ol

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.)