Cholinesterase

Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., Cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline-huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at Fa = 0.5. Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer's patients [1].

Cholinesterase Activity Assay [1]
The extracts and alkaloids of STR were tested for cholinesterase activity using a 96-well microtiter plate with a final volume of 200 μL, according to the Ellman method. The mouse brain was lysate was prepared by homogenizing with precooled low-salt lysis buffer (1:10 w/v; containing 1 mM ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA), 0.5% triton X-100, 150 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.4), 50 μg/mL benzamidine HCL, 10 μg/mL aprotinin, leupeptin, and pepstatin) for the AChE activity assay. The AChE assay medium consisted of brain lysate (5 mg/mL) with testing drugs, 80 mM Na2HPO4 (pH 7.4), 0.1 mM tetraisopropylpyro-phosphoramide (iso-OMPA), 0.5 mM DTNB (5′-dithiobis (2-nitrobenzenoic acid)), and 0.625 mM ATCh. The mixture of brain lysate, Na2HPO4 buffer, and iso-OMPA solution was incubated at 37 °C for 10 min, and then the solutions of DNTB and ATCh were added. After re-incubating at 37 °C for 30 min, AChE activity was tested by determining the absorbance at 405 nm.
Mouse serum (1: 50 v/v, diluted with precooled low-salt lysis buffer) was prepared for the BChE activity assay. The BChE assay medium consisted of the same composition as the AChE assay medium, except that the brain lysate and iso-OMPA were replaced with diluted serum solution and 0.1 μM BW284c51. The mixture of diluted serum, Na2HPO4 buffer, and BW284c51 solution was incubated at 37 °C for 10 min, and then the solutions of DNTB and ATCh were added. After re-incubating at 37 °C for 30 min, the BChE activity was tested by determining the absorbance at 405 nm. The concentration of DMSO in the reaction system of AChE/BChE assay was controlled at 0.5%. The background contrast without brain lysate or serum was set to eliminate the color interference at 405 nm. The AChE/BChE inhibitory activity of drugs was calculated as a percentage of the control absorbance value, AChE/BChE inhibition (%) = (1 − absorbance with inhibitor/absorbance without inhibitor) × 100%. Galanthamine was used as a positive control. The protein concentrations were measured using the Bio-Rad Protein Assay kit).
The Michaelis constant (Km), maximum velocity (Vmax), and inhibitory constant (Ki) were used to evaluate the kinetics of fangchinoline and Cyclanoline. Lineweaver–Burk plots were then plotted using initial velocities, from which the Km and Vmax values were calculated (the absolute values of horizontal and vertical intercepts were 1/Km and 1/Vmax, respectively). The slopes of reciprocal plots were then plotted against the doses of fangchinoline and Cyclanoline, and Ki values were determined as the absolute value of the x-axis intercept. Donepezil was used as a positive control. All experiments were performed in triplicate.

[1]. The Cholinesterase Inhibitory Properties of Stephaniae Tetrandrae Radix. Molecules. 2020;25(24):5914.

Cyclanoline alkaloid is a charged berberine alkaloid obtained by N-methylation of (S)-scoulerine. It is an EC 3.1.1.7 (acetylcholinesterase) inhibitor and a plant metabolite. It is a quaternary ammonium ion compound and also a berberine alkaloid. Its function is related to (S)-scoulerine. Cyclanoline alkaloid has been reported to exist in Stephania tetrandra, Cyclea tonkinensis, and several other organisms with relevant data. The chemical components of natural plants and herbs are the effective material basis of traditional Chinese medicine. This paper proposes that alkaloids are the main active components of STR (Synthetic Alpha-ChE) and BChE inhibitors. Enzyme activity results show that the AChE inhibitory activity of STR extracts increases with increasing alkaloid content. Bisbenzyloisoquinoline and protoberberine are the two main types of alkaloids that inhibit AChE/BChE activity in STR. Among them, bisbenzylisoquinoline alkaloids inhibit AChE, while water-soluble protoperine alkaloids inhibit both AChE and BChE activities. Fangchinolin is a representative bisbenzylisoquinoline alkaloid that inhibits AChE, while its structural analog tetrahydroberberine does not have AChE inhibitory activity. Cyclanoline bases are the main protoperine alkaloids, and their inhibitory activity on AChE is comparable to their inhibitory activity on BChE. In addition, both fangchinolin and Cyclanoline bases exhibit non-competitive AChE inhibitory effects. The Ki value of fangchinolin is much lower than that of Cyclanoline bases. Molecular docking simulations also support this result. Furthermore, fangchinolin combined with huperzine A or donepezil has a good synergistic effect on acetylcholinesterase inhibition. [1]

C20H24NO4+

342.40886

342.169

C, 70.15; H, 7.07; N, 4.09; O, 18.69

18556-27-9

17472-50-3 (Cl-);18556-27-9;

3082134

Typically exists as Off-white to light brown solid at room temperature

-2.19

2

4

2

25

488

2

COC1=C(O)C=C2[C@@H]3CC4C=CC(=C(O)C=4C[N@+]3(C)CCC2=C1)OC

LKLWVKCEYSPQHL-KKSFZXQISA-O

InChI=1S/C20H23NO4/c1-21-7-6-13-9-19(25-3)17(22)10-14(13)16(21)8-12-4-5-18(24-2)20(23)15(12)11-21/h4-5,9-10,16H,6-8,11H2,1-3H3,(H-,22,23)/p+1/t16-,21-/m0/s1

(7S,13aS)-3,10-dimethoxy-7-methyl-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinolin-7-ium-2,9-diol

Cyclanoline; Cissamine; 18556-27-9; 77QC59KBD2; (-)-Cyclanoline; (7S,13aS)-3,10-dimethoxy-7-methyl-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinolin-7-ium-2,9-diol; (-)-Cissamine; UNII-77QC59KBD2;

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.)