Phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway (activator) [1]

In mouse hippocampal slices, treatment with β-Amyrin (1, 10, 100 μM) did not affect basal long-term potentiation (LTP) under normal conditions. [1]
- Incubation of hippocampal slices with amyloid β (Aβ, 1 μM) for 2 hours impaired high-frequency stimulation (HFS)-induced LTP. Pretreatment with β-Amyrin (1, 10, 100 μM) for 30 minutes before Aβ exposure ameliorated this LTP impairment in a concentration-dependent manner. The effect was significant at 100 μM. [1]
- In an experimental paradigm where hippocampal slices were first incubated with Aβ (1 μM) for 2 hours to establish LTP impairment, delayed treatment with β-Amyrin (100 μM) for an additional 2 hours significantly restored LTP. [1]
- Western blot analysis of hippocampal slices showed that Aβ (1 μM, 2h) decreased the phosphorylation levels of PI3K and Akt, indicating suppression of this signaling pathway. Co-treatment with β-Amyrin (100 μM) significantly ameliorated this Aβ-induced reduction in pPI3K and pAkt levels, without affecting the total protein levels of PI3K and Akt. [1]
- The protective effect of β-Amyrin (100 μM) on Aβ-induced LTP impairment was blocked by the PI3K inhibitor LY294002 (50 μM), but not by the MAPK inhibitor U0126 (20 μM). This confirms that the effect is mediated through the PI3K/Akt pathway. [1]

In an Alzheimer's disease mouse model generated by intracerebroventricular (i.c.v.) injection of Aβ (10 μM, 5 μL), oral administration of β-Amyrin (4 mg/kg/day, p.o.) for 5 days (starting 1 day post-Aβ injection) significantly ameliorated memory impairments. [1]
- In the object recognition test, β-Amyrin-treated Aβ-injected mice showed a significantly higher discrimination ratio for the displaced object compared to vehicle-treated Aβ-injected mice, indicating improved spatial memory. Total exploration time was not different between groups, ruling out non-specific effects on locomotion or motivation. [1]
- In the passive avoidance test, β-Amyrin-treated Aβ-injected mice showed a significantly longer step-through latency in the test trial compared to vehicle-treated Aβ-injected mice, indicating improved fear memory. [1]
- Immunohistochemical analysis of the hippocampus revealed that Aβ injection reduced the number of doublecortin (DCX)-positive immature neurons and Ki67-positive proliferating cells in the subgranular zone, indicating impaired neurogenesis. Treatment with β-Amyrin (4 mg/kg/day, p.o.) significantly ameliorated this Aβ-induced reduction in both DCX-positive and Ki67-positive cells. [1]

Hippocampal Slice Preparation and Electrophysiology: Mouse brains were rapidly removed, and the hippocampus was isolated. Hippocampal slices (400 μm thick) were prepared using a tissue chopper and incubated in artificial cerebrospinal fluid (ACSF) at 20-25°C for 1 hour before experiments. For drug treatments, slices were incubated in ACSF containing vehicle or β-Amyrin for 30 minutes, then further incubated in ACSF containing Aβ (1 μM) with or without β-Amyrin for 2 hours prior to recording. For the delayed treatment experiment, slices were first incubated with Aβ for 2 hours, followed by a further 2-hour incubation with β-Amyrin. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the Schaffer collateral-commissural pathway in the CA1 region. LTP was induced by two trains of high-frequency stimulation (HFS: 100 Hz, 100 pulses in 1 s, 30 s interval). The fEPSP slope was monitored and the magnitude of LTP was quantified as the percentage change from baseline at 80 minutes post-HFS. [1]
- Western Blot Analysis in Hippocampal Slices: Hippocampal slices were incubated in ACSF containing β-Amyrin (100 μM) for 30 minutes, then further incubated in ACSF containing Aβ (1 μM) and β-Amyrin (100 μM) for 2 hours. Slices were homogenized in lysis buffer with protease and phosphatase inhibitors. Proteins (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with primary antibodies against pPI3K, PI3K, pAkt, Akt, and GAPDH (loading control), followed by HRP-conjugated secondary antibodies. Protein bands were detected and quantified. [1]

Animals:** Male CD-1 mice (6 weeks old, 26-28 g) were used. They were housed under standard conditions with free access to food and water and a 12h light/dark cycle. [1]
- **Aβ Injection Model:** Aβ₁₋₄₂ was prepared by dissolving in 1.0% NH₄OH, diluting with PBS to 1 mg/mL, and incubating at 37°C for 24 hours to obtain oligomeric species. Mice were anesthetized and 5 μL of Aβ (10 μM) or vehicle (PBS) was acutely injected into the left lateral ventricle (i.c.v.) by hand. [1]
- **Drug Administration:** β-Amyrin (4 mg/kg) was suspended in 10% Tween 80 solution and administered orally (p.o.) once daily for 5 days, starting one day after the Aβ injection. Minocycline (30 mg/kg, i.p.) was used as a positive control. The control group received vehicle (10% Tween 80). [1]
- **Behavioral Tests:** Behavioral tests were conducted 5 days after Aβ injection.
- **Object Recognition Test:** Mice were habituated to an open field for 10 min. In the training phase, they were placed in the same box with two distinct objects for 10 min. After 2 h, in the test phase, one object was displaced to a novel location, and mice were allowed to explore for 5 min. Time spent exploring the displaced and non-displaced objects was measured, and a preference ratio was calculated. [1]
- **Passive Avoidance Test:** In the training trial, mice were placed in an illuminated room. When they entered the dark room, a guillotine door closed, and an electric shock (0.5 mA for 3 s) was delivered. The next day, in the test trial, mice were re-introduced to the illuminated room, and the step-through latency to enter the dark room was measured (max 300 s). [1]
- **Immunohistochemistry:** After behavioral tests, brains were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and frozen. Coronal sections (30 μm) were cut using a cryostat. Sections were incubated with goat anti-doublecortin (DCX, immature neuron marker) or rat anti-Ki67 (proliferation marker) antibodies, followed by biotinylated secondary antibodies and avidin-biotin-peroxidase complex. Staining was visualized with DAB. DCX- and Ki67-positive cells in the subgranular zone of the hippocampal dentate gyrus were counted. [1]

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Animals: Male CD-1 mice (6 weeks old, 26-28 g) were used. They were housed under standard conditions with free access to food and water and a 12h light/dark cycle. [1]
- Aβ Injection Model: Aβ₁₋₄₂ was prepared by dissolving in 1.0% NH₄OH, diluting with PBS to 1 mg/mL, and incubating at 37°C for 24 hours to obtain oligomeric species. Mice were anesthetized and 5 μL of Aβ (10 μM) or vehicle (PBS) was acutely injected into the left lateral ventricle (i.c.v.) by hand. [1]
- Drug Administration: β-Amyrin (4 mg/kg) was suspended in 10% Tween 80 solution and administered orally (p.o.) once daily for 5 days, starting one day after the Aβ injection. Minocycline (30 mg/kg, i.p.) was used as a positive control. The control group received vehicle (10% Tween 80). [1]
- Behavioral Tests: Behavioral tests were conducted 5 days after Aβ injection.
- Object Recognition Test: Mice were habituated to an open field for 10 min. In the training phase, they were placed in the same box with two distinct objects for 10 min. After 2 h, in the test phase, one object was displaced to a novel location, and mice were allowed to explore for 5 min. Time spent exploring the displaced and non-displaced objects was measured, and a preference ratio was calculated. [1]
- Passive Avoidance Test: In the training trial, mice were placed in an illuminated room. When they entered the dark room, a guillotine door closed, and an electric shock (0.5 mA for 3 s) was delivered. The next day, in the test trial, mice were re-introduced to the illuminated room, and the step-through latency to enter the dark room was measured (max 300 s). [1]
- Immunohistochemistry: After behavioral tests, brains were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and frozen. Coronal sections (30 μm) were cut using a cryostat. Sections were incubated with goat anti-doublecortin (DCX, immature neuron marker) or rat anti-Ki67 (proliferation marker) antibodies, followed by biotinylated secondary antibodies and avidin-biotin-peroxidase complex. Staining was visualized with DAB. DCX- and Ki67-positive cells in the subgranular zone of the hippocampal dentate gyrus were counted. [1]

[1]. β-Amyrin Ameliorates Alzheimer's Disease-Like Aberrant Synaptic Plasticity in the Mouse Hippocampus. Biomol Ther (Seoul). 2019 Jul 30.

β-Amyrinol is a pentacyclic triterpenoid compound with a structure in which oleanane is substituted at the 3β position with a hydroxyl group, forming a double bond between the 12 and 13 positions. It is one of the most common triterpenoids in higher plants, with metabolites found in both plants and Aspergillus fungi. It is a pentacyclic triterpenoid and a secondary alcohol derived from the hydride of oleanane. β-Amyrinol has been reported in tea (Camellia sinensis), elderberry (Sambucus chinensis), and other organisms with relevant data. See also: Calendula (partial); Viburnum bark (partial); Cornflower (partial).

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β-Amyrin is a natural pentacyclic triterpene found in various plants, including the surface wax of tomato fruit and dandelion coffee. It is a component of glycyrrhizin. [1]
- Previous studies have reported that β-Amyrin possesses anti-fibrotic, anti-inflammatory, anti-diabetic, anti-hyperglycemic, hypolipidemic, and angiogenic effects. It also has neurological effects, including regulating sleep, memory, and nociception. [1]
- This study demonstrates for the first time the anti-Alzheimer's disease potential of β-Amyrin. It shows that β-Amyrin can ameliorate Aβ-induced synaptic dysfunction, memory impairment, and neurogenesis deficits. The mechanism involves the activation of the PI3K/Akt signaling pathway. The ability of β-Amyrin to restore already-established LTP impairment and improve memory with delayed administration suggests it might have disease-modifying properties, making it a potential candidate for treating moderate to severe AD. Based on dose conversion guidelines, the effective dose of 4 mg/kg in mice would translate to approximately 0.325 mg/kg in humans. [1]

C30H50O

426.73

426.386

559-70-6

73145

White to off-white solid powder

1.0±0.1 g/cm3

490.7±44.0 °C at 760 mmHg

187-190°C

217.7±20.7 °C

0.0±2.8 mmHg at 25°C

1.539

11.06

1

1

0

31

790

8

C[C@@]12CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)O)C)C)[C@@H]1CC(CC2)(C)C)C

JFSHUTJDVKUMTJ-QHPUVITPSA-N

InChI=1S/C30H50O/c1-25(2)15-16-27(5)17-18-29(7)20(21(27)19-25)9-10-23-28(6)13-12-24(31)26(3,4)22(28)11-14-30(23,29)8/h9,21-24,31H,10-19H2,1-8H3/t21-,22-,23+,24-,27+,28-,29+,30+/m0/s1

(3S,4aR,6aR,6bS,8aR,12aR,14aR,14bR)-4,4,6a,6b,8a,11,11,14b-octamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-ol

beta-Amyrin beta-Amyrenol Amyrinβ-amyrin

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)

DMF : 10 mg/mL (~23.43 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.

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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.)