Albiflorin inhibits the p38 mitogen-activated protein kinase (p38 MAPK) pathway in spinal microglia.
It also inhibits the c-Jun N-terminal kinase (JNK) pathway, suppressing the overelevated expression of phosphorylation of JNK (p-JNK) in spinal cord astrocytes. [2]

Treatment with albiflorin (50-200 μM; 3 hours pretreatment; PC12 cells) can greatly ameliorate the reduction in cell viability caused by glutamate (Glu) [1]. Reactive oxygen species accumulation, B-cell lymphoma 2 (Bcl-2)/Bax ratio reduction, and Glu-induced nuclear and mitochondrial apoptotic alterations were all markedly alleviated by albiflorin (100 μM; 3 hours pretreatment; PC12 cells) treatment[1]. The treatment of PC12 cells with 100 μM albiflorin three hours prior to treatment increases the phosphorylation of AKT and its downstream component GSK-3β [1].

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Albiflorin (50‑200 μM) pretreatment for 3 h significantly reduced glutamate (20 mM, 24 h)-induced cell death in differentiated PC12 cells. At 50 μM and 100 μM, AF significantly improved cell viability (MTT assay); the optimal dose of 100 μM was used for further experiments. [1]
- Pretreatment with 100 μM Albiflorin for 3 h followed by 20 mM glutamate for 24 h almost completely inhibited glutamate-induced apoptotic nuclear alteration (Hoechst 33342 staining), with a survival rate >93% compared to 84% in untreated controls. [1]
- Intracellular reactive oxygen species (ROS) levels increased by approximately 30% after glutamate exposure; 100 μM Albiflorin pretreatment suppressed this increase back to control level (DCFH-DA staining). [1]
- Glutamate exposure caused a striking increase in mitochondrial permeability (60% reduction of Rh123 fluorescence intensity). Pretreatment with 100 μM Albiflorin restored Rh123 fluorescence intensity to 87% of unexposed control value, indicating protection of mitochondrial membrane potential. [1]
- Albiflorin (100 μM, 3 h pretreatment) reversed the glutamate-induced decrease in Bcl‑2/Bax ratio. Glutamate reduced the Bcl‑2/Bax ratio to 79.2% of control; AF pretreatment increased it to 110.1% (Western blot). [1]
- Albiflorin (100 μM) significantly upregulated the phosphorylation of AKT and GSK‑3β in a time‑dependent manner (30, 60, 180, 360 min after glutamate exposure). This effect was completely abolished by the PI3K inhibitor LY294002 (10 μM). [1]
- Albiflorin did not significantly suppress glutamate‑induced intracellular calcium overload (Fluo‑4‑AM assay): after AF pretreatment, intracellular Ca²⁺ level was 134.8% of unexposed control (vs. 150.5% with glutamate alone, not significant). [1]
- Albiflorin did not produce significant suppression of glutamate‑induced CaMKII phosphorylation (Western blot) at any time point (30, 60, 180 min). [1]
- The cell viability protection by Albiflorin (100 μM) against glutamate toxicity was significantly abrogated by the PI3K inhibitor LY294002 (10 μM) (MTT assay). [1]

Albiflorin (50 mg/kg; i.p.; once daily; for 15 days; Wistar rats) therapy significantly enhanced the paw withdrawal threshold (PWT) on postoperative days 11 and 15 in rats. Albiflorin suppresses the activation of the p38 MAPK pathway in spinal microglia and therefore upregulates IL-1β and TNF-α. Albiflorin has strong effects in decreasing astrocyte activation, preventing the phosphorylation and overexpression of c-JNK (p-JNK) in astrocytes, and reducing the concentration of the chemokine CXCL1 in the spinal cord [2].

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Albiflorin (50 mg/kg, intraperitoneal injection once daily for 15 days) significantly increased the paw withdrawal threshold (PWT) in CCI rats on the 11th and 15th days after surgery, indicating relief of mechanical allodynia; however, it had no significant effect on the decreased paw withdrawal latency (PWL) induced by CCI, indicating no effect on thermal hyperalgesia. [2]
Administration of Albiflorin decreased the activation of microglia (Iba-1 immunoreactivity) in the spinal cord dorsal horn of CCI rats at both 11th and 15th days after surgery, and also suppressed the activation of astrocytes (GFAP immunoreactivity) at the 15th day after surgery. [2]
Albiflorin treatment significantly decreased the elevated levels of proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord of CCI rats at the 15th day after surgery. Additionally, Albiflorin remarkably decreased the elevated level of chemokine CXCL1 in the spinal cord, whereas it exerted no effect on the CCI-induced upregulation of IL-6. [2]
Albiflorin decreased the elevated protein level of phosphorylated p38 (p-p38) and phosphorylated JNK (p-JNK) in the spinal cord of CCI rats at the 15th day after surgery, as shown by immunofluorescence and Western blotting. Double immunofluorescence staining revealed that p-p38 was colocalized with spinal microglia, and p-JNK was colocalized with spinal astrocytes. [2]

Cell viability assay [1]
Cell Types: PC12 Cell
Tested Concentrations: 50 μM, 100 μM, 200 μM
Incubation Duration: 3 hrs (hours) of pretreatment
Experimental Results: Dramatically improved Glu-induced decrease in cell viability.

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Apoptosis analysis [1]
Cell Types: PC12 Cell
Tested Concentrations: 100 μM
Incubation Duration: 3 hrs (hours) of pretreatment
Experimental Results: Significant improvement in the reduction of Glu-induced apoptotic changes in the nucleus and mitochondria.

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Western Blot Analysis[1]
Cell Types: PC12 Cell
Tested Concentrations: 100 μM
Incubation Duration: 3 hrs (hours) of pretreatment
Experimental Results: Enhanced phosphorylation of AKT and its downstream component GSK-3β.

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Cell viability assay (MTT): Differentiated PC12 cells (2×10⁴ per well) were seeded in 96‑well plates, pretreated with 50‑200 μM Albiflorin for 3 h, then co‑treated with 20 mM glutamate for 24 h. MTT solution (0.5 mg/ml) was added for 4 h at 37 °C in darkness, and absorbance was measured at 540 nm. Viability values were expressed as percentage of corresponding control cells. [1]
- Apoptosis analysis (Hoechst 33342 staining): PC12 cells (1×10⁵ per well) in 12‑well plates were differentiated, pretreated with 100 μM Albiflorin for 3 h, then co‑treated with 20 mM glutamate for 24 h. Cells were incubated with Hoechst 33342 (5 μg/ml) for 30 min at 37 °C in darkness, washed three times with PBS, and fluorescence intensity was determined by laser scanning confocal microscopy (excitation 350 nm, emission 460 nm). The percentage of survival cells was calculated. [1]
- Measurement of intracellular ROS levels (DCFH-DA staining): Differentiated PC12 cells (1×10⁵ per well) in 12‑well plates were pretreated with 100 μM Albiflorin for 3 h, then co‑treated with 20 mM glutamate for 24 h. Cells were incubated with 5 μM DCFH-DA for 30 min at 37 °C in darkness, washed three times with PBS, and fluorescence intensity was monitored by fluorescence microscope (excitation 485 nm, emission 530 nm). Mean fluorescent intensity of each group was calculated. [1]
- Measurement of mitochondrial membrane potential (Rh123 staining): Differentiated PC12 cells (1×10⁵ per well) in confocal dishes were pretreated with 100 μM Albiflorin for 3 h, then exposed to 20 mM glutamate for 24 h, followed by incubation with 5 μM Rh123 for 30 min at 37 °C. After three PBS washes, fluorescent intensity was determined by laser scanning confocal microscopy (excitation 525 nm, emission 590 nm). Average fluorescent intensity from 30 cells per group was calculated. [1]
- Measurement of intracellular Ca²⁺ concentration (Fluo‑4‑AM): Differentiated PC12 cells (1×10⁵ per well) in confocal dishes were pretreated with 100 μM Albiflorin for 3 h, then co‑treated with 20 mM glutamate for 3 h. Cells were incubated with 5 μM Fluo‑4‑AM for 30 min at 37 °C in darkness, washed three times with PBS, and fluorescence intensity was determined by laser scanning confocal microscopy (excitation 488 nm, emission 520 nm). Total fluorescent intensity from nearly 60 cells per group was calculated, and Ca²⁺ concentration was expressed as percentage of corresponding control. [1]
- Western blot analysis: Whole cell lysates were prepared from treated PC12 cells using lysis buffer containing protease inhibitor cocktail and PMSF. After centrifugation (12,000 rpm, 30 min, 4 °C), protein concentration was determined by Bradford assay. Samples (30 μg protein) were fractioned on 12% SDS‑PAGE, transferred to PVDF membranes, blocked with 5% BSA, and incubated overnight at 4 °C with primary antibodies against P‑AKT, T‑AKT, P‑CaMKII, T‑CaMKII, P‑GSK3β, T‑GSK3β, Bcl‑2, Bax, and GAPDH (1:1,000). After TBST washes, membranes were incubated with HRP‑conjugated secondary antibodies (1:2,000) for 2 h at 37 °C, and proteins were visualized using ECL detection kit. Band intensity was quantified by densitometry. For Albiflorin experiments, cells were pretreated with 100 μM AF for 3 h then exposed to glutamate; cells were collected at 30, 60, 180, or 360 min after exposure. [1]

Animal/Disease Models: Wistar rats (7 weeks old; 200-220 g), suffering from chronic constrictive injury (CCI) [2]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; intraperitoneal (ip) injection. one time/day; for 15 days
Experimental Results: Paw withdrawal threshold (PWT) increased Dramatically on postoperative days 11 and 15.

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Adult male Wistar rats (7-week-old, 200-220 g) were used. Chronic constriction injury (CCI) of the right sciatic nerve was performed under chloral hydrate anesthesia (300 mg/kg intraperitoneal). The right sciatic nerve was exposed at mid-thigh level, and four ligatures (chromic catgut 4.0) were tied loosely at 1.0 mm intervals around the nerve. Sham surgery involved exposure of the right sciatic nerve without ligation. [2]
Albiflorin was dissolved in normal saline solution and administered by intraperitoneal injection once daily at a dose of 50 mg/kg, starting on the first day after CCI surgery and continuing for 15 days. The vehicle control group received normal saline solution. [2]
Mechanical withdrawal threshold (PWT) was assessed using calibrated von Frey filaments applied to the plantar surface of the right hind paw by Chaplan's up-down method. Tests were performed one day before CCI surgery and on days 3, 7, 11, and 15 after CCI surgery. Thermal withdrawal latency (PWL) was measured using a thermal pain stimulator (radiant heat applied to the plantar surface). A cutoff time of 25 seconds was used. Each measurement was repeated three times at 15-minute intervals. [2]
On the 11th and 15th days after CCI surgery, 60 minutes after the last drug dose, rats were deeply anesthetized with chloral hydrate (350 mg/kg intraperitoneal). The L4-L5 spinal cord segments ipsilateral to the nerve injury were removed for immunohistofluorescence analysis. On the 15th day after surgery, spinal cords were also collected for enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. [2]

[1]. Neuroprotective effects of paeoniflorin, but not the isomer albiflorin, are associated with the suppression of intracellular calcium and calcium/calmodulin protein kinase II in PC12 cells. J Mol Neurosci. 2013 Oct;51(2):581-90.

[2]. Paeoniflorin and Albiflorin Attenuate Neuropathic Pain via MAPK Pathway in Chronic Constriction Injury Rats. Evid Based Complement Alternat Med. 2016;2016:8082753.

Albiflorin is a monoterpenoid glycoside with the molecular formula C23H28O11, originally isolated from the roots of peony (Paeonia lactiflora). It is a plant metabolite and neuroprotective agent. It is a benzoate, γ-lactone, β-D-glucoside, monoterpenoid glycoside, secondary alcohol, and bridging compound. Albiflorin has been reported to exist in peony (Paeonia suffruticosa), peony (Paeonia lactiflora), and other organisms with relevant data.

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Albiflorin is an isomer of paeoniflorin (PF), both being major constituents of Paeonia lactiflora Pall. In this CCI rat model of neuropathic pain, Albiflorin exhibited differential mechanisms compared to PF: while both isomers inhibited microglial activation and the p38 MAPK pathway, only Albiflorin further inhibited astrocytic activation, suppressed p-JNK expression in astrocytes, and decreased CXCL1 levels in the spinal cord. This suggests that Albiflorin may have analgesic effects in both acute and late phases of neuropathic pain. The study indicates that Albiflorin is a potential therapeutic agent for neuropathic pain via inhibiting neuroinflammation. [2]

C23H28O11

480.4618

480.163

39011-90-0

24868421

White to off-white solid powder

1.6±0.1 g/cm3

722.1±60.0 °C at 760 mmHg

248.9±26.4 °C

0.0±2.5 mmHg at 25°C

1.662

-0.97

5

11

7

34

830

10

C[C@]12C[C@H]([C@@H]3C[C@]1([C@@]3(C(=O)O2)COC(=O)C4=CC=CC=C4)O[C@H]5[C@@H]([C@H]([C@@H]([C@H](O5)CO)O)O)O)O

QQUHMASGPODSIW-ICECTASOSA-N

InChI=1S/C23H28O11/c1-21-8-13(25)12-7-23(21,33-19-17(28)16(27)15(26)14(9-24)32-19)22(12,20(30)34-21)10-31-18(29)11-5-3-2-4-6-11/h2-6,12-17,19,24-28H,7-10H2,1H3/t12-,13+,14+,15+,16-,17+,19-,21-,22-,23-/m0/s1

[(1R,3R,4R,6S,9S)-4-hydroxy-6-methyl-8-oxo-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-7-oxatricyclo[4.3.0.03,9]nonan-9-yl]methyl benzoate

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 : ≥ 100 mg/mL (~208.13 mM)
H2O : ~100 mg/mL (~208.13 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.20 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.20 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (208.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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