NLRP3 inflammasome (NLRP3, CASPASE-1, TXNIP, ASC). [1]

NF-κB pathway (mentioned in discussion as inhibited by PA). [1]

Patchouli alcohol (5-20 μg/mL; 24 h) not only markedly increased cell viability and decreased cellular metabolic dehydrogenase (LDH) leakage, but it also markedly increased mitochondrial membrane potential and significantly disconnected GES-1. Furthermore, patchouli alcohol can prevent inflammatory factors like MCP-1, TNF-α, and IL-6 from damaging cell mitochondria[1].

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Patchouli alcohol (5, 10, 20 μg/ml) significantly increased the viability of GES-1 cells co-cultured with H. pylori NCTC11637 at MOI=100:1 for 24 h, as measured by trypan blue assay (viable cells: 58.2% in model vs. 80.2% and 75.2% at 10 and 20 μg/ml PA, respectively; P<0.01 or P<0.05). [1]

Patchouli alcohol (10 and 20 μg/ml) reduced plasma membrane damage as indicated by LDH leakage (model: 48.4%; PA 10 μg/ml: 38.8%; PA 20 μg/ml: 41.3%; P<0.01 or P<0.05). [1]

Patchouli alcohol (5, 10, 20 μg/ml) significantly reduced H. pylori-induced apoptosis of GES-1 cells measured by flow cytometry with annexin V/PI staining (model: 22.3% apoptotic cells; PA 5 μg/ml: 10.8%; 10 μg/ml: 9.3%; 20 μg/ml: 15.3%; P<0.01). [1]

Patchouli alcohol (5, 10, 20 μg/ml) restored mitochondrial membrane potential (MMP) in H. pylori-infected GES-1 cells, as shown by increased rhodamine 123 fluorescence intensity (P<0.01). [1]

Patchouli alcohol (5, 10, 20 μg/ml) significantly attenuated H. pylori-induced intracellular ROS production measured by DCFH-DA staining and flow cytometry (P<0.01). [1]

Patchouli alcohol (5, 10, 20 μg/ml) significantly reduced the production of TNF-α and MCP-1 in H. pylori-infected GES-1 cells; PA (10 and 20 μg/ml) also reduced IL-6 production (P<0.01 or P<0.05). [1]

Patchouli alcohol (5, 10, 20 μg/ml) downregulated H. pylori-induced gene expression levels of NLRP3 and CASPASE-1, and protein levels of NLRP3, pro-CASPASE-1, cle-CASPASE-1, and TXNIP as determined by RT-qPCR and Western blot (P<0.05). ASC gene and protein expression were not significantly changed. [1]

Patchouli alcohol (5, 10, 20 μg/ml) decreased the colocalization of NLRP3 with CASPASE-1 and NLRP3 with ASC in H. pylori-infected GES-1 cells observed by immunofluorescence microscopy; Pearson’s correlation coefficient was reduced. [1]

Patchouli alcohol reduced the release of IL-1β in infected cells, indicating inhibition of NLRP3 inflammasome activation. [1]

Patchouli alcohol (5-20 mg/kg; lateral; for 2 weeks) significantly protects the gastric mucosa from damage caused by Helicobacter pylori. Patchouli alcohol can effectively reduce oxidative stress by reducing the levels of intracellular ROS and MDA and increasing the levels of NP-SH and GSH/GSSG. Patchouli alcohol significantly inhibits the production of IL-1β, a cosmetic cell-forming chemoattractant, and IL-6 [1].

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In a C57BL/6 mouse model of H. pylori SS1-induced gastritis, oral administration of Patchouli alcohol (5, 10, 20 mg/kg) for 2 weeks significantly attenuated chronic inflammation in the gastric antrum and body as evaluated by HE staining (20 mg/kg PA, P<0.05 vs. model). [1]

Patchouli alcohol (5, 10, 20 mg/kg) restored CAT activity and NP-SH content in gastric tissue compared to model group. [1]

Patchouli alcohol (20 mg/kg) decreased MDA content and increased GSH/GSSG ratio in gastric tissue. [1]

Patchouli alcohol (5, 10, 20 mg/kg) significantly decreased serum levels of IL-1β, KC (IL-8), and IL-6 (P<0.01 or P<0.05). [1]

Patchouli alcohol (20 mg/kg) significantly protected the gastric mucosa from H. pylori-induced damage. MPO and SOD activities did not significantly change. [1]

Cell Viability Assay[1]
Cell Types: GES-1 cells treated with Helicobacter pylori
Tested Concentrations: 5 μg/mL, 10 μg/mL and 20 μg/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: Dramatically increased cell viability and diminished cell viability Lactate dehydrogenase (LDH) leakage.

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GES-1 cells were cultured in high-glucose DMEM with 10% FBS at 37°C in 5% CO2. Cells were passaged at 80% confluency. For treatment, GES-1 cells were co-cultured with H. pylori NCTC11637 at MOI=100:1 and treated with Patchouli alcohol (5, 10, 20 μg/ml) or DMSO (control and model) for 24 h. [1]

Cell viability was assessed by trypan blue exclusion: cells were collected after 24 h incubation and trypan blue-negative cells were counted. LDH leakage was measured from the supernatant according to kit instructions. [1]

Mitochondrial membrane potential (MMP) was measured using rhodamine 123: cells were incubated with 1 μM rhodamine 123 for 20 min, washed, and then fluorescence intensity was measured by fluorescence microscopy and flow cytometry (excitation 507 nm, emission 529 nm). [1]

Intracellular ROS was measured using DCFH-DA: cells were stained with DCFH-DA (1:1000) for 20 min, then fluorescence was measured by microscopy and flow cytometry (excitation 488 nm, emission 525 nm). [1]

Apoptosis was assessed by flow cytometry after staining with annexin V-FITC and propidium iodide: adherent cells were digested with 0.25% trypsin (without EDTA), resuspended in PBS (5×10^5 cells/500 μl), incubated with 5 μl annexin V-FITC for 20 min, then mixed with 5 μl PI and analyzed immediately. [1]

Pro-inflammatory factors (IL-6, TNF-α, MCP-1) in cell culture supernatant were measured by ELISA following the manufacturer’s instructions. [1]

Total RNA was extracted using Trizol reagent, reverse transcribed using PrimeScript RT reagent kit with gDNA eraser, and real-time PCR performed using SYBR Green with primers for NLRP3, CASPASE1, ASC, and GAPDH. Cycling conditions: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s and 60°C for 1 min. Results quantified by 2^-ΔΔCT method. [1]

Western blot: total protein extracted with RIPA buffer, centrifuged at 10,000g for 15 min at 4°C, protein concentration measured by BCA assay. Samples denatured with Laemmli buffer at 95°C for 5 min, separated by 12% SDS-PAGE, transferred to PVDF membrane. Primary antibodies: anti-TXNIP (1:1000), anti-NLRP3 (1:1000), anti-CASPASE-1 (1:5000), anti-ASC (1:5000), anti-β-ACTIN (1:1000). Secondary antibodies: anti-rabbit IgG (1:2000) or anti-mouse IgG (1:2000) for 2 h at room temperature. Bands detected and analyzed with Image J software. [1]

Immunofluorescence: GES-1 cells on coverslips fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, incubated with primary antibodies (anti-NLRP3 1:200, anti-CASPASE-1 1:200, anti-ASC 1:100), then with Alexa Fluor-488 and Alexa Fluor-555 conjugated secondary antibodies, visualized under confocal microscope. Colocalization analyzed by Pearson’s correlation coefficient using Image Pro Plus. [1]

Animal/Disease Models: Five to sixweeks old male C57BL/6 mice injected with Helicobacter pylori [1]
Doses: 5, 10 and 20 mg/kg
Route of Administration: Oral; continued for 2 weeks
Experimental Results: By reducing intracellular reactive oxygen species (ROS) and MDA, as well as increasing the levels of non-protein sulfhydryl (NP-SH), catalase and glutathione (GSH)/glutathione disulfide (GSSG), effectively alleviating oxidative stress. Excited.

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Male C57BL/6 mice (5-6 weeks old) were used. H. pylori SS1 strain was cultured in Columbia agar with 7% sheep blood or BHI with 10% FBS under 10% CO2, 5% O2, 85% N2 at 37°C for 48-72 h. For infection, logarithmic-phase bacteria (1.5×10^7 CFU/ml, 0.25 ml) were orally gavaged three times at 2-day intervals over 5 days. Mice were fasted for 12 h before gavage and resumed diet after 2 h. 2% salt was added to drinking water. After 1 week, infection was confirmed by PCR and rapid urease test. Mice were further fed for 10 weeks then randomly divided into groups: control (sterile BHI + poloxamer 407), model (H. pylori + poloxamer 407), triple therapy (omeprazole 400 μmol/kg/day + metronidazole 14.2 mg/kg/day + clarithromycin 7.15 mg/kg/day), and Patchouli alcohol (5, 10, 20 mg/kg) groups. [1]

Patchouli alcohol solid dispersion was prepared using poloxamer 407: PA and poloxamer 407 were mixed at ratio 1:5, melted at 70°C, then rapidly solidified by dropping into 10°C water. After 30 min, diluted water was added to final concentration. Oral administration was performed for 2 weeks. After treatment, mice were anesthetized, whole blood collected from orbit and centrifuged for serum. Stomach pylorus and antrum were isolated; one part fixed in 4% paraformaldehyde for HE and BAMB staining, the other part homogenized in Tris buffer (20 mM, pH 7.5) on ice, centrifuged at 12,000g at 4°C for 10 min, supernatant used for oxidative indices (CAT, GSH, MPO, NP-SH, SOD, MDA) measured by kits, protein concentration by Bradford method. Serum pro-inflammatory factors (IL-6, KC, IL-1β) detected by flow cytometry using CBA kit. [1]

The oral LD50 of Patchouli alcohol in mice was 4.693 g/kg, which is much higher than the effective doses used in this study (5-20 mg/kg). [1]

[1]. Unraveling the Novel Protective Effect of Patchouli Alcohol Against Helicobacter pylori-Induced Gastritis: Insights Into the Molecular Mechanism in vitro and in vivo. Front Pharmacol. 2018 Nov 22;9:1347.

Patchouli alcohol is a tricyclic carbon-3-cyclic compound and a tertiary sesquiterpene alcohol with the chemical formula tricyclic [5.3.1.0(3,8)]undecane-3-ol, with methyl groups substituted at positions 2, 2, 6, and 8 (1R,3R,6S,7S,8S-diastereomers). It is a tertiary alcohol, a sesquiterpene, and a tricyclic carbon-3-cyclic compound. Patchouli alcohol has been reported to be found in valerian (Valeriana fauriei), Celtic valerian (Valeriana celtica), and other organisms with relevant data.

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Patchouli alcohol has been used in Asian countries for hundreds of years as a component of Pogostemon cablin to clinically treat gastrointestinal diseases such as dyspepsia, gastritis, and ulcer. Previous research indicated that PA exerts specific anti-H. pylori effect and a wide range of antioxidant activities. It also has anti-inflammatory effects against xylene-induced oedema in mouse ear, carrageenan-induced oedema in rat paw, and H. pylori urease-induced cell cytotoxicity. The anti-inflammatory mechanism of PA involves inhibition of the NF-κB pathway and NLRP3 inflammasome activation signaling. PA also inhibits bacterial virulence factors such as urease. [1]

C15H26O

222.3663

222.198

5986-55-0

10955174

White to off-white solid

1.0±0.1 g/cm3

287.4±8.0 °C at 760 mmHg

56°; mp (racemate) 39-40° (Danishevsky, Dumas); mp 46-47° (Mirrington, Schmalzl)

120.2±10.9 °C

0.0±1.3 mmHg at 25°C

1.515

4.73

1

1

0

16

321

5

O([H])[C@@]12C([H])([H])C([H])([H])[C@]([H])(C([H])([H])[H])[C@]3([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[C@]13C([H])([H])[H])C2(C([H])([H])[H])C([H])([H])[H]

GGHMUJBZYLPWFD-CUZKYEQNSA-N

InChI=1S/C15H26O/c1-10-5-8-15(16)13(2,3)11-6-7-14(15,4)12(10)9-11/h10-12,16H,5-9H2,1-4H3/t10-,11+,12-,14-,15+/m0/s1

(1R,3R,6S,7S,8S)-2,2,6,8-tetramethyltricyclo[5.3.1.03,8]undecan-3-ol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~449.70 mM)
Ethanol : ~20 mg/mL (~89.94 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (28.11 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 62.5 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: ≥ 6.25 mg/mL (28.11 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 62.5 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: ≥ 6.25 mg/mL (28.11 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2 mg/mL (8.99 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2 mg/mL (8.99 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH 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 6: ≥ 2 mg/mL (8.99 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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