Flavoring, perfume additives

Under starvation conditions, sabinene (10-300 μM; 18 hours) induces a significant restoration of myotube diameter reduction [2]. In starved myotubes, sabinene (300 μM; 18 hr) sever extracellular ligases, reduces elevated E3 ubiquitin ligase ring finger protein-1 (MuRF-1) expression, and phosphorylates Signal regulatory switch 1/2 (ERK1/2) [2]. Sabinene (180 hours at 300 μM)

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Sabinene, at concentrations ranging from 10 to 300 µM, did not affect the viability of differentiated L6 myotubes. However, at higher concentrations of 1000 and 2000 µM, it inhibited myotube viability. [2]
- In a starvation-induced atrophy model (L6 myotubes incubated in serum-free DMEM for 18 hours), treatment with sabinene at 10-300 µM inhibited the decrease in myotube diameter in a dose-dependent manner. The maximum inhibitory effect was observed at 300 µM, where the myotube diameter was approximately two times greater than that in starved myotubes without sabinene treatment. [2]
- Sabinene (300 µM) treatment for 18 hours in starved L6 myotubes diminished the elevated protein expression of the E3 ubiquitin ligase MuRF-1. [2]
- Sabinene (300 µM) treatment for 18 hours in starved L6 myotubes attenuated the increased phosphorylation levels of p38 MAPK and ERK1/2. [2]
- Sabinene (300 µM) treatment for 18 hours in starved L6 myotubes inhibited the upregulation of intracellular reactive oxygen species (ROS) levels, as shown by both fluorescence microscopy and fluorometric analysis. [2]

By modifying ROS-mediated activation of MAPK/MuRF-1 starvation in starved myotubes, sabinene (6.4 mg/kg; oral; once daily; during a two-day fasting) reduces the likelihood of eventual muscle atrophy and may even reverse reduced amputation.

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Chrysanthemum boreale Makino essential oil (CBMEO) has diverse biological activities including a skin regenerating effect. However, its role in muscle atrophy remains unknown. This study explored the effects of CBMEO and its active ingredients on skeletal muscle atrophy using in vitro and in vivo models of muscle atrophy. CBMEO reversed the size decrease of L6 myoblasts under starvation. Among the eight monoterpene compounds of CBMEO without cytotoxicity for L6 cells, sabinene induced predominant recovery of reductions of myotube diameters under starvation. Sabinene diminished the elevated E3 ubiquitin ligase muscle ring-finger protein-1 (MuRF-1) expression and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations in starved myotubes. Moreover, sabinene decreased the increased level of reactive oxygen species (ROS) in myotubes under starvation. The ROS inhibitor antagonized expression of MuRF-1 and phosphorylation of MAPKs, which were elevated in starved myotubes. In addition, levels of muscle fiber atrophy and MuRF-1 expression in gastrocnemius from fasted rats were reduced after administration of sabinene. These findings demonstrate that sabinene, a bioactive component from CBMEO, may attenuate skeletal muscle atrophy by regulating the activation mechanism of ROS-mediated MAPK/MuRF-1 pathways in starved myotubes, probably leading to the reverse of reduced muscle fiber size in fasted rats.[2]

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In a fasting-induced muscle atrophy rat model (8-week-old male SD rats fasted for 2 days), oral administration of sabinene (6.4 mg/kg body weight, once daily for 2 days) reversed the decrease in gastrocnemius muscle fiber cross-sectional area caused by fasting. [2]
- The same administration of sabinene (6.4 mg/kg body weight, once daily for 2 days) in fasted rats resulted in increased gastrocnemius muscle weights, which were decreased in fasted animals (data not shown in the provided text, but mentioned). [2]
- Sabinene administration (6.4 mg/kg body weight, once daily for 2 days) decreased the elevated MuRF-1 expression levels in the gastrocnemius muscles of fasted rats. [2]

cytotoxicity assay [2]
Cell Types: IL-6 Myoblast
Tested Concentrations: 300 μM
Incubation Duration: 18 and 48 hrs (hours)
Experimental Results: Failed to affect reactive oxygen species (ROS) in myotubes under starvation conditions levels increase[2]. IL-6 myoblast viability after 48 hrs (hours) of culture restored more than 50% of starvation-induced myoblast atrophy after 18 hrs (hours).

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Western Blot Analysis[2]
Cell Types: IL-6 Myoblasts
Tested Concentrations: 300 μM
Incubation Duration: 18 hrs (hours)
Experimental Results: Phosphorylation of muscle atrophy-related signaling proteins is diminished in starved myotubes.

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Cell Viability Assay: L6 myoblast cells were seeded into 96-well plates. For myotube viability analysis, L6 myoblasts were differentiated for 7 days. Differentiated myotubes were then incubated with sabinene at 37°C for 48 hours. Cell viability was measured using an XTT assay. XTT solution was added, and after a 2-hour incubation to allow formazan dye formation, the absorbance was measured at 450 nm. [2]
- Cell Atrophy Model & Morphologic Analysis: L6 myoblasts were differentiated into myotubes over 7 days. To induce atrophy, myotubes were incubated in serum-free low-glucose DMEM in the presence or absence of sabinene (10-300 µM) for 18 hours. Myotubes were fixed, blocked, and incubated with anti-MYH-2 primary antibody, followed by Alexa Fluor 488-conjugated secondary antibody, and stained with DAPI. Images were captured with a fluorescence microscope. Myotube diameters were measured from five points along the length of each myotube using ImageJ software. The average diameter was calculated and expressed as a percentage relative to the control group. [2]
- Western Blot Analysis: L6 myotubes were starved in serum-free DMEM with or without sabinene (300 µM) for 18 hours. Cells were lysed, and proteins were separated by SDS-PAGE, then transferred to membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies against MuRF-1, p-p38 MAPK, p38 MAPK, p-ERK1/2, ERK1/2, and β-actin. After incubation with HRP-conjugated secondary antibodies, protein bands were visualized using chemiluminescence and quantified with ImageJ software. [2]
- Reactive Oxygen Species (ROS) Measurement: L6 myotubes, differentiated in 24-well or 96-well plates, were starved in serum-free DMEM with or without sabinene (300 µM) for 18 hours. Cells were then stained with H2DCFDA for 30 minutes. Fluorescence was visualized using an inverted fluorescence microscope for image analysis, and quantified using a microplate reader at excitation/emission wavelengths of 488/519 nm for fluorometric analysis. [2]

Animal/Disease Models: Rat fasted animal model [2]
Doses: 6.4 mg/kg
Route of Administration: po (oral gavage); fasted fiber size [2]. one time/day; fasted for 2 days.
Experimental Results: diminished gastrocnemius muscle fiber atrophy levels and MuRF-1 expression in fasted rats.

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Fasting-Induced Muscle Atrophy Model & Drug Administration:** Male Sprague-Dawley rats (8-week-old, 250-300 g) were used. Animals were divided into three groups (n=10 per group). The sabinene-treated group was orally administered sabinene (6.4 mg/kg body weight) once daily for 2 days. The vehicle control groups (fasted and non-fasted) were orally treated with a saline solution containing 1% Tween-80. Food pellets were removed from the cages of the fasted groups (both sabinene-treated and vehicle control) for the 2-day fasting period. [2]
- **Tissue Collection and Processing:** After 2 days, rats were anesthetized intraperitoneally with Zoletil (40 mg/kg body weight) and Rompun (10 mg/kg body weight). Adequacy of anesthesia was confirmed by lack of reflex response to foot pinching. Gastrocnemius muscles were dissected and isolated. The isolated muscles were washed with ice-cold PBS, fixed in 4% paraformaldehyde, segmented, and embedded in paraffin. [2]

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Fasting-Induced Muscle Atrophy Model & Drug Administration: Male Sprague-Dawley rats (8-week-old, 250-300 g) were used. Animals were divided into three groups (n=10 per group). The sabinene-treated group was orally administered sabinene (6.4 mg/kg body weight) once daily for 2 days. The vehicle control groups (fasted and non-fasted) were orally treated with a saline solution containing 1% Tween-80. Food pellets were removed from the cages of the fasted groups (both sabinene-treated and vehicle control) for the 2-day fasting period. [2]
- Tissue Collection and Processing: After 2 days, rats were anesthetized intraperitoneally with Zoletil (40 mg/kg body weight) and Rompun (10 mg/kg body weight). Adequacy of anesthesia was confirmed by lack of reflex response to foot pinching. Gastrocnemius muscles were dissected and isolated. The isolated muscles were washed with ice-cold PBS, fixed in 4% paraformaldehyde, segmented, and embedded in paraffin. [2]

In vitro cytotoxicity: Sabinene did not affect the viability of L6 myoblasts at a concentration of 300 µM. In differentiated L6 myotubes, sabinene did not affect viability at concentrations of 10, 30, 100, and 300 µM. Cytotoxicity was observed in myotubes at higher concentrations of 1000 and 2000 µM. [2]

[1]. Biosynthesis and production of sabinene: current state and perspectives. Appl Microbiol Biotechnol. 2018 Feb;102(4):1535-1544.

[2]. Sabinene Prevents Skeletal Muscle Atrophy by Inhibiting the MAPK-MuRF-1 Pathway in Rats. Int J Mol Sci. 2019 Oct 8;20(19):4955.

Sabinene is a thujene-type bicyclic monoterpene that can be isolated from the essential oils of various plants. It is a plant metabolite. Sabinene has been reported in hops (Humulus lupulus), Serbian anise (Pimpinella serbica), and other organisms with relevant data. See also: black pepper (note moved to); carrot seed oil (note moved to); nutmeg (note moved to)... Sabinene is an important natural bicyclic monoterpene that can be used as a flavoring agent, spice additive, fine chemical, and advanced biofuel. Currently, this valuable terpene has not been commercialized due to the lack of suitable production processes. Microbial synthesis may be a promising route for Sabinene production. This article reviews the metabolic pathways and key enzymes involved in Sabinene biosynthesis and highlights recent advances in the production of Sabinene using microbial fermentation. In these studies, researchers have identified general synthetic pathways for Sabinene from simple intermediate metabolites. Sabinene synthases from different sources have also been cloned and characterized. In addition, heterologous systems of model microorganisms Escherichia coli and Saccharomyces cerevisiae were constructed to produce sine. This review also proposes new directions and attempts in order to gain some insights into the industrial production of sine. Combining traditional molecular biology with new genomics and proteomics analysis tools will help to better understand the biosynthesis of sine and improve the potential of microbial production. [1]

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Source: Sabinene is a monoterpene compound identified as a bioactive component of Chrysanthemum boreale Makino essential oil (CBMEO). [2]
- Background Biological Activities: Accumulated evidence indicates that sabinene has potential therapeutic applications for various diseases due to its biological properties, including anti-fungal, anti-inflammatory, and antioxidant activities. It has been shown to possess anti-radical activity against DPPH radicals. [2]
- Proposed Mechanism in This Study: The study suggests that sabinene attenuates skeletal muscle atrophy by regulating the ROS-mediated MAPK/MuRF-1 pathway. It inhibits ROS generation, which in turn reduces the phosphorylation of p38 MAPK and ERK1/2, leading to decreased expression of the E3 ubiquitin ligase MuRF-1 and ultimately preventing muscle fiber size reduction. [2]
- Potential Therapeutic Application: The findings imply that sabinene and CBMEO may be promising agents with therapeutic potential for treating or preventing disorders related to skeletal muscle atrophy, such as those caused by cachexia, malnutrition, denervation, bedding, and aging. [2]

C10H16

136.23

136.125

C, 88.16; H, 11.84

3387-41-5

18818

Colorless to light yellow liquid

0.9±0.1 g/cm3

164.0±0.0 °C at 760 mmHg

36.7±0.0 °C

2.6±0.1 mmHg at 25°C

1.484

4.13

0

0

1

10

179

0

CC(C12CCC(C1C2)=C)C

NDVASEGYNIMXJL-UHFFFAOYSA-N

InChI=1S/C10H16/c1-7(2)10-5-4-8(3)9(10)6-10/h7,9H,3-6H2,1-2H3

4-methylidene-1-propan-2-ylbicyclo[3.1.0]hexane

SABINENE; 3387-41-5; Sabinen; 4(10)-Thujene; 4-methylidene-1-(propan-2-yl)bicyclo[3.1.0]hexane; 1-Isopropyl-4-methylenebicyclo[3.1.0]hexane; Bicyclo[3.1.0]hexane, 4-methylene-1-(1-methylethyl)-; CHEBI:50027;

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 : ~33.33 mg/mL (~244.66 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.)