Endogenous Metabolite

The development of the baby brain's growth and functionality depends on docosahexaenoic acid (DHA). Adults' regular brain function also requires DHA. Memory and learning capacity can be enhanced by consuming a diet high in DHA [1]. Every stage of brain development, including nerve cell proliferation, migration, differentiation, and synaptogenesis, depends fundamentally on DHA. Due to its distinct structure and numerous double bonds, DHA can impart specific membrane characteristics that are beneficial for cell signaling. Reduced DHA levels have been linked to a number of developmental diseases, including dyslexia, autism spectrum disorder, attention deficit hyperactivity disorder, schizophrenia, and others [2]. Strong RXR activation can be induced at low micromolar concentrations by the powerful RXR ligand DHA. RXRα activation by DHA has an EC50 of 5–10 μM fatty acid [3].

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Recently, evidence was provided for the existence of a different natural RXR ligand in mouse brain, the highly enriched polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) (Mata de Urquiza et al. (2000) Science 290, 2140-2144). However, the results suggested that supra-physiological levels of DHA were required for efficient RXR activation. Using a refined method for ligand addition to transfected cells, the current study shows that DHA is a more potent RXR ligand than previously observed, inducing robust RXR activation already at low micromolar concentrations. Furthermore, it is shown that other naturally occurring PUFAs can activate RXR with similar efficiency as DHA. In additional experiments, the binding of fatty acid ligands to RXRalpha is directly demonstrated by electrospray mass spectrometry of the noncovalent complex between the RXR ligand-binding domain (LBD) and its ligands. Data is presented that shows the noncovalent interaction between the RXR LBD and a number of PUFAs including DHA and arachidonic acid, corroborating the results in transfected cells[3].

Over the course of ten weeks, taking docosahexaenoic acid dramatically raised the amount of docosahexaenoic acid in the cerebral cortex and hippocampus and significantly decreased the number of reference memory errors without increasing the number of working memory errors. ratio of dodecahexaenoic acid to arachidonic acid[4]. In experimental mice models of Parkinson's disease, DHA therapy has neuroprotective effects. The brains of MPTP mice show a tendency toward slightly lower levels of lipid oxidation [5].

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Wistar rats were fed a fish oil-deficient diet through three generations. The young (five-week-old) male rats of the third generation were randomly divided into two groups. Over 10 weeks, one group was perorally administered docosahexaenoic acid/DHA dissolved in 5% gum Arabic solution at 300 mg/kg/day; the other group received a similar volume of vehicle alone. Five weeks after starting the administration, the rats were tested for learning ability related to two types of memory, reference memory and working memory, with the partially (four of eight) baited eight-arm radial maze. Reference memory is information that should be retained until the next trial. Working memory is information that disappears in a short time. Entries into unbaited arms and repeated entries into visited arms were defined as reference memory errors and working memory errors, respectively. Docosahexaenoic acid administration over 10 weeks significantly reduced the number of reference memory errors, without affecting the number of working memory errors, and significantly increased the docosahexaenoic acid content and the docosahexaenoic acid/arachidonic acid ratio in both the hippocampus and the cerebral cortex. In addition, the ratio demonstrated a significantly negative correlation with the number of reference memory errors. These results suggest that chronic administration of docosahexaenoic acid is conducive to the improvement of reference memory-related learning ability, and that the docosahexaenoic acid/arachidonic acid ratio in the hippocampus or the cerebral cortex, or both, may be an indicator of learning ability.[4]

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This study aimed to investigate the effects of docosahexaenoic acid (DHA) on the oxidative stress that occurs in an experimental mouse model of Parkinson's disease (PD). An experimental model of PD was created by four intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (4 × 20 mg/kg, at 12h intervals). Docosahexaenoic acid was given daily by gavage for 4 weeks (36 mg/kg/day). The motor activity of the mice was evaluated via the pole test, and the dopaminergic lesion was determined by immunohistochemical analysis for tyrosine hydroxylase (TH)-immunopositive cells. The activity of antioxidant enzymes in the brain were determined by spectrophotometric assays and the concentration of thiobarbituric acid-reactive substances (TBARS) were measured as an index of oxidative damage. The number of apoptotic dopaminergic cells significantly increased in MPTP-treated mice compared to controls. Although DHA significantly diminished the number of cell deaths in MPTP-treated mice, it did not improve the decreased motor activity observed in the experimental PD model. Docosahexaenoic acid significantly diminished the amount of cell death in the MPTP+DHA group as compared to the MPTP group. TBARS levels in the brain were significantly increased following MPTP treatment. Glutathione peroxidase (GPx) and catalase (CAT) activities of brain were unaltered in all groups. The activity of brain superoxide dismutase (SOD) was decreased in the MPTP-treated group compared to the control group, but DHA treatment did not have an effect on SOD activity in the MPTP+DHA group. Our current data show that DHA treatment exerts neuroprotective actions on an experimental mouse model of PD. There was a decrease tendency in brain lipid oxidation of MPTP mice but it did not significantly [5].

Lipid Extraction and ES Analysis of Ligands Bound to RXRα LBD—[3]
Desalted protein fractions from the affinity capture experiments above were thawed, and bound lipids were extracted using Lipidex-1000 gel. Before use, Lipidex-1000 was washed with: 1) 50% methanol, 2) 100% methanol, and 3) water, and then stored as a 50% (v/v) slurry in water. For each extraction, 300 μl of Lipidex-1000 slurry was transferred to a 1.5-ml Eppendorf tube, and excess water was removed after centrifugation. One milliliter of the thawed desalted protein fraction from the affinity capture experiment above and 1432.2 ng of [14C]DHA in 2 μl were added to the gel bed ([14C]DHA in ethanol, specific activity 55 mCi/mmol). The concentration of [14C]DHA was determined by counting 1 μl of the ethanol solution and calculated to be 716.1 ng/μl using the specific activity value. The slurry was acidified to pH 2–3 using acetic acid and incubated for 30 min at 37 °C. The supernatant was removed, and the Lipidex bed was washed with 1.2 ml of water, which was discarded. The lipids bound to the Lipidex bed were extracted using 3 × 0.5 ml of methanol. The methanol phase was evaporated under a stream of nitrogen, and the residue was reconstituted in 0.1 ml of methanol. The resulting sample was analyzed by negative ion nano-ES mass spectrometry. The amount of unlabelled extracted DHA was calculated from the intensity ratio of unlabelled-to-labeled DHA ions (i.e. m/z 327/329, DHA [M-H]−: m/z 327 and [14C]DHA [M-H]−: m/z 329) and a knowledge of the amount of [14C]DHA added (1432.2 ng). The m/z 327/329 intensity ratio was corrected for the contribution of the 327 + 2 isotope ion to the intensity of the 329 ion and for unlabelled DHA in the [14C]DHA standard using the following equation:
where RS is the ratio of unlabelled (327) to labeled (329) ions in the sample, RL is the ratio of unlabelled to labeled ions in the labeled standard (measured to be 0.167), and RU is the ratio of “labeled” (i.e. the [M-H]− + 2 isotope ion) to unlabelled ions for the unlabelled reference compound (theoretically calculated for DHA to be 0.0264).
The amounts of oleic and arachidonic acids extracted were determined from the relative intensity of their respective deprotonated molecules (m/z 281 and 303, respectively) as compared with deprotonated DHA. To determine the fatty acid composition of the original brain-conditioned medium, 1 ml of brain-conditioned medium was extracted using the Lipidex-1000 procedure described above. The ions at m/z 281, 303, and 327 were identified by low-energy collision-induced dissociation of their respective dilithiated adducts ([M-H+2Li]+).

Docosahexaenoic acid administration [4]
G3 male rats were weaned on to the F-1® and randomly divided into two groups of eight rats each. One group (DHA group) was perorally administered DHA-95E emulsified in 5% gum Arabic solution at 300 mg/kg/day; the other group (control group) was administered a similar volume of vehicle alone. The administration was started when the G3 rats were five weeks of age and maintained until all the experiments ended.

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The eight-arm radial maze task [4]
An eight-arm radial maze, used for the estimation of learning ability, set at 60 cm elevation above floor level, consisted of an octagonal center platform surrounded by eight equally spaced radial arms (50 cm long×11 cm wide). Food cups, 1.5 cm deep and 2.5 cm in diameter, were located at the end of each arm. The maze was placed in a closed room with a number of visual cues: fluorescent ceiling lights, curtained door and windows, a chair for the observer and some boxes. The experimenter maintained a constant position beside the maze and observed the behavior of the rats.
Three weeks after starting the DHA administration, each rat was placed on a limited-food schedule designed to maintain weight at 80–85% of the free-feeding weight and was handled 5 min daily for five consecutive days. Then, for five days, the rats were familiarized with the apparatus, where 45 mg of reward pellets (made with F-1®) was scattered throughout the maze.

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Experimental design [5]
Mice were randomly divided into four experimental groups as follows: control (n = 15); DHA-treated (DHA) (n = 15); MPTP-injected (MPTP) (n = 15), and DHA-treated + MPTP-injected (MPTP + DHA) (n = 15). DHA/Docosahexaenoic acid was dissolved in corn oil at a concentration of 0.046 M and was given to the treatment groups for 30 days (36 mg/kg/day) by gavage (Hacioglu et al., 2006, Hacioglu et al., 2007, Kremer et al., 1990, Simopoulos, 1989, Tanriover et al., 2010). To eliminate the effects of a daily gavage and vehicle, other groups received a similar volume of corn oil alone. Food and water were provided ad libitum throughout the experiments.

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Treatment of mice with MPTP [5]
On the 23rd day of gavage treatment, animals in the MPTP and MPTP + DHA groups received intraperitoneal (i.p.) injections of either freshly prepared 20 mg/kg MPTP hydrochloride dissolved in saline or an equivalent volume of saline (pH 7.4) every 12 h with a total of four doses (Date et al., 1990). All four animal groups continued on their normal diets for an additional week after treatment.

Absorption, Distribution and Excretion
Like other omega-3 fatty acids, DHA is hydrolyzed from the intestines and delivered through the lymphatic circulation. Plasma DHA concentrations increase in a dose-dependent and saturable manner.
DHA is the most abundant n−3 fatty acid in membranes and is present in all organs. It is also the most variable among organs and is particularly abundant in neural tissue, such as brain and retina, where it is several hundred-fold more abundant than EPA.

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Metabolism / Metabolites
DHA can be metabolized into DHA-derived specialized pro-resolving mediators (SPMs), DHA epoxides, electrophilic oxo-derivatives (EFOX) of DHA, neuroprostanes, ethanolamines, acylglycerols, docosahexaenoyl amides of amino acids or neurotransmitters, and branched DHA esters of hydroxy fatty acids, among others. It is converted to 17-hydroperoxy-DHA derivatives via COX-2 and 15-LOX and 5-LOX activity. These derivatives are further converted into D-series resolvins and protectins with potent anti-inflammatory potential and potent neuroprotective effect. DHA may also be metabolized to 19,20-epoxydocosapentaenoic acids (EDPs) and isomers via CYP2C9 activity. Epoxy metabolites are reported to mediate anti-tumor activity by inhibiting angiogenesis, tumor growth, and metastasis.

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Biological Half-Life
Approximately 20 hours.

[1]. Health benefits of docosahexaenoic acid (DHA). Pharmacol Res. 1999 Sep;40(3):211-25.

[2]. Essential role of docosahexaenoic acid towards development of a smarter brain. Neurochem Int. 2015 Oct;89:51-62.

[3]. Polyunsaturated fatty acids including docosahexaenoic and arachidonic acid bind to the retinoid Xreceptor alpha ligand-binding domain. Mol Cell Proteomics. 2004 Jul;3(7):692-703.

[4]. Chronic administration of docosahexaenoic acid improves reference memory-related learning ability in young rats. Neuroscience. 1999;93(1):237-41.

[5]. The influence and the mechanism of docosahexaenoic acid on a mouse model of Parkinson's disease. Neurochem Int. 2011 Oct;59(5):664-70.

All-cis-docosa-4,7,10,13,16,19-hexaenoic acid is a docosahexaenoic acid having six cis-double bonds at positions 4, 7, 10, 13, 16 and 19. It has a role as a nutraceutical, an antineoplastic agent, a human metabolite, a Daphnia tenebrosa metabolite, a mouse metabolite and an algal metabolite. It is a docosahexaenoic acid and an omega-3 fatty acid. It is a conjugate acid of a (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate.
A mixture of fish oil and primrose oil, doconexent is used as a high-docosahexaenoic acid (DHA) supplement. DHA is a 22 carbon chain with 6 cis double bonds with anti-inflammatory effects. It can be biosythesized from alpha-linolenic acid or commercially manufactured from microalgae. It is an omega-3 fatty acid and primary structural component of the human brain, cerebral cortex, skin, and retina thus plays an important role in their development and function. The amino-phospholipid DHA is found at a high concentration across several brain subcellular fractions, including nerve terminals, microsomes, synaptic vesicles, and synaptosomal plasma membranes.
Docosahexaenoic acid has been reported in Homo sapiens, Sarcophyton trocheliophorum, and other organisms with data available.
Doconexent is a polyunsaturated very long-chain fatty acid with a 22-carbon backbone and 6 double bonds, originating from the 3rd, 6th, 9th, 12th, 15th and 18th positions from the methyl end.
Docosahexaenoic Acid is a polyunsaturated very long-chain fatty acid with a 22-carbon backbone and 6 double bonds. Four separate isomers can be called by this name.
C22-unsaturated fatty acids found predominantly in FISH OILS.
See also: Fish Oil (is active moiety of); Cod Liver Oil (part of); Krill oil (part of) ... View More ...

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Drug Indication
Used as a high-docosahexaenoic acid (DHA) oral supplement.
Treatment of Retinitis Pigmentosa

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Mechanism of Action
DHA and its conversion to other lipid signalling moleccules compete with the arachidonic acid cascade from endogenous phospholipids and shift the inflammatory state to being more anti-inflammatory. DHA inhibits endotoxin-stimulated production of IL-6 and IL-8 in human endothelial cells. Derivatives of DHA are anti-inflammatory lipid mediators. Lipid mediators resolvin D1 and protectin D1 all inhibit transendothelial migration of neutrophils, so preventing neutrophilic infiltration at sites of inflammation, resolvin D1 inhibits IL-1β production, and protectin D1 inhibits TNF and IL-1β production. Monoxydroxy derivative of DHA converted by LOX inhibit thromboxane-induced platelet aggregation. DHA supplementation has also shown to reduce the levels of serum C-reactive protein (CRP) and other circulating markers of inflammation such as neutrophils in hypertriglyceridemic men. DHA acts as a ligand at peroxisome proliferator-activated receptor (PPAR) gamma and alpha that regulate lipid signalling molecule-mediated transduction pathways and modulate inflammation. As a natural ligand, DHA induces a protective effect in retinal tissues by activating retinoid x receptors and subsequent ERK/MAPK signaling pathway in photoreceptors to promote their survival and differentiation, stimulating the expression of antiapoptotic proteins such as Bcl-2 and preserving mitochondrial membrane potential.

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Pharmacodynamics
DHA in the central nervous system is found in the phospholipid bilayers where it modulates the physical environment and increase the free volume within the membrane bilayer. It influences the G-protein coupled receptor activity and affects transmembrane transport and cell interaction with the exterior world. It is also reported to promote apoptosis, neuronal differentiation and ion channel activity. Like other polyunsaturated fatty acids, DHA acts as a ligand at PPARs that plays an anti-inflammatory effect and regulate inflammatory gene expression and NFκB activation. DHA also gives rise to resolvins and related compounds (e.g., protectins) through pathways involving cyclooxygenase and lipoxygenase enzymes to resolve the inflammatory responses.

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Docosahexaenoic acid (DHA) is essential for the growth and functional development of the brain in infants. DHA is also required for maintenance of normal brain function in adults. The inclusion of plentiful DHA in the diet improves learning ability, whereas deficiencies of DHA are associated with deficits in learning. DHA is taken up by the brain in preference to other fatty acids. The turnover of DHA in the brain is very fast, more so than is generally realized. The visual acuity of healthy, full-term, formula-fed infants is increased when their formula includes DHA. During the last 50 years, many infants have been fed formula diets lacking DHA and other omega-3 fatty acids. DHA deficiencies are associated with foetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, and adrenoleukodystrophy. Decreases in DHA in the brain are associated with cognitive decline during aging and with onset of sporadic Alzheimer disease. The leading cause of death in western nations is cardiovascular disease. Epidemiological studies have shown a strong correlation between fish consumption and reduction in sudden death from myocardial infarction. The reduction is approximately 50% with 200 mg day(-1)of DHA from fish. DHA is the active component in fish. Not only does fish oil reduce triglycerides in the blood and decrease thrombosis, but it also prevents cardiac arrhythmias. The association of DHA deficiency with depression is the reason for the robust positive correlation between depression and myocardial infarction. Patients with cardiovascular disease or Type II diabetes are often advised to adopt a low-fat diet with a high proportion of carbohydrate. A study with women shows that this type of diet increases plasma triglycerides and the severity of Type II diabetes and coronary heart disease. DHA is present in fatty fish (salmon, tuna, mackerel) and mother's milk. DHA is present at low levels in meat and eggs, but is not usually present in infant formulas. EPA, another long-chain n-3 fatty acid, is also present in fatty fish. The shorter chain n-3 fatty acid, alpha-linolenic acid, is not converted very well to DHA in man. These longchain n-3 fatty acids (also known as omega-3 fatty acids) are now becoming available in some foods, especially infant formula and eggs in Europe and Japan. Fish oil decreases the proliferation of tumour cells, whereas arachidonic acid, a longchain n-6 fatty acid, increases their proliferation. These opposite effects are also seen with inflammation, particularly with rheumatoid arthritis, and with asthma. DHA has a positive effect on diseases such as hypertension, arthritis, atherosclerosis, depression, adult-onset diabetes mellitus, myocardial infarction, thrombosis, and some cancers. [1]

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Evolution of the high order brain function in humans can be attributed to intake of poly unsaturated fatty acids (PUFAs) of which the ω-3 fatty acid, docosahexaenoic acid (DHA) has special significance. DHA is abundantly present in the human brain and is an essential requirement in every step of brain development like neural cell proliferation, migration, differentiation, synaptogenesis etc. The multiple double bonds and unique structure allow DHA to impart special membrane characteristics for effective cell signaling. Evidences indicate that DHA accumulate in areas of the brain associated with learning and memory. Many development disorders like dyslexia, autism spectrum disorder, attention deficit hyperactivity disorder, schizophrenia etc. are causally related to decreased level of DHA. The review discusses the various reports of DHA in these areas for a better understanding of the role of DHA in overall brain development. Studies involving laboratory animals and clinical findings in cases as well as during trials have been taken into consideration. Additionally the currently available dietary source of DHA for supplementation as nutraceutics with general caution for overuse has been examined.[2]

C₂₂H₃₂O₂

328.4883

328.24

6217-54-5

Docosahexaenoic acid-d5;1197205-71-2;Docosahexaenoic acid-13C22

445580

Colorless to light yellow liquid

0.9±0.1 g/cm3

446.7±24.0 °C at 760 mmHg

-44ºC

343.4±18.0 °C

0.0±2.3 mmHg at 25°C

1.521

6.78

1

2

14

24

462

0

O([H])C(C([H])([H])C([H])([H])/C(/[H])=C(/[H])\C([H])([H])/C(/[H])=C(/[H])\C([H])([H])/C(/[H])=C(/[H])\C([H])([H])/C(/[H])=C(/[H])\C([H])([H])/C(/[H])=C(/[H])\C([H])([H])/C(/[H])=C(/[H])\C([H])([H])C([H])([H])[H])=O

MBMBGCFOFBJSGT-KUBAVDMBSA-N

InChI=1S/C22H32O2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20-21-22(23)24/h3-4,6-7,9-10,12-13,15-16,18-19H,2,5,8,11,14,17,20-21H2,1H3,(H,23,24)/b4-3-,7-6-,10-9-,13-12-,16-15-,19-18-

cis-4,7,10,13,16,19-Docosahexaenoic acid

DHA; Cervonic Acid; Docosahexaenoic acid; Doconexent; Doconexento; Doconexentum; Doxonexent; AquaGrow Advantage; Martek DHA HM; 6217-54-5; Doconexent

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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 (~304.42 mM)
Ethanol : ~50 mg/mL (~152.21 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (15.22 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 50.0 mg/mL clear EtOH 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: 5 mg/mL (15.22 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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 3: ≥ 5 mg/mL (15.22 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 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (7.61 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 5: ≥ 2.5 mg/mL (7.61 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 6: ≥ 2.5 mg/mL (7.61 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 7: ≥ 2.5 mg/mL (7.61 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 8: 2.5 mg/mL (7.61 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 9: 33.33 mg/mL (101.46 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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