Natural product

D-pinitol stimulates p53 and Bax while suppressing NF-κB and Bcl-2 to induce MCF-7 cell apoptosis [3].

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Development of drugs from natural products has been undergoing a gradual evoluation. Many plant derived compounds have excellent therapeutic potential against various human ailments. They are important sources especially for anticancer agents. A number of promising new agents are in clinical development based on their selective molecular targets in the field of oncology. D-pinitol is a naturally occurring compound derived from soy which has significant pharmacological activitites. Therefore we selected D-pinitol in order to evaluate apoptotic potential in the MCF-7 cell line. Human breast cancer cells were treated with different concentrations of D-pinitol and cytotoxicity was measured by MTT and LDH assays. The mechanism of apoptosis was studied with reference to expression of p53, Bcl-2, Bax and NF-kB proteins. The results revealed that D-pinitol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, while upregulating the expression of p53, Bax and down regulating Bcl-2 and NF-kB. Thus the results obtained in this study clearly vindicated that D-pinitol induces apotosis in MCF-7 cells through regulation of proteins of pro- and anti-apoptotic cascades.[3]

D-pinitol, a compound isolated from Pinaceae and Leguminosae plants, has been reported to possess insulin-like properties. Although the hypoglycemic activity of D-pinitol was recognized in recent years, the molecular mechanism of D-pinitol in the treatment of diabetes mellitus remains unclear. In this investigation, a model of type 2 diabetes mellitus (T2DM) with insulin resistance was established by feeding a high-fat diet (HFD) and injecting streptozocin (STZ) to Sprague-Dawley (SD) rats, targeting the exploration of more details of the mechanism in the therapy of T2DM. D-pinitol was administrated to the diabetic rats as two doses [30, 60 mg/(kg·body weight·day)]. The level of fasting blood glucose (FBG) was decreased 12.63% in the high-dosage group, and the ability of oral glucose tolerance was improved in D-pinitol-treated groups. The biochemical indices revealed that D-pinitol had a positive effect on hypoglycemic activity. Western boltting suggested that D-pinitol could promote the expression of the phosphatidylinositol-3-kinase (PI3K) p85, PI3Kp110, as well as the downstream target protein kinase B/Akt (at Ser473). Besides, D-pinitol inhibited the expression of glycogen synthesis kinase-3β (GSK-3β) protein and regulated the expression of glycogen synthesis (GS) protein and then accelerated the glycogen synthesis. Above all, D-pinitol played a positive role in regulating insulin-mediated glucose uptake in the liver through translocation and activation of the PI3K/Akt signaling pathway in T2DM rats.[1]

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D-pinitol is a cyclitol present in several edible plant species and extensively investigated for the treatment of metabolic diseases in humans, as food supplement, and demonstrated protective effects in the cardiovascular system. For these reasons, the present work aimed at investigating the mechanisms involved in the vascular effects of D-pinitol in mouse mesenteric artery. Mesenteric arteries from male C57BL/6 mice were mounted in a wire myograph. Nitrite was measured by the 2,3-diaminonaphthalene (DAN) method. Protein expression and phosphorylation were measured by Western blot. The systolic blood pressure (SBP) was measured by tail-cuff plethysmography. D-pinitol induced a concentration-dependent vasodilatation in endothelium-intact, but not in endothelium-denuded arteries. Nω-Nitro-L-arginine methyl ester (300 μM) abolished the effect of D-pinitol, while 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM) shifted the concentration-response curve to the right. KN-93 (1 μM) blunted the vasodilator effect of D-pinitol, but H-89 (0.1 μM) did not change it. 1-[2-(Trifluoromethyl) phenyl]imidazole (300 μM), indomethacin (10 μM), celecoxib (5 μM), wortmannin (1 μM), ruthenium red (10 μM), tiron (10 μM), MnTMPyP (30 μM), MPP (0.1 μM), PHTPP (0.1 μM), and atropine (1 μM) did not change the effect of D-pinitol. D-pinitol increased the concentration of nitrite, which was inhibited by L-NAME and calmidazolium (10 μM). D-pinitol increased the phosphorylation level of eNOS activation site at Ser1177 and reduced the phosphorylation level of its inactivation site at Thr495. In normotensive mice, the intraperitoneal administration of D-pinitol (10 mg/kg) induced a significant reduction of the SBP after 30 min. The present results led us to conclude that D-pinitol has an endothelium- and NO-dependent vasodilator effect in mouse mesenteric artery through a mechanism dependent on the activation of eNOS by the calcium-calmodulin complex, which can explain its hypotensive effect in mice.[2]

Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-kappaB (NF-kappaB) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-kappaB activation pathway. We found that pinitol suppressed NF-kappaB activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-kappaB activation noted in most tumor cells. The suppression of NF-kappaB activation by pinitol occurred through inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Pinitol also suppressed the NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-beta-activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IkappaBalpha kinase but not that induced by p65. The inhibition of NF-kappaB activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-kappaB activation pathway, which may explain its ability to suppress inflammatory cellular responses.[4]

Maintenance of michigan cancer foundation-7 (MCF-7) cell lines [3]
Human mammary carcinoma cell lines MCF-7 were grown as mono layers in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% v/v heat inactivated fetal bovine serum (FBS), antibiotics, such as penicillin 50 U/mL, streptomycin 50µg/mL and 1 mmol/L sodium pyruvate under standard conditions containing in a humidified incubator with 5% CO2 at 37ºC. The medium was changed for every three days.

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Viability assay [3]
Cell viability was assessed as per the standard protocol by MTT (3-4,5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide) method of Mossmann (1983). The cell viability is calculated using the formula: %growth inhibition=(A570nm of treated cells/A570nm of control cells)×100.

Vascular Reactivity[2]
Sixty-six male C57BL/6 mice, aged 10–12 weeks, were used in the present study. Mice were euthanized by decapitation, the abdomen was cut, and the mesenteric bed was quickly removed and placed in a dissecting plate with physiological salt solution (PSS) with the following composition (mM): NaCl 119.0; KCl 4.7; KH2PO4 0.4; NaHCO3 14.9; MgSO4.7H2O 1.17; CaCl2.2H2O 2.5; and glucose 5.5. A segment of the second branch of the mesenteric artery was dissected, and the adipose and connective tissues were removed. The arteries were sectioned into rings (1.6–2.0 mm long) with an internal diameter ranging from 150 to 250 μm. The rings were mounted in a wire myograph (620M, DMT, Denmark), kept in carbogen aerated PSS at 37°C. After mounting, the artery was stretched to a length that yielded a circumference equivalent to 90% of that given by an internal pressure of 100 mmHg; this required a load of approximately 200 mg. The vessel was maintained for an equilibration period of 60 min. The mechanical activity was recorded isometrically as previously described (Silva et al., 2016). The functionality of the arteries was observed by the contraction induced by phenylephrine (3 μM) and by the vasodilator effect induced by acetylcholine (ACh, 10 μM) in arteries pre-contracted with phenylephrine. Arteries with ACh-induced vasodilatation higher than 70% were considered with functional endothelium. In some experimental procedures, the endothelium was removed by rubbing the lumen slightly with the tungsten wire. The removal of the endothelium was confirmed by the absence of vasodilatation induced by ACh in precontracted arteries. The vasodilator effect of D-pinitol was evaluated by concentration-response curves (1 nM to 100 μM) in mesenteric arteries in the presence and the absence of a functional endothelium pre-contracted with phenylephrine (3 μM). The participation of nitric oxide synthase (NOS) was investigated in arteries pretreated with Nω-nitro-L-arginine-methyl-ester (L-NAME; 300 μM), a non-selective inhibitor of NOS, and 1-(2-trifluoromethylphenyl) imidazole (TRIM; 300 μM), a selective inhibitor of neuronal NOS (nNOS). The activation of guanylate cyclase was investigated with 1H- [1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM). The involvement of cyclooxygenase (COX) 1 and 2, phosphatidylinositol-3-kinase (PI3K), Ca2+/calmodulin-dependent kinase II (CaMKII), and non-selective cationic channels was verified in arteries pretreated with indomethacin (10 μM), celecoxib (5 μM), wortmannin (1 μM), KN-93 (1 μM), and ruthenium red (10 μM), respectively. Tiron (10 μM) and MnTMPyP (30 μM), a cell-permeable analog of superoxide dismutase, were used to investigate the action of antioxidant drugs on the vasodilator effect of D-pinitol. The participation of muscarinic receptors and α and β estrogen receptors was investigated in arteries pretreated with atropine (1 μM), MPP (0.1 μM), and PHTPP (0.1 μM), respectively.

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Nitrite Measurement in Mouse Mesenteric Artery[2]
The assessment of NO production in the mesenteric artery was performed indirectly by the measurement of nitrite (NO2-) using the fluorescence method with 2,3-diaminonaphthalene (DAN), according to Silva et al. (2016). The mesenteric artery branches were placed in PSS, at 37°C in 5% CO2 atmosphere. Samples were collected in the absence (basal) or the presence of D-pinitol (20 μM) or ACh (10 μM). The involvement of NOS and calmodulin in the production of nitrite was evaluated in the presence of L-NAME (300 μM) and calmidazolium (10 μM), respectively. 150 μl samples were collected, added to 150 μl of purified water, followed by the immediate addition of 15 μl fresh DAN solution (0.05 mg/l in 0.62 M HCl) in 96-well opaque black plates (Costar®, United States). The reaction proceeded for 10 min at room temperature and protected from light. After this period, the reaction was stopped with 5 μl of NaOH (2.8 N) and the absorbance determined using a spectrofluorometer (Fluoroskan Ascent FL, Thermo Scientific) at 365 and 415 nm, as respective excitation and emission wavelengths. The nitrite concentration in the samples was calculated using a standard curve with predetermined concentrations of sodium nitrite in each experiment and normalized by the amount of protein in the branches. The results were expressed in [nitrite] nM/μg of protein.

[1]. Effects of D-Pinitol on Insulin Resistance through the PI3K/Akt Signaling Pathway in Type 2Diabetes Mellitus Rats. J Agric Food Chem. 2015 Jul 8;63(26):6019-26.

[2]. Activation of eNOS by D-pinitol Induces an Endothelium-Dependent Vasodilatation in MouseMesenteric Artery. Front Pharmacol. 2018 May 22;9:528.

[3]. D-pinitol promotes apoptosis in MCF-7 cells via induction of p53 and Bax and inhibition of Bcl-2 and NF-κB. Asian Pac J Cancer Prev. 2014;15(4):1757-62.

[4]. Pinitol targets nuclear factor-kappaB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis. Mol Cancer Ther. 2008 Jun;7(6):1604-14.

D-pinitol is the D-enantiomer of pinitol. It has a role as a geroprotector and a member of compatible osmolytes. It is functionally related to a 1D-chiro-inositol. It is an enantiomer of a L-pinitol.
Methylinositol has been used in trials studying the treatment of Dementia and Alzheimer's Disease.
D-Pinitol has been reported in Abies pindrow, Glycine max, and other organisms with data available.
See also: Ononitol, (+)- (annotation moved to).

C7H14O6

194.1825

194.079

C, 43.30; H, 7.27; O, 49.43

10284-63-6

164619

White to off-white solid powder

1.6±0.1 g/cm3

317.2±42.0 °C at 760 mmHg

178-185ºC

145.6±27.9 °C

0.0±1.5 mmHg at 25°C

1.588

-0.74

5

6

1

13

158

4

COC1[C@@H]([C@H](C([C@@H]([C@@H]1O)O)O)O)O

DSCFFEYYQKSRSV-KLJZZCKASA-N

InChI=1S/C7H14O6/c1-13-7-5(11)3(9)2(8)4(10)6(7)12/h2-12H,1H3/t2-,3-,4-,5-,6+,7+/m0/s1

3-O-methyl-D-chiro-inositol

10284-63-6; Pinitol; 3-O-Methyl-D-chiro-inositol; (+)-Pinitol; Inzitol; D-ononitol; Methylinositol;

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 : ~125 mg/mL (~643.73 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (10.71 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 20.8 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: ≥ 2.08 mg/mL (10.71 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 20.8 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: ≥ 2.08 mg/mL (10.71 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 20.8 mg/mL clear DMSO 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.)