Natural occurring flavonoid; Nitric oxide (NO); PKC

In vitro activity: Myricitrin, a flavonoid compound isolated from the root bark of Myrica cerifera, which exerts antinociceptive effect. Myricitrin produces pronounced antinociception against chemical and mechanical models of pain in rodents via preventing the protein kinase C (PKC) alpha and PKC epsilon activation by phorbol myristate acetate (PMA). Another study proves that opening of voltage- and small-conductance calcium-gated K(+) channels and reduction of calcium influx leads to the antinociceptive of myricitrin. Myricitrin also decreases H2O2-induced apoptosis in vascular endothelial cells via inhibition of p53 gene expression, activation of caspase-3 and the MAPK signaling pathway, and alteration of the patterns of pro-apoptotic and anti-apoptotic gene expression.

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Myr/Myricitrin treatment inhibited the lamellipodia formation, migration, and invasion, but not the apoptosis and proliferation, of RA FLSs. Myr also reduced the expression of CCL2, IL-6, IL-8, MMP-1, MMP-3, and MMP-13 induced by TNF-α. The RNA-seq results indicated that AIM2 may be a target gene of Myr in RA FLSs. Furthermore, compared to healthy controls, AIM2 expression showed higher levels in synovial tissues and FLSs from RA patients. AIM2 knockdown also inhibited RA FLS migration, invasion, cytokine, and MMP expression. In addition, either Myr treatment or AIM2 knockdown reduced the phosphorylation of AKT induced by TNF-α stimulation [3].

In mice, Myricitrin (i.p. 10 mg/kg or 30 mg/kg) blocks apomorphine-induced stereotypy and climbing, and increases hindlimb retraction time (HRT).

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The present study investigated the antinociceptive effects of the flavonoid myricitrin in chemical behavioral models of pain in mice and rats. Myricitrin given by i.p. or p.o. routes produced dose-related antinociception when assessed on acetic acid-induced visceral pain in mice. In addition, the i.p. administration of Myricitrin exhibited significant inhibition of the neurogenic pain induced by intraplantar (i.pl.) injection of capsaicin. Like-wise, myricitrin given by i.p. route reduced the nociception produced by i.pl. injection of glutamate and phorbol myristate acetate (PMA). Western blot analysis revealed that myricitrin treatment fully prevented the protein kinase C (PKC) alpha and PKCepsilon activation by PMA in mice hind paws. Myricitrin given i.p. also inhibited the mechanical hyperalgesia induced by bradykinin, without affecting similar responses caused by epinephrine and prostaglandin E(2). The antinociception caused by myricitrin in the acetic acid test was significantly attenuated by i.p. treatment of mice with the nitric oxide precursor, L-arginine. In contrast, myricitrin antinociception was not affected by naloxone (opioid receptor antagonist) or neonatal pretreatment of mice with capsaicin and myricitrin antinociceptive effects is not related to muscle relaxant or sedative action. Together, these results indicate that myricitrin produces pronounced antinociception against chemical and mechanical models of pain in rodents. The mechanisms involved in their actions are not completely understood but seem to involve an interaction with nitric oxide-L-arginine and protein kinase C pathways. [1]

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The present study was designed to investigate the mechanisms involved in the antinociception afforded by Myricitrin in chemical models of nociception in mice. Myricitrin given by intrathecal (i.t.) or intracerebroventricular (i.c.v.) route produced dose-related antinociception when evaluated against acetic acid-induced visceral pain in mice. In addition, the intraperitoneal administration of myricitrin caused significant inhibition of biting behaviour induced by i.t. injection of glutamate, substance P, capsaicin, interleukin 1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). The antinociception caused by myricitrin in the acetic acid test was fully prevented by i.t. pre-treatment with pertussis toxin, a Gi/o protein inactivator, and by i.c.v. injection of calcium chloride (CaCl(2)). In addition, the i.t. pre-treatment of mice with apamin, a blocker of small (or low)-conductance calcium-gated K(+) channels and tetraethylammonium, a blocker of voltage-gated K(+) channels significantly reversed the antinociception induced by myricitrin. The charybdotoxin, a blocker of large (or fast)-conductance calcium-gated K(+) channels and glibenclamide, a blocker of the ATP-gated K(+) channels had no effect on myricitrin-induced antinociception. Calcium uptake analysis revealed that myricitrin inhibited (45)Ca(2+) influx under a K(+)-induced depolarization condition. However, calcium movement was modified in a non-depolarizing condition only when the highest concentration of myricitrin was used. In summary, our findings indicate that myricitrin produces consistent antinociception in chemical models of nociception in mice. These results clearly demonstrate an involvement of the Gi/o protein dependent mechanism on antinociception caused by myricitrin. The opening of voltage- and small-conductance calcium-gated K(+) channels and the reduction of calcium influx led to the antinociceptive of myricitrin [2].

RA FLS viability assay [3]
Cell Counting Kit-8 (CCK8) assay kit was used to assess RA FLS viability. Briefly, RA FLSs were incubated with Myricitrin/Myr at different concentrations (0–800 μM) for 24 h. Cells were washed with DMEM. Subsequently, a culture medium containing 10% Cell Counting Kit-8 (CCK-8) reagent was added to each well and incubated for another 4 h. Afterward, the absorbance of the plate was measured at 450 nm by a microplate reader.

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FLS migration and invasion assay [3]
FLS migration was performed using a transwell assay with a filter (6.5 mm in diameter, 8.0 μm pore size). Briefly, FLSs were suspended in serum-free DMEM in the upper compartments of the chambers at a concentration of 6×104 cells/ml. Medium containing 10% FBS as a chemoattractant was added to the lower compartments of the chambers. After incubation for 8 h, the cells on the top surface of the membrane were scraped using a cotton swab. RA FLSs that migrated to the lower side of the filter were fixed in methanol for 15 min and stained with 0.1% crystal violet for 15 min. The number of stained FLSs is the average number of cells from 5 random fields. To measure cell invasion, we performed a similar experiment in chambers coated with BD Matrigel basement membrane matrix.

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FLS proliferation assays [3]
RA FLS proliferation was measured by using a Cell-Light EdU DNA Cell Proliferation Kit following the manufacturer’s protocol. The cells were pretreated with different concentrations of Myricitrin/Myr (0–400 μM) for 24 h. EdU was added to measure the cell proliferation and incubated for another 12 h. DAPI was used to stain cell nuclei. EdU-positive cells were quantified by microscopy.

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FLS apoptosis assays [3]
An Annexin V-APC/PI Apoptosis Detection kit was used to assess RA FLS apoptosis following the manufacturer’s protocol. Briefly, 1 × 105 FLSs were suspended in 0.1 ml 1× binding buffer, stained with 5 μL PI and 5 μL annexin V and incubated for another 15 min in darkness at room temperature. The samples were then analyzed within 1 h by flow cytometry.

Abdominal constriction induced by acetic acid [2]
The abdominal constrictions were induced according to procedures described previously (Collier et al., 1968) and resulted in the contraction of the abdominal muscle together with a stretching of the hind limbs in response to an intraperitoneal (i.p.) injection of acetic acid (0.6%, 0.45 ml/mouse) at the time of the test. Mice were lightly anesthetized with ether and a volume of 5 μl of sterile phosphate buffer saline (PBS) or Myricitrin (0.1–10 μg/site) was injected directly into the lateral ventricle (i.c.v.; coordinates from bregma: 1 mm lateral, 1 mm rostral, 3 mm vertical) or between the L5 and L6 vertebrae (intrathecal, i.t.) using a microsyringe connected to polyethylene tubing, as described previously (Laursen and Belknap, 1986, Hylden and Wilcox, 1980). The mice were treated with PBS or mMyricitrin 10 min before acetic acid injection. After the challenge, the mice were individually placed into glass cylinders of 20 cm diameter, and the abdominal constrictions were counted cumulatively over a period of 20 min.

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Algogen-induced overt nociception in mice [2]
In another set of experiments we examined Myricitrin effects on the nociception induced by glutamate, substance P, capsaicin and pro-inflammatory cytokines. Animals received an i.p. injection of myricitrin (30 mg/kg) 30 min before i.t. injection of 5 μl of drug. Injections were given to fully conscious mice awake using the method described by Hylden and Wilcox (1980). Briefly, the animals were restrained manually and a 30 gauge needle, attached to a 50 μl microsyringe, was inserted through the skin and between the vertebrae into the subdural space of the L5–L6 spinal segments. Injections were given over a period of 5 s. The nociceptive response was elicited by glutamate (30 μg/site) (Scheidt et al., 2002); substance P (135 ng/site) (Sakurada et al., 1990); capsaicin (30 ng/site) (Sakurada et al., 1996); IL-1β (1 pg/site) and TNF-α (0.1 pg/site) (Choi et al., 2003) with minor modifications. A group of mice received vehicle (PBS) by i.t. route. The amount of time the animal spent biting was evaluated following local post-injections of one of the following agonists: glutamate (3 min); substance P and capsaicin (6 min); IL-1β, TNF-α and PBS (15 min). A bite was defined as a single head movement directed at the flanks or hind limbs, resulting in contact of the animal's snout with the target organ.

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Analysis of the mechanism of action of Myricitrin [2]
In this set of experiment Myricitrin was administered intraperitoneally in mice. This provides a wide distribution of the compound that allows the evaluation of the effects of myricitrin at peripheral and central levels.

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Participation of Gi/o protein [2]
To determine the involvement of Gi/o protein in the antinociceptive action of Myricitrin, mice were pre-treated with pertussis toxin (0.5 μg/site), an inactivator of Gi/o protein. A control group was pre-treated with PBS (5 μl /site) by intrathecal route. The experiment was carried out as described by Sánchez-Blázquez and Garzón (1991). Seven days after the pre-treatment, mice received vehicle (10 ml/kg), Myricitrin (1 mg/kg, i.p.) or morphine (2.5 mg/kg, s.c.) (Santos et al., 1999). After 30 min, the animals were injected with 0.6% acetic acid. The number of abdominal constrictions was recorded during the 20 min following acetic acid administration.

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Involvement of K+ channels in the antinociceptive action of Myricitrin We next investigated the involvement of K+ channels on the antinociceptive action of Myricitrin. Mice were pre-treated with K+ channels blockers: apamin (50 ng/site, i.t.; a blocker of small (or low)-conductance calcium-gated K+ channels); charybdotoxin (250 pg/site, i.t.; a blocker of large (or fast)-conductance calcium-gated K+ channels); tetraethylammonium (1 μg/site, i.t.; a blocker of voltage-gated K+ channels); or glibenclamide (80 μg/site, i.t.; a blocker of ATP-gated K+ channels) and after 15 min they received Myricitrin (1 mg/kg, i.p.), morphine (2.5 mg/kg, s.c.) or vehicle (10 ml/kg, i.p.) (Strong, 1990, Aronson, 1992, Welch and Dunlow, 1993, Santos et al., 1999). The nociceptive response was evaluated through the number of abdominal constrictions in 20 min, which was caused by an i.p. injection of acetic acid (0.6%, 0.45 ml) 30 min after Myricitrin, morphine or vehicle administration.

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Involvement of calcium channels in the antinociceptive action of Myricitrin [2]
Mice received 5 μl of CaCl2 (200 nmol/site) or PBS (vehicle) by i.c.v. route as described by Liang et al. (2003). The i.c.v. injection was carried out as described above (Section 2.3). After 10 min animals were treated with Myricitrin (1 mg/kg, i.p.), morphine (2.5 mg/kg, s.c.) or saline (10 ml/kg). Thirty min following drugs administration, the animals received 0.45 ml of acetic acid (0.6%) by i.p. route, and the number of abdominal constrictions was recorded over 20 min.

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Modulation of 45Ca2+ influx by Myricitrin [2]
To investigate Myricitrin effects on calcium movement, the 45Ca2+ influx into cortical slices of rats was assessed. 45Ca2+ uptake was carried out essentially as described by Eason and Aronstam (1984), with some modifications. Two salt solutions were used in these studies: (1) Krebs buffer containing 127 mM NaCl, 1.2 mM Na2HPO4, 0.44 mM KH2PO4, 0.95 mM MgCl2, 0.70 mM CaCl2, 10 mM glucose, and 0.50 mM Hepes, pH 7.4 with KCl 5.36 mM for a baseline analysis or 80 mM for the K+-stimulated assay; (2) lanthanium solution containing 127 mM NaCl, 0.95 mM MgCl2, 10 mM LaNO3, 10 mM glucose, and 0.60 mM Hepes, pH 7.4 with KCl 5.0 mM for a baseline analysis or 80 mM for the K+-stimulated assay. To measure 45Ca2+ uptake, rats were killed by decapitation, the cerebral cortex was dissected, isolated and the parietal cortex was cut into 400 μm slices, which were washed with Krebs buffer (solution 1). The slices (0.8–1.3 mg protein) were pre-incubated in 96-well polycarbonate plates for 22 min at 32 °C in the absence (control group) or presence of Myricitrin (100 and 200 μM). The slices were then transferred to medium containing solution 1 plus 21 pmol of 45Ca2+. The 45Ca2 uptake was monitored for 15 s at 32 °C. The reaction was stopped by five times of 2 min washes with ice-cold lanthanium solution (solution 2). Immediately after washing, aliquots were lysed with 0.25 ml of a solution containing 0.5 M NaOH plus 0.2% SDS and maintained at 60 °C for 5 min. An aliquot was taken for determination of the intracellular calcium content by liquid scintillation counting. Nonspecific calcium uptake (20–30% of the total uptake) was determined by carrying out the same experiment using solution 2, which contained the nonspecific voltage-dependent calcium channel blocker, lanthanium. Specific uptake was considered as the difference between total uptake and nonspecific uptake.

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CIA model and treatment [3]
The CIA mouse model was established as previously described (Wang C. et al., 2020). Briefly, 7–8 weeks old male DBA/1J mice were intradermally injected with 100 μg of bovine type II collagen and Freund’s complete adjuvant in a 1:1 ratio (vol/vol) emulsion (100 μL) at the base of the tail. DBA/1J mice were intraperitoneally injected with 200 μg bovine type II collagen on Day 21 after the first immunization. In the therapeutic treatment study, the mice were randomly divided into groups. Myricitrin group (100 mg × kg−1, every day, n = 5) or DMSO group (vehicle, n = 5) was intragastrically administered for 14 days. Arthritis progression was monitored every other day according to previously described scoring system ranging from 0 to 4 (0, normal; 1, swelling or redness of paw or a single digit; 2, 2 joints involved; 3, 3 joints involved; 4, severe arthritis of the entire paw and digits) (Thornton et al., 2017). The arthritic score was independently calculated in a blinded manner and defined as the combining scores of all four paws. All mice were raised under specific pathogen-free (SPF) conditions.

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Dissolved in Tween 80 followed by saline; 10, 30 mg/kg; i.p. injection

Male adult Swiss albino mice

[1]. J Pharmacol Exp Ther.2006 Feb;316(2):789-96.
[2]. Eur J Pharmacol.2007 Jul 19;567(3):198-205.
[3]. Front Pharmacol. 2022 Aug 31:13:905376.

Myricitrin is a glycosyloxyflavone that consists of Myricitrin attached to a alpha-L-rhamnopyranosyl residue at position 3 via a glycosidic linkage. Isolated from Myrica cerifera, it exhibits anti-allergic activity. It has a role as an anti-allergic agent, an EC 1.14.13.39 (nitric oxide synthase) inhibitor, an EC 2.7.11.13 (protein kinase C) inhibitor and a plant metabolite. It is a pentahydroxyflavone, a glycosyloxyflavone, an alpha-L-rhamnoside and a monosaccharide derivative. It is functionally related to a myricetin. It is a conjugate acid of a myricitrin(1-).
Myricitrin has been reported in Castanopsis fissa, Phyllanthus tenellus, and other organisms with data available.

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In conclusion, the present results are in agreement with previous data and demonstrate that Myricitrin produces antinociception when administered at peripheral or central levels. Furthermore, these results showed that myricitrin antinociception is closely related to pathways activated by glutamate, substance P, capsaicin and pro-inflammatory cytokines. The mechanisms of antinociception are dependent of the Gi/o protein activation; opening of specific K+ channels (voltage- and small-conductance Ca2+-gated) and inhibition of calcium influx.[2]

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Objective: To explore the effect and underlying mechanism of Myricitrin (Myr) in regulating fibroblast-like synoviocyte (FLS)-mediated synovitis and joint destruction in RA. Methods: FLSs were isolated from synovial tissues from patients with RA. Gene expression was measured using quantitative RT-qPCR. Protein expression was detected by immunohistochemistry or Western blot. Cell apoptosis was performed by an Annexin-PI staining assay. EdU incorporation was used to assess the proliferation of RA FLS. Transwell assay was used to characterize the cell migration and invasion ability of RA FLS. The potential target of Myr was identified by RNA sequencing analysis. The in vivo effect of Myr was assessed in a collagen-induced arthritis (CIA) model. Results: Myr treatment inhibited the lamellipodia formation, migration, and invasion, but not the apoptosis and proliferation, of RA FLSs. Myr also reduced the expression of CCL2, IL-6, IL-8, MMP-1, MMP-3, and MMP-13 induced by TNF-α. The RNA-seq results indicated that AIM2 may be a target gene of Myr in RA FLSs. Furthermore, compared to healthy controls, AIM2 expression showed higher levels in synovial tissues and FLSs from RA patients. AIM2 knockdown also inhibited RA FLS migration, invasion, cytokine, and MMP expression. In addition, either Myr treatment or AIM2 knockdown reduced the phosphorylation of AKT induced by TNF-α stimulation. Importantly, Myr administration relieved arthritis symptoms and inhibited AIM2 expression in the synovium of CIA mice. Conclusion: Our results indicate that Myr exerts an anti-inflammatory and anti-invasion effect in RA FLSs and provide evidence of the therapeutic potential of Myr for RA.[3]

C21H20O12

464.38

464.095

C, 54.32; H, 4.34; O, 41.34

17912-87-7

5281673

White to yellow solid powder

1.9±0.1 g/cm3

896.6±65.0 °C at 760 mmHg

197 °C

315.7±27.8 °C

0.0±0.3 mmHg at 25°C

1.805

1.98

8

12

3

33

760

5

C[C@H]1[C@@H]([C@H]([C@H]([C@@H](O1)OC2=C(OC3=CC(=CC(=C3C2=O)O)O)C4=CC(=C(C(=C4)O)O)O)O)O)O

DCYOADKBABEMIQ-OWMUPTOHSA-N

InChI=1S/C21H20O12/c1-6-14(26)17(29)18(30)21(31-6)33-20-16(28)13-9(23)4-8(22)5-12(13)32-19(20)7-2-10(24)15(27)11(25)3-7/h2-6,14,17-18,21-27,29-30H,1H3/t6-,14-,17+,18+,21-/m0/s1

3-[(6-deoxy-α-L-mannopyranosyl)oxy]-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.38 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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 (Please use freshly prepared in vivo formulations for optimal results.)