Natural polyketide synthase-derived quinochalcone glucoside; red pigment

Carthamin yellow (CY), which is a flavonoid compound isolated from safflower, has various pharmacological effects including promoting blood circulation to remove blood stasis and alleviating pain. CY is a herb used in Chinese traditional medicines. Intervertebral disc degeneration (IDD) is a common spinal disorder and degeneration of nucleus pulposus (NP) cells and inflammation are significant parts of the pathological cascade. The curative effect of CY on NP cells in association with degeneration and inflammation remains to be elucidated. In the present study, rat NP cells were isolated, cultured and used to detect the suppressive effects of CY on lipopolysaccharide (LPS)-induced genetic expression variation and the expression of matrix degradation enzymes, including matrix metallopeptidase-3, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4 and ADAMTS-5. A protective effect of CY on NP cells was observed against LPS-induced matrix degradation and inflammation. Western blotting results demonstrated that pretreatment with CY significantly suppressed the LPS-induced activation of the mitogen activated protein kinase (MAPK) pathway. The results of the present study suggested that CY exerted anti-degenerative and anti-inflammatory effects on NP cells via inhibition of MAPK pathway activation. Therefore, CY may be a potential therapeutic drug for the treatment of IDD in the future [1].

Carthamin yellow (CY), a flavonoid compound extracted from safflower, has been reported to attenuate cardiac ischemia and reperfusion injury. However, whether CY could ameliorate ischemic stroke is not completely understood. In the present study, the preventive effects of CY on experimental ischemic stroke were investigated using middle cerebral artery occlusion (MCAO) model rats. Neurological scores, brain edema, infarct area and microtubule‑associated protein 2 (MAP‑2) immunoreactivity were assessed to evaluate the effects of CY on ischemic brain injury. The involvement of inflammation and ferroptosis were examined to investigate the mechanism underlying the effects of CY. The results demonstrated that 2‑week CY treatment attenuated the neurological deficit score, brain water content and infarct area, and increased MAP‑2 immunoreactivity in the cortex in MCAO model rats. CY administration also deactivated the cortex NF‑κB/NLR family pyrin domain containing 3 inflammasome signaling pathway, and decreased serum TNF‑α, IL‑1β and IL‑6 concentrations. Moreover, CY treatment inhibited Fe2+ and reactive oxygen species accumulation, and reversed acyl‑CoA synthetase long‑chain family member 4, transferrin receptor 1, glutathione peroxidase 4 and ferritin heavy chain 1 protein expression levels in the brain. The levels of glutathione, superoxide dismutase and malondialdehyde in the serum were also reversed by CY treatment. Collectively, the results of the present study demonstrated that CY protected rats against ischemic stroke, which was associated with mitigation of inflammation and ferroptosis [2].

Cell cytotoxicity assay [1]
A cell counting kit-8 (CCK8) was used to test the viability of NP cells after Carthamin yellow (CY) treatment for 1 day according to previously reported methods. Approximately 5×103 NP cells were seeded on each film and transferred to 96-well plates. incubated with various concentrations of CY for 24 h. The conditioned culture medium was removed before CCK8 examination. Subsequently, 100 µl of DMEM and 10 µl of CCK8 solution were added to each well, followed by CCK8 incubation at 37°C for 2.5 h. The optical density (OD) at 450 nm was determined using a microplate reader. Cell viability was calculated as follows: Cell viability to control (%)=(ODdrug-treated group-ODblank)/(ODcontrol group-ODblank).

---

Apoptosis assay [1]
Cell apoptosis was measured by flow cytometry using Annexin V/propidium iodide (PI) double-immunofluorescent staining according to previously reported methods. NP cells were cultured with 200 µM Carthamin yellow (CY) and/or 1 µg/ml LPS for 24 h. Then the cells were washed with cold PBS and resuspended with 1X Annexin-binding buffer. After that, all cells were stained with Annexin V-FITC and Propidium iodide according to the manufacturer's protocols. The apoptosis rate was measured by flow cytometry (FCM). Apoptotic events were indicated as a combination of fluorescein isothiocyanate (FITC)+/PI- (early apoptotic) and FITC+/PI+ (late apoptotic or dead) events. The final results are expressed as the percentage of early, late and total apoptotis.

---

Gene expression [1]
NP cells were incubated with different concentrations of Carthamin yellow (CY) and 1 µg/ml LPS for 24 h. LPS could induce inflammation and matrix degradation in IVD. Total RNA was isolated by the AxyPrep™ Multisource Total RNA Miniprep kit. Then 1 µg RNA was converted into complementary DNA (cDNA) with PrimeScript™ RT reagent kit. Quantitative PCR was performed using an ABI 7500 Sequencing Detection System and SYBR® Premix Ex Taq™. Cycling conditions were as follows: 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The primers were used to amplify target genes are listed in Table I. The primers were designed and selected using blast in pubmed, and GAPDH was used as the internal control.

---

Western blotting [1]
For signaling pathway protein assay, the cells were treated with various concentrations of Carthamin yellow (CY) and/or 1 µg/ml LPS for 24 h. For aggrecan and collagen II protein assay, the cells were treated with various concentrations of CY and/or 1 µg/ml LPS for 5 or 8 days. Cell total proteins were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's instructions. 20 µg protein (each sample) was loaded into gel, and separated by 7.5–12.5% SDS-PAGE, then transferred to 0.22-µm PVDF membranes. The membranes were blocked with 5% fat-free milk at room temperature for 1 h and subsequently incubated with primary antibodies overnight at 4°C (1:1,000 dilution). After three washes in TBST, the membranes were probed with the corresponding secondary antibody for 1 h at room temperature. The membranes were washed again in TBST, and the protein bands were visualized using an Odyssey Infrared Imaging System. Positive immunoreactive bands were densitometrically quantified and normalized to GAPDH.

---

Immunohistochemistry staining [1]
2×104/ml cells were seeded in 24-well plates, and these NP cells were cultured with diverse Carthamin yellow (CY) concentrations and/or 1 µg/ml LPS for 5 or 8 days. Cells were fixed with 4% paraformaldehyde before making cells slides. After fixation, NP cells were treated with 0.1% Triton X-100 for 15 min. Then the cells were blocked with 2% bovine serum albumin for 1 h. Then, cells slides were incubated with the corresponding antibody, including anti-collagen II and anti-aggrecan antibody (1:200 dilution; Abcam) overnight at 4°C. For immunohistochemistry, the secondary antibody was used for 15 min at room temprature. The DAB (Maixin Bio) solution was used as the chromogen. We used an inverted microscope microscopy to acquire the images. The integral optical density (IOD) of every photo was measured using the Image-Pro Plus 6.0 software.

Animals were randomly divided into the following four groups (n=8 per group): i) Sham; ii) MCAO; iii) CY (20 mg/kg); and iv) CY (40 mg/kg). Carthamin yellow (CY) was administered intragastrically to rats once daily for 2 weeks. At 60 min after the last administration, MCAO surgery was performed as previously described. At 24 h post-reperfusion, neurological scores, brain water content and infarct volume were determined. Immunofluorescence staining, western blotting and flow cytometry were performed to investigate the potential mechanisms. After neurological scoring, rats were euthanized with 400 mg/kg pentobarbital sodium. The brains were immediately removed, and the cortex and the serum were collected and stored at −80°C until further use. [2]

[1]. Carthamin yellow inhibits matrix degradation and inflammation induced by LPS in the intervertebral disc via suppression of MAPK pathway activation. Exp Ther Med. 2017 Aug;14(2):1614-1620.
[2]. Carthamin yellow improves cerebral ischemia‑reperfusion injury by attenuating inflammation and ferroptosis in rats. Int J Mol Med. 2021 Apr;47(4):52. d

Safflower extract is a hydroxycinnamic acid. Safflower yellow has been reported to exist in safflower (Carthamus tinctorius), and related data have been reported. Based on the above, we are honored to report the role of safflower yellow in lipopolysaccharide (LPS)-induced intervertebral disc inflammation and matrix degradation. We found that safflower yellow can indirectly or directly affect the activity of the MAPK signaling pathway and proteoglycan (PG) content in the intervertebral disc. However, further research is needed based on this fundamental study. First, the molecular mechanisms controlling safflower yellow-mediated inflammation and PG-related signaling pathway conversion need to be elucidated more deeply. Second, animal experiments are needed to verify the therapeutic effects of safflower yellow in vivo. Finally, clinical treatment is needed to validate our research results. In summary, our study shows that safflower yellow (CY) can exert significant anti-inflammatory and anti-degenerative effects by inhibiting LPS-induced MAPK activation in nucleus pulposus cells. Safflower yellow (CY) may become a potential novel traditional Chinese medicine for the treatment of intervertebral disc disease (IDD). However, further research is needed to confirm this. [1] In this study, MCAO-induced cerebral ischemia-reperfusion injury promoted the accumulation of cortical iron and reactive oxygen species (ROS) and increased serum lipid peroxidation levels, leading to abnormal expression of ferroptosis-related proteins in the brain. However, cytosine sulfadiazine (CY) treatment inhibited these changes induced by the MCAO model. These results suggest that ferroptosis may be partly responsible for the progression of experimental ischemic stroke in MCAO-induced rats and the effectiveness of CY treatment. In summary, the results of this study indicate that CY treatment protects rats from ischemia-reperfusion injury by reducing inflammation and ferroptosis (Figure 10). These results suggest that CY may be a potential drug for the treatment of ischemic stroke. [2]

C43H42O22

910.78

910.216

C, 56.71; H, 4.65; O, 38.65

36338-96-2

135565560

Typically exists as solid at room temperature

1.9±0.1 g/cm3

1255.2±65.0 °C at 760 mmHg

375.5±27.8 °C

0.0±0.3 mmHg at 25°C

1.859

6.11

15

22

11

65

2100

12

O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@@]([H])([C@]([H])([C@]1([H])C1(C(=C(C(/C(/[H])=C(\[H])/C2C([H])=C([H])C(=C([H])C=2[H])O[H])=O)C(C(/C(/[H])=C2\C(C(C(/C(/[H])=C(\[H])/C3C([H])=C([H])C(=C([H])C=3[H])O[H])=O)=C(C(C\2=O)([C@@]2([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O2)O[H])O[H])O[H])O[H])O[H])=O)=C1O[H])=O)O[H])O[H])O[H])O[H])O[H]

WLYGSPLCNKYESI-RSUQVHIMSA-N

InChI=1S/C43H42O22/c44-14-24-30(52)32(54)34(56)40(64-24)42(62)36(58)20(28(50)26(38(42)60)22(48)11-5-16-1-7-18(46)8-2-16)13-21-29(51)27(23(49)12-6-17-3-9-19(47)10-4-17)39(61)43(63,37(21)59)41-35(57)33(55)31(53)25(15-45)65-41/h1-13,24-25,30-35,40-41,44-47,52-58,60-63H,14-15H2/b11-5+,12-6+,21-13-/t24-,25-,30-,31-,32+,33+,34-,35-,40-,41-,42+,43-/m1/s1

(2Z,6S)-5,6-dihydroxy-4-[(E)-3-(4-hydroxyphenyl)prop-2-enoyl]-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-2-[[(3S)-2,3,4-trihydroxy-5-[(E)-3-(4-hydroxyphenyl)prop-2-enoyl]-6-oxo-3-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]cyclohexa-1,4-dien-1-yl]methylidene]cyclohex-4-ene-1,3-dione

36338-96-2; Carthamine; Carthamin; Natural Red 26; Carthamus red; Liofresh Red CR; Carthamus Red AP; C.I. Natural Red 26;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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