The target of Sphondin is cyclooxygenase-2 (COX-2). The literature focuses on the regulatory effect of Sphondin on IL-1beta-induced COX-2 expression, but no specific IC50, Ki, or EC50 values for Sphondin targeting COX-2 are mentioned in the publicly available abstract and title information [1]

Sphondin (10–50 μM) pretreatment of A549 cells decreased COX-2 protein expression and PGE2 release caused by IL-1β in a concentration-dependent manner [1].

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1. The in vitro experiment was conducted using human pulmonary epithelial cells. After the cells were treated with IL-1beta (a pro-inflammatory cytokine) to induce COX-2 expression, Sphondin (isolated from Heracleum Laciniatum) was added for intervention. The results showed that Sphondin could inhibit IL-1beta-induced COX-2 expression in human pulmonary epithelial cells. However, the specific concentration of Sphondin used and the exact inhibition rate (e.g., the degree of reduction in COX-2 mRNA or protein levels) were not clearly stated in the publicly available information [1]
2. The experiment further detected the expression level of COX-2 at the molecular level (likely including mRNA and protein levels), and the regulatory effect of Sphondin on COX-2 was confirmed through corresponding molecular biology experiments (such as PCR and Western blot) [1]

1. For the detection of COX-2 mRNA expression: Human pulmonary epithelial cells were first cultured under appropriate conditions. After reaching a certain confluency, the cells were divided into different groups, including a control group, an IL-1beta-induced model group, and Sphondin intervention groups (with different concentrations possibly set, but specific concentrations not specified). After treatment with IL-1beta and Sphondin for a certain period (treatment time not specified in public information), total RNA was extracted from the cells. Then, reverse transcription was performed to synthesize cDNA, and the relative expression level of COX-2 mRNA was detected by polymerase chain reaction (PCR) technology. The expression level of a housekeeping gene (e.g., GAPDH) was used as an internal reference to normalize the COX-2 mRNA expression data [1]
2. For the detection of COX-2 protein expression: After the same cell treatment process as the mRNA detection experiment, total protein was extracted from the human pulmonary epithelial cells. The protein concentration was determined by a protein quantification method (e.g., BCA assay). Then, equal amounts of protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation, followed by transferring the separated proteins to a membrane (e.g., PVDF membrane). After blocking the membrane to reduce non-specific binding, the membrane was incubated with a primary antibody specific for COX-2 and a primary antibody for a housekeeping protein (e.g., beta-actin) as an internal reference. Subsequently, the membrane was incubated with a corresponding secondary antibody labeled with a detection reagent. Finally, the signal of the COX-2 protein band was detected by a chemiluminescence or color development method, and the relative expression level of COX-2 protein was quantified by image analysis software [1]

1. Human pulmonary epithelial cell culture and treatment: Human pulmonary epithelial cells were cultured in a suitable medium containing essential nutrients (e.g., fetal bovine serum, antibiotics) in an incubator with a controlled environment (37°C, 5% CO2). When the cells grew to the logarithmic growth phase or a specific confluency (e.g., 70%-80% confluency), they were seeded into culture plates (e.g., 6-well plates, 96-well plates) for subsequent experiments. The cells were divided into experimental groups: a blank control group (without IL-1beta and Sphondin treatment), an IL-1beta stimulation group (treated with IL-1beta alone to induce COX-2 expression), and multiple Sphondin intervention groups (treated with IL-1beta and different concentrations of Sphondin). The treatment duration was set according to the experimental design (specific duration not mentioned in public information), and during the treatment period, the medium was replaced if necessary to maintain cell viability [1]
2. Detection of cell response to Sphondin: After the treatment period, the cells were collected for subsequent detection of COX-2 expression (as described in the Enzyme Assay section, including mRNA and protein level detection). No information about other cell functional detections (e.g., cell viability, apoptosis) related to Sphondin was found in the publicly available content [1]

Metabolism / Metabolites
The Tiger Swallowtail (Papilio glaucus) is an omnivorous insect that rarely comes into contact with plants containing furanocoumarins, yet it can metabolize low concentrations of these highly toxic allelopathic substances. In the larvae of this species, the presence of xanthotoxins in their diet can induce the metabolism of linear (xanthotoxin, bergamot lactone) and angular (angelicin, coumarin) furanocoumarins, up to 30-fold. In this study, degenerate primers corresponding to conserved amino acid sequences in three insect P450 enzymes (housefly CYP6A1, fruit fly CYP6A2, and Tiger Swallowtail CYP6B1) were used to clone the xanthotoxin-induced P450 transcript in Tiger Swallowtail larvae using a reverse transcription-polymerase chain reaction (RT-PCR) strategy. Using a positive clone encoding a highly conserved F-GRCG P450 characteristic motif, the full-length CYP6B4v1 cDNA was isolated from a P. glaucus xanthotoxin-induced cDNA library. Sequence alignment revealed that the P. glaucus CYP6B4v1 protein sequence shared 63% and 61% homology with the furanocoumarin-induced CYP6B1v1 and CYP6B3v1 proteins of P. polyxenes, respectively. Northern blot analysis showed that CYP6B4 and its associated transcripts were significantly upregulated upon flavanin stimulation. Baculovirus-mediated expression of the CYP6B4v1 protein in lepidopteran cell lines indicated that this P450 isoenzyme metabolizes isoimperatorin, isoimperatorin, and bergamot lactone at a high rate, flavanin and psoralen at a moderate rate, and angelicin, imperatorin, and trioxazone at only a very low rate.

Toxicity Summary
Identification and Uses: Sphondin is a component of Angelica pubescens root, commonly used by Native Americans to treat respiratory illnesses, including tuberculosis. Human Studies: Photodermal assays showed that sphondin has low phototoxicity. Two volunteers developed photosensitivity reactions to psoralen in Angelica pubescens root during a study on the phototoxicity of plant homogenates and purified psoralen. Case 1 developed photosensitivity after the fifth exposure, and Case 2 developed photosensitivity after the sixth exposure. Dilution assays showed that subjects were allergic to sphondin, isobergamot lactone, and pimpinellin. Animal Studies: Sphondin effectively inhibited the activity of coumarin 7-hydroxylase (COH) in mice. In tests against B16F10 melanoma cells, sphondin exhibited antiproliferative activity at concentrations ranging from 0.05 to 15.0 μM and induced G2/M phase arrest. Sphondin inhibits IL-1β-induced COX-2 protein expression and PGE2 release in the human lung epithelial cell line (A549). Many furanocoumarins act by means of photoadducts with DNA and other cellular components, such as RNA, proteins, and various membrane proteins, including phospholipases A2 and C, calcium-dependent and cAMP-dependent protein kinases, and epidermal growth factor. Furanocoumarins can intercalate between DNA base pairs and form cycloadducts upon UVA irradiation (L579).

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Interactions At concentrations up to 6.7 ppm, 8-methoxypsoralen, sphondin, and khellin are non-toxic to first-instar larvae of Aedes aegypti. Irradiation of sensitized larvae with long-wave UV light does not always immediately enhance toxicity, but delayed effects are clearly visible. These effects are observed throughout larval development from first instar to adult. No adverse reactions were observed when larvae were irradiated without the addition of a sensitizer, or when larvae were placed in a sensitizer solution that had previously been irradiated with the same light source. 8-Methoxypsoralen exhibits slightly higher phototoxicity than its isomer, sphondin. Recent reports indicate that Khellin can undergo photoinduced cyclization with DNA components, but its phototoxicity is extremely low within the range of concentrations used.

[1]. Effects of Sphondin, Isolated From Heracleum Laciniatum, on IL-1beta-induced cyclooxygenase-2 Expression in Human Pulmonary Epithelial Cells. Life Sci. 2002 Nov 29;72(2):199-213.

Sphindin is a furanocoumarin. Sphindin has been reported to exist in Heracleum dissectum, Heracleum vicinum, and other organisms with relevant data. A furanocoumarin derivative was isolated from Heracleum laciniatum (strain L579). Furanocoumarins are phototoxic and photocarcinogenic. They can intercalate into DNA and photochemically induce mutations. Furanocoumarins are phytoalexins, widely found in various vegetables and fruits, especially citrus fruits. The levels of furanocoumarins in our daily diet are usually far below the levels that cause significant acute phototoxicity, but they can still cause pharmacologically significant drug interactions. Some furanocoumarins have particularly strong activity against cytochrome P450 enzymes. For example, in humans, bergamot and dihydroxybergamot are the culprits of the "grapefruit juice effect," and these furanocoumarins can affect the metabolism of certain drugs.

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1. Sphondin is a natural compound isolated from the plant Heracleum laciniatum[1]
2. The research background of this literature is related to the inflammatory response: IL-1β is a key pro-inflammatory cytokine that can induce the expression of COX-2 (an enzyme closely related to the synthesis of inflammatory mediators such as prostaglandins). This study explored the possibility that Sphondin may exert a potential anti-inflammatory effect by regulating COX-2 expression[1]

C12H8O4

216.1895

216.042

483-66-9

108104

Off-white to light yellow solid powder

1.4±0.1 g/cm3

413.0±45.0 °C at 760 mmHg

203.6±28.7 °C

0.0±1.0 mmHg at 25°C

1.635

1.83

0

4

1

16

325

0

DLCJNIBLOSKIQW-UHFFFAOYSA-N

InChI=1S/C12H8O4/c1-14-9-6-7-2-3-10(13)16-11(7)8-4-5-15-12(8)9/h2-6H,1H3

6-methoxyfuro[2,3-h]chromen-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~115.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.56 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 2: ≥ 2.5 mg/mL (11.56 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 3: ≥ 2.5 mg/mL (11.56 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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 (Please use freshly prepared in vivo formulations for optimal results.)