NF-κB; MAPK; Caspase-3/9

Isopsoralen treatment stimulates the accumulation of cartilage nodules in a dose-dependent manner. Isopsoralen increases the expression of chondrogenic marker genes like collagen II, collagen X, OCN, Smad4 and Sox9 in a time-dependent manner. Additionally, isopsoralen induces the activation of p38 MAP kinase and extracellular signal-regulated kinase (ERK), but not of c-jun N-terminal kinase (JNK). Isopsoralen significantly increases BMP-2 protein expression in a time-dependent manner and, through BMP-2 or MAPK signaling pathways, mediates a chondromodulating effect.

---

Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for its antiproliferative activity in treatment of different skin diseases. To verify the activity of angelicin, we employed human SH-SY5Y neuroblastoma cells to investigate its cytotoxicity, although its mechanism of action has not yet been fully elucidated. Here, we examined the cellular cytotoxicity of angelicin by cell viability assay, DNA fragmentation by DNA ladder assay, and activation of caspases and Bcl-2 family proteins by western blot analyses. The results of our investigation suggest that Angelicin increased cellular cytotoxicity in a dose- and time-dependent manner with IC(50) of 49.56 μM at 48 h of incubation. In addition, angelicin dose-dependently downregulated the expression of anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1 suggesting the involvement of the intrinsic mitochondria-mediated apoptotic pathway which did not participate in Fas/FasL-induced caspase-8-mediated extrinsic, MAP kinases, and PI3K/AKT/GSK-3β pathway. Furthermore, we clarified the dose-dependent upregulation of caspase-9 and caspase-3 which indicated that angelicin-induced apoptosis is mediated primarily through the intrinsic caspase-mediated pathway. In particular, the caspase-3 inhibitor, DEVD-fmk, induced a reduction in angelicin-induced cytotoxicity which confirmed that the intrinsic caspase-dependent pathway during this apoptosis which did not prevent cytotoxicity using MAP kinases and GSK-3 inhibitor. Taken together, our data shows that angelicin is an effective apoptosis-inducing natural compound of human SH-SY5Y neuroblastoma cells which suggests that this compound may have a role in future therapies for human neuroblastoma cancer. [1]

---

Human gammaherpesviruses including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are important pathogens as they persist in the host and cause various malignancies. However, few antiviral drugs are available to efficiently control gammaherpesvirus replication. Here we identified the antiviral activity of Angelicin against murine gammaherpesvirus 68 (MHV-68), genetically and biologically related to human gammaherpesviruses. Angelicin, a furocoumarin naturally occurring tricyclic aromatic compound, efficiently inhibited lytic replication of MHV-68 in a dose-dependent manner following the virus entry. The IC50 of angelicin antiviral activity was estimated to be 28.95μM, while the CC50 of angelicin was higher than 2600μM. Furthermore, incubation with angelicin efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lytic replication of human gammaherpresviruses in both EBV- and KSHV-infected cells. Taken together, these results suggest that MHV-68 can be a useful tool to screen novel antiviral agents against human gammaherepsviruses and that angelicin may provide a lead structure for the development of antiviral drug against gammaherpesviruses.[2]

---

Effects of Angelicin on cell viability [3]
To determine whether Angelicin has potential cytotoxicity on RAW 264.7 cells, the cell viability was evaluated by MTT assay (Fig. 2A) after incubating cells for 18 h in the absence or presence of LPS. The results showed that angelicin had no effect on the viability of RAW 264.7 cells at concentrations of 0, 12.5, 25, and 50 μM. However, concentrations of angelicin up to 100 μM were found to reduce the cell viability. Therefore, these observations showed that the effects of angelicin were not due to cytotoxicity.

---

Effects of Angelicin on the production of proinflammatory cytokines in LPS-stimulated RAW 264.7 cells [3]
In this study, we used ELISA kits to measure the level of TNF-α (Fig. 2B-a) and IL-6 (Fig. 2B-b) in the culture supernatants. Compared with the control group, treatment of RAW 264.7 cells with LPS alone led to a significant increase in cytokine production. However, compared with the LPS group, the levels of TNF-α and IL-6 in angelicin-pretreated, LPS-stimulated cells were reduced significantly in a dose-dependent manner.

---

Effects of Angelicin on inflammatory cell count in BALF of LPS-induced ALI mice Six hours after LPS injection, the numbers of total cells (Fig. 3A), neutrophils (Fig. 3B), and macrophages (Fig. 3C) were significantly higher compared with the control group (P < 0.01). Furthermore, pretreatment with angelicin (5, 10 mg/kg) decreased the number of total cells (P < 0.05) and neutrophils (P < 0.05 or P < 0.01) compared with those in the LPS group. In contrast, angelicin (1 mg/kg) did not decrease the number of macrophages (P < 0.05) compared with those in the LPS group.

In mice with sex hormone deprivation, isopsoralen has a significant osteoprotective effect on both male and female mice. Isopsoralen treatment for 8 weeks results in improved trabecular bone microstructure and increased bone strength. The elimination half-life of isopsoralen after intravenous administration to wistar rats is 5.35 hours. Kidney > lung > liver > heart > spleen > brain is the order in which the area under the tissues' curves for isopsoralen decrease. The elimination half-life of isopsoralen after oral administration to Wistar rats is 5.56 h, and its bioavailability is 70.35%.

---

Effect of Angelicin on cytokine production in LPS-induced ALI mice [3]
Six hours after LPS challenge, BALF was collected to investigate the levels of cytokine. Compared with the control group, the levels of TNF-α (Fig. 4A) and IL-6 (Fig. 4B) in BALF were significantly increased by LPS challenge. We found that, compared with the LPS group, angelicin dramatically reduced TNF-α and IL-6 production.

---

Effects of Angelicin on histologic changes in lung from LPS-induced ALI mice [3]
To assess the histologic changes (Fig. 5) after angelicin treatment in LPS-treated mice, the lungs were collected at 6 h after LPS injection for H&E staining. In the control group, no obvious histologic changes were observed in lung tissues. In contrast, in the LPS group, the lung specimens showed the most dramatic changes in lung architecture, such as alveolar wall thickening, alveolar hemorrhage, interstitial edema, and inflammatory cell infiltration. However, pretreatment with angelicin (1, 5, and 10 mg/kg) markedly attenuated these pathologic changes (Fig. 5).

---

Effects of Angelicin on LPS-induced lung W/D ratio [3]
Pulmonary edema, a typical symptom of inflammation, is indicated by the lung W/D ratios (Fig. 6). Compared with the control group, the lung W/D ratios were markedly increased after LPS stimulation. In contrast, in the angelicin groups, there were significant reductions in the lung edema formation.

---

Effects of Angelicin on MPO activity in mice with LPS-induced ALI [3]
Neutrophils are an important component of the inflammatory response during ALI. To investigate whether LPS-induced increase in polymorphonuclear accumulation in the lung was effectively prevented by angelicin, the activity of MPO was assessed (Fig. 7). Compared with the control group, we found that MPO activity was markedly increased 6 h after LPS instillation. However, intraperitoneal injection of angelicin led to significant inhibition of MPO activity.

---

Effect of Angelicin on NF-κB and MAPK activation in mice with LPS-induced ALI [3]
NF-κB (Fig. 8A) and MAPK (Fig. 8B) signaling pathways are known to play an important role in the regulation of inflammatory mediator production. Thus, we investigated the effects of angelicin on the activation of phosphor-ERK, phosphor-JNK, phosphor-p38, phosphor-IκBα, and phosphor-NF-κB p65. Western blotting results showed that LPS stimulation increased the phosphorylation levels of MAPK, phosphor-IκBα, and phosphor-NF-κB p65. This LPS-induced protein phosphorylation, except ERK phosphorylation, was significantly blocked by angelicin (1, 5, and 10 mg/kg).

Promoter reporter analysis [2]
For promoter activity analysis, 293T cells (1.2 × 105/well) were seeded in 24-well plates 24 h before transfection. A total of 650 ng of plasmid DNA per well was co-transfected using polyethylenimine, and treated with Angelicin at 1 h post-transfection. A luciferase reporter plasmid containing the upstream 210 bp-sequence of RTA cloned into pGL3-basic (pGL3-RTAp) was a kind gift from Dr. Ebrahimi at Liverpool University, UK. The plasmid DNA mixture included the luciferase reporter plasmid pGL3-RTAp (50 ng), pCMV-β-gal (80 ng), and Flag-mRTA (0.04–10 ng). The plasmid pCMV2-flag (260–269.96 ng) was added as a filler DNA to make 650 ng of total DNA. The luciferase assay system was used according to manufacturer’s instructions. The cells were washed with 1 × PBS and lysed with the 1 × passive lysis buffer 48 h after transfection. After one cycle of freeze–thaw, the cells were centrifuged to separate cell debris. Cell lysates (20 μl) were added to luciferase substrate buffer (40 μl) and luciferase activities were measured by Victor 3. Cell lysates (20 μl) were added to β-galactosidase substrate (30 μl) and incubated for 20 min. The activities of β-galactosidase were measured by PowerWave XS (Bio-Tek) and used as an internal control.

---

Plaque reduction assays [2]
To determine IC50 for MHV-68 antiviral activity, plaque reduction assay using MHV-68 WT was performed as previously described with minor modification (Harper, 2000). Briefly, Vero cells were seeded at 5 × 104 cells/well in 12 well plates. Next day, the cells were incubated with MHV-68 (∼100 pfu/well) for 90 min and the overlay media of DMEM containing 10% FBS and 0.6% methylcellulose was added upon removal of the virus inoculum. Tested compounds were diluted at the indicated concentrations into the normal growth media for pre-treatment (3 h prior to virus adoption) or the overlay media for post-treatment and added into the cells. After 5 days of incubation, the cells were fixed and stained with 0.2% crystal violet in 20% ethanol. Plaques were then counted and IC50 value was determined as the quantity of Angelicin required to reduce the plaque number by 50% using Prism 6.

---

Cytokine assays [3]
The concentrations of TNF-α and IL-6 in the supernatants of the BALF were measured using sandwich ELISA kits (Biolegend) according to the protocol recommended by the manufacturers. The optical density of the microplate was read at 450 nm, and the levels of TNF-α and IL-6 were expressed as picto- or nanogram per milliliter of BALF.

---

MPO assay [3]
MPO activity in lung was determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute. Six hours after LPS treatment, mice under diethyl ether anesthesia were killed and the right lungs were excised. One hundred milligrams of lung tissue were homogenized and fluidized in extraction buffer to obtain 5% of homogenate. The homogenate was centrifuged at 13,000g for 30 min at 4°C, and the cell-free extracts were stored at −20°C until further use. The enzymatic activity was determined by measuring the changes in absorbance at 460 nm using a 96-well plate reader.

ATDC5 cells are plated in 96-well plates at a density of 5×103 cells per well, incubated for an overnight period in media containing 10% FBS, and then subjected to various isopsoralen concentrations. Following a 24-hour incubation period, cells are treated with media containing 100 g/ml MTT for 2 hours at 37°C after being washed with phosphate-buffered saline (PBS). Following a PBS wash, the cells are dissolved in 200 l of DMSO. By using a spectrophotometer to measure the absorbance at a wavelength of 540 nm, the resulting solubilized purple formazan is quantified.

---

Cell viability assay [1]
Cell viability was determined using a cytotoxicity assay kit, CCK-8 according to the manufacturer’s protocol. The cells were plated into 96 wells to a density of 50–60 % confluence. After 24 h incubation in starvation media, the cells were treated with various concentrations of Angelicin as described in the figure legends. After chemical treatment of 48 h, CCK-8 (10 μl) was added to each well of the plates and incubated for 3 h. A 96-well microtiter plate reader (molecular devices) was used to determine the absorbance at 450 nm for CCK-8. The mean concentrations in each set of three wells were measured.

---

Detection of DNA fragmentation [1]
For the detection of apoptotic DNA cleavage, the DNA fragmentation assay was performed using the ladder DNA fragmentation assay. In brief, cells were collected after treatment at various concentrations of Angelicin as described in the figure legends and washed in PBS. The cells were then lysed with 500 μl of genomic DNA extraction buffer (0.1 M NaCl, 10 mM EDTA, 0.3 M Tris–HCl, 0.2 M sucrose, pH 8.0). The lysate was incubated with 20 μl of 10 % SDS solution and incubated at 65 °C for 30 min. 120 μl of potassium acetate (pH 5.3) was added and stored on ice for 1 h after centrifugation for 10 min at 4 °C and 12,000 rpm. 2 μl (10 mg/ml) of RNase was added to the supernatant, and incubated for 30 min at room temperature. The DNA was extracted by washing the resultant pellet in phenol/chloroform extraction and precipitation by ethanol, and then dissolved with distilled water. DNA fragmentation was visualized by electrophoresis in a 0.8 % agarose gel containing ethidium bromide.

---

Western blot analysis [1]
Human SH-SY5Y neuroblastoma cells were starved on 60-mm culture dishes in DMEM with 0.5 % FBS for 24 h. Cells were pretreated with various concentrations of Angelicin as indicated in each figure legend, and then washed twice with ice-cold PBS. Cells were lysed in lysis buffer (2 % SDS, Na3VO4, and protease inhibitor cocktail). After incubation on ice for 10 min and sonication for 10 s in 10 % amplitude, the lysates were centrifuged (13,000 rpm, 20 min). The supernatants were collected and protein concentrations were determined by Bradford assay. Equal amounts of proteins were separated by SDS–PAGE (8 %, 10 %, or 15 % reducing gels), transferred to polyvinylidene difluoride membranes, and blocked with 5 % non-fat milk. Membranes were incubated overnight in primary antibody at 4 °C. Membranes were then washed in TBST (10 mM Tris, 140 mM NaCl, 0.1 % Tween-20, pH 7.6), incubated with appropriate secondary antibody, and washed again in TBST. Bands were visualized by enhanced chemiluminescence and exposed to X-ray film. The relative abundance of each band was quantified via the Bio-profile Bio-1 D application, and the expression levels were normalized to β-actin.

---

Cytotoxicity assays [2]
The cytotoxicity of Angelicin was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, as previously described (Cho et al., 2008). Briefly, 104 cells were seeded in 100 μl of complete medium into each well of 96-well plates and incubated with various concentrations of angelicin in a volume of 2 μl for 24 h. After treatment, 10 μl of MTT solution (5 mg/ml in 1 × PBS) was added to each well and the mixtures were incubated for an additional 3 h at 37 °C. The plates were then centrifuged, the supernatants were discarded, and 100 μl of DMSO was added to each well. After the crystals were dissolved, the amount of reduced MTT was measured at 570 nm.

---

Cell culture and treatment [3]
The RAW 264.7 mouse macrophage cell line was purchased from the China Cell Line Bank. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, at 37°C under a humidified atmosphere of 5% CO2 in air. In all experiments, cells were incubated in the presence or absence of various concentrations of Angelicin, which were always added 1 h prior to LPS (1 μg/mL) treatment.

---

MTT assay for cell viability [3]
MTT was used to evaluate the cell viability. RAW 264.7 cells were mechanically scraped and 100 μL cells were plated at a density of 4 × 105 cells/mL in 96-well plates in a 37°C, 5% CO2 incubator for 1 h. Then the cells were treated with 50 μL of different concentrations of Angelicin (0–200 μg/mL) for 1 h, followed by stimulation with 50 μL LPS (4 μg/mL). After 18 h of LPS stimulation, 20 μL MTT (5 mg/mL) was added to each well, and the cells were further incubated for an additional 4 h. The supernatants were then removed and resolved with 150 μL/well DMSO. The optical density was measured at 570 nm on a microplate reader.

---

Measurement of proinflammatory cytokine (TNF-α and IL-6) production [3]
Angelicin was dissolved in DMSO prior to any treatment. RAW 264.7 cells (4 × 105 cells/mL) were seeded into 24-well plates and pretreated with 12.5, 25, and 50 μg/mL Angelicin for 1 h prior to treatment with 1 mg/L LPS for 24 h. Cell-free supernatants were collected and analyzed by ELISA for TNF-α and IL-6, following the manufacturer's protocol.

LPS-induced ALI model [3]
All mice were randomly divided into five groups: the control group, LPS group, and Angelicin (1, 5, and 10 mg/kg) + LPS groups. Before inducing acute lung inflammation, Angelicin (at doses of 1, 5, and 10 mg/kg) was given intraperitoneally. One hour after pretreatment with Angelicin, the BALB/c mice were diethyl ether anesthetized and 10 μg LPS was instilled intranasally (i.n.) in 50 μL phosphate-buffered saline (PBS) to induce lung injury. Control mice were given 50 μL PBS i.n. without LPS. Six hours after LPS exposure, the mice were killed by diethyl ether asphyxiation. The lungs were lavaged three times through a tracheal cannula with 0.5 mL autoclaved PBS, instilled up to a total volume of 1.3 mL. The excised lungs were placed into a sterile centrifuge tube and stored at −80°C. The bronchoalveolar lavage fluid (BALF) recovered from each sample was centrifuged (4°C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemocytometer, and cytospins were prepared for differential cell counts by staining with the Wright−Giemsa staining method.

Absorption, Distribution and Excretion
Objective: To investigate the nasal absorption patterns of psoralen and isopsoralen at different concentrations. Methods: An orthotopic nasal circulation model was established in rats, and the contents of psoralen and isopsoralen were determined by high-performance liquid chromatography (HPLC). Results: The nasal absorption of psoralen and isopsoralen followed zero-order kinetics and reached saturation with increasing concentration. Conclusion: Appropriate concentrations are required for the preparation of nasal formulations of psoralen and isopsoralen. Psoralen compounds are a new class of drugs, with psoralen and isopsoralen being their active ingredients. This study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the pharmacokinetics, tissue distribution, and excretion of these two compounds after intravenous administration to Wistar rats. Elimination half-lives of psoralen and isopsoralen were 4.88 hours and 5.35 hours, respectively. Following administration, the area under the pharmacokinetic curve (AUC) of psoralen in various tissues was as follows: liver > lung > heart > kidney > spleen > brain; for isopsoralen, the AUC was as follows: kidney > lung > liver > heart > spleen > brain. After administration, 51.27% of psoralen and 56.25% of isopsoralen were excreted unchanged, with urine being the primary excretion route. Furthermore, the pharmacokinetics of psoralen and isopsoralen in Wistar rats after oral administration were investigated. The elimination half-lives of psoralen and isopsoralen were 4.13 hours and 5.56 hours, respectively, with relative bioavailabilities of 61.45% and 70.35%, respectively. Overall, the results indicate that psoralen compounds have high oral bioavailability and are rapidly and widely distributed in tissues after intravenous injection, but clearance and excretion are slow.

---

Biological Half-Life
...After intravenous injection of psoralen and isopsoralen, the elimination half-lives in Wistar rats were 4.88 hours and 5.35 hours, respectively. ...After oral administration of psoralen and isopsoralen, the elimination half-lives in Wistar rats were 4.13 hours and 5.56 hours, respectively...

Toxicity Summary
Identification and Uses: Isopsoralen is a natural furanocoumarin. It has been used as an experimental therapy. Human Studies: Isopsoralen acts as an estrogen receptor α agonist in vitro and significantly promotes the proliferation of MCF-7 cells. Isopsoralen forms DNA monoadducts upon photoactivation. In human cells, isopsoralen promotes sister chromatid exchange. Animal Studies: Intraperitoneal and oral administration of isopsoralen have potent sedative, anticonvulsant, and central muscle relaxant effects in rats, mice, and rabbits. Isopsoralen is photomutable in bacteria and animal cells, forming DNA monoadducts. Many furanocoumarins' mechanisms of action are based on their ability to form photoadducts with DNA and other cellular components, such as RNA, proteins, and membrane proteins, such as phospholipases A2 and C, calcium-dependent and cAMP-dependent protein kinases, and epidermal growth factor. Furanocoumarins can insert between DNA base pairs and form cycloadducts upon UVA irradiation. (L579)

---

Interactions
Photochemical genotoxicity can be detected by appropriately modifying most standard in vitro genotoxicity assays. The most sensitive method for detecting potential photogenotoxic substances appears to be the in vitro study of DNA damage (DNA strand breaks, chromosomal aberrations, micronuclei) in mammalian cells. In a previous paper, we proposed a micronucleus assay using Chinese hamster V79 cells to study photogenotoxic compounds. This assay is suitable for detecting a variety of photogenotoxic compounds with different photoactivation mechanisms. To expand the experimental experience of this assay, this paper further presents data from a screening assay of 16 potential photosensitizers. We also investigated the photofracture and photocytotoxicity of these compounds. To date, all substances detected as photogenotoxic in the photomicronucleus assay have also exhibited photocytotoxicity, and vice versa. Among the compounds tested in this study, thiamethoxam, 5-mercaptopurine (5-MOP), angelicin, nitrazepam, benzylfluoxazine, and dacarbazine all exhibited both photogenotoxicity and photocytotoxicity. Furthermore, 6-mercaptopurine, a metabolite of azathioprine, was positive for both endpoints, while azathioprine itself was negative. Azathioprine appears to be a compound lacking photogenotoxicity in vitro but which can be enzymatically metabolized into a photosensitizer. The results of this study expand the database of photomicronucleus assays to 35 compounds, all tested using the same protocol and irradiation conditions. This paper summarizes and discusses the photogenotoxicity results of all these compounds and explores their different photoactivation mechanisms, photocytotoxicity, and photocarcinogenicity. This study found that crude extract of Psoralea corylifolia inhibited acetylcholinesterase activity in vitro and improved scopolamine-induced impairment of inhibitory avoidance response and spatial behavior in the water maze in rats. Among all components, the chloroform component showed the strongest inhibitory effect on acetylcholinesterase activity and alleviated scopolamine-induced impairment of inhibitory avoidance response. High-performance liquid chromatography (HPLC) identified psoralen and isopsoralen as the two main components of the chloroform extract of psoralen, which also alleviated the damage caused by inhibitory avoidance responses. The results indicate that psoralen and isopsoralen are the main active components of psoralen, capable of gradually reversing scopolamine-induced amnesia, with their action partly related to the inhibition of acetylcholinesterase (AChE) activity and activation of the central cholinergic nervous system. Monofunctional psoralen combined with UVA radiation did not induce erythema and its mutagenicity was lower than that of bifunctional psoralen combined with UVA radiation. Therefore, in recent years, they have received widespread attention as potential drugs for treating various skin diseases. This study aimed to investigate the immunological side effects in mice treated with monofunctional psoralen combined with UVA radiation. We found that angelicin combined with UVA radiation could inhibit the contact hypersensitivity response to dinitrofluorobenzene in mice. This reduction in immune response was related to the presence of splenic suppressor cells, which can transmit the inhibitory effect to normal receptors. Treatment with angelica dahurica and UVA radiation also reduced the number of Thy-1+ and Ia+ dendritic epidermal cells at the treatment site. We concluded that although this monofunctional psoralen is not phototoxic, it has immunosuppressive activity in mice. Due to the adverse side effects of treating skin diseases such as psoriasis with 8-methoxypsoralen and long-wave ultraviolet A (UVA) radiation, the use of monofunctional psoralens, which are less erythematous, less mutagenic, and generally non-phototoxic, has received considerable attention. However, little is known about the immunosuppressive properties of monofunctional psoralens. This study aimed to investigate the effects of parenteral administration of monofunctional psoralen-angelica dahurica combined with UVA radiation on the immune response. UVA irradiation following angelica dahurica injection significantly suppressed delayed-type hypersensitivity to allogeneic antigens in a dose-dependent manner. Similarly, the proliferative capacity of spleen cells in animals treated with angelica dahurica and UVA was significantly reduced in response to allogeneic antigens. This inhibitory effect is specific to the alloantigens used in animals sensitized with angelica and UVA treatment, and is accompanied by the appearance of antigen-specific suppressor T lymphocytes in the spleen. These data indicate that the effect of systemic administration of monofunctional psoralen followed by UVA irradiation on the immune response is similar to that observed after injection of bifunctional psoralen. These findings also suggest that the severe skin phototoxicity induced by bifunctional psoralen and UVA irradiation is not a necessary condition for inducing systemic immunosuppression. Furthermore, angelica combined with UVA induces systemic antigen-specific immunosuppression without significant skin phototoxicity, suggesting that this compound and related compounds may be used to specifically suppress unwanted immune responses.
For more complete data on interactions of isopsoralens (12 in total), please visit the HSDB record page.

---

Non-human toxicity values
Mouse intraperitoneal LD50: 254 mg/kg
Rat intraperitoneal LD50: 165 mg/kg
Rat oral LD50: 322 mg/kg

[1]. Angelicin induces apoptosis through intrinsic caspase-dependent pathway in human SH-SY5Y neuroblastoma cells. Molecular and Cellular Biochemistry, 2012 Oct;369(1-2):95-104.

[2]. Antiviral activity of angelicin against gammaherpesviruses. Antiviral Res . 2013 Oct;100(1):75-83.

[3]. Angelicin regulates LPS-induced inflammation via inhibiting MAPK/NF-κB pathways. J Surg Res . 2013 Nov;185(1):300-9.

Angelicin is a furocoumarin. It has been reported to be found in Hoita macrostachya, Mandragora autumnalis, and other organisms with relevant data. Angelicin is found in coriander. It is a component of the roots and leaves of parsley (Angelica archangelica). It is also found in the roots and surface of parsnip and diseased celery. Angelicin is a furocoumarin. It is also found in Bituminaria bituminosa. It is listed as a Group 3 carcinogen by the International Agency for Research on Cancer (IARC) (Angelicin and ultraviolet A radiation). (Wikipedia). See also: Parsley root (part); Cullen corylifolium fruit (part). Mechanism of Action: The combined action of 365 nm ultraviolet light and Angelicin inhibits the injection of λ phage into host cells. This article explores the injection inhibition phenomenon from the perspective of photochemically induced cross-linking of DNA within the phage head. Based on the PPP method, the first excited-state electronic structures of xanthotoxin, psoralen, and angelicin obtained by quantum chemical calculations are very similar.

---

Therapeutic Uses
/Exploring Treatments/ Background: Modern research shows that psoralen has a significant inhibitory effect on tumor growth in various animals and humans. Objective: To isolate coumarin compounds—psoralen and isopsoralen—from the traditional Chinese medicine Psoralea corylifolia L. using chromatographic techniques and separation and purification methods, and to observe the inhibitory effects and adverse reactions of psoralen and isopsoralen on the growth of osteosarcoma xenografts in nude mice. Methods: Crude extracts of Psoralea corylifolia L. were prepared using the dried, mature fruit as raw material via ethanol reflux. Crude extracts were separated by column chromatography. The compounds were structurally identified by ¹H-NMR and ¹³C-NMR, revealing psoralen and isopsoralen as compounds with contents of 99.7% and 99.6%, respectively. A nude mouse osteosarcoma model was established. Nude mice were randomly divided into four groups: saline group, low-dose psoralen group, high-dose psoralen group, low-dose isopsoralen group, high-dose isopsoralen group, and cisplatin group. The inhibition rate of osteosarcoma volume and weight in each group was observed. Serum alkaline phosphatase activity was measured using radioimmunoassay. Peripheral blood cell and bone marrow nucleated cell counts were determined. Histopathology of the heart, liver, spleen, lungs, kidneys, and tumors was observed using optical microscopy. The fine structure of tumor cells was observed using electron microscopy. Results: The tumor volume inhibition rates of the low-dose psoralen and isopsoralen groups were 43.75% and 40.18%, respectively, and the tumor weight inhibition rates were 38.83% and 37.77%, respectively. The tumor volume inhibition rates of the high-dose psoralen and isopsoralen groups were 67.86% and 66.96%, respectively, and the tumor weight inhibition rates were 49.47% and 47.87%, respectively. Both psoralen and isopsoralen significantly reduced serum alkaline phosphatase (AKP) levels. Psoralen and isopsoralen induced osteosarcoma cell apoptosis or necrosis. After administration of high doses of psoralen and isopsoralen, toxic reactions such as writhing, fatigue, and decreased activity were observed. Histopathological examination of the kidney tissue showed dilation and congestion of the renal tubules and interstitium, as well as inflammatory cell aggregation in the intercellular spaces of the kidneys. Psoralen and isopsoralen did not cause significant toxic side effects on bone marrow or other organs such as the heart, lungs, liver, and spleen. Conclusion: Psoralen and isopsoralen inhibited the growth of osteosarcoma xenografts in nude mice and induced tumor cell apoptosis or necrosis without significant toxicity.
/EXPL THER/ Angelica sinensis, a furanocoumarin compound found in the fruit of Psoralea corylifolia L., has been reported to possess anti-inflammatory activity. This study aimed to investigate the protective effect of angelica sinensis against ovalbumin (OVA)-induced allergic asthma in mice. Mice were sensitized with OVA on days 0 and 14, and challenged with OVA three times from days 21 to 23. Angelica sinensis (2.5, 5, and 10 mg/kg) was injected intraperitoneally one hour before OVA challenge after the first OVA sensitization. The levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid (BALF) and serum IgE were detected using enzyme-linked immunosorbent assay (ELISA). Lung histological changes were detected using hematoxylin-eosin (H&E) staining. Results showed that angelica extract significantly inhibited the infiltration of inflammatory cells into the lungs. Histological studies indicated that angelica extract significantly reduced OVA-induced lung injury. Simultaneously, angelica extract treatment dose-dependently inhibited the production of IL-4, IL-5, and IL-13 in ovalbumin (OVA)-induced bronchoalveolar lavage fluid (BALF) and serum IgE levels. Furthermore, angelica extract was found to inhibit airway hyperresponsiveness and NF-κB activation. In summary, our results indicate that angelica extract inhibits allergic airway inflammation and hyperresponsiveness by suppressing NF-κB activation.
/EXPL THER/ Isopsoralen is a furanocoumarin compound with estrogen-like activity. This study aimed to investigate the estrogen-like neuroprotective effects of isopsoralen in an animal model of spinal cord injury (SCI). The results showed that intraperitoneal injection of isopsoralen (5 and 10 mg/kg/day for two weeks) significantly improved hindlimb motor function in SCI mice, as evidenced by BMS scores and tilt plane angle tests. Morphological data showed that isopsoralen significantly alleviated spinal cord gray matter injury and induced upregulation of estrogen receptor α (ERα) levels. The ERα antagonist MPP (0.3 mg/kg) blocked the neuroprotective effect of isopsoralen, while the estrogen receptor β (ERβ) antagonist PHTPP (0.3 mg/kg) had no such effect. Isopsoralen treatment increased the levels of phosphorylated PI3K and AKT (P-PI3K and P-AKT) in the spinal cord of SCI mice and exhibited significant anti-apoptotic activity. These results indicate that isopsoralen exerts estrogen-like neuroprotective effects by activating ERα and regulating the PI3K/AKT pathway, thus counteracting SCI-induced apoptosis. Previous studies have reported that angelica extract has anti-proliferative effects on various tumor cells. However, to our knowledge, the efficacy of angelica extract monotherapy in human liver cancer remains to be investigated. This study evaluated the antitumor activity of angelica extract in vitro and in vivo and explored its molecular mechanism of action. The results showed that angelica extract induced apoptosis in liver cancer cells in a dose- and time-dependent manner. Furthermore, in HepG2 and Huh-7 cells, angelica extract-induced apoptosis was confirmed to be mitochondrial-dependent, involving the phosphatidylinositol-4,5-bisphosphate 3-kinase/RAC serine/threonine protein kinase signaling pathway. In addition, in a liver-bearing tumor xenograft mouse model, administration of angelica extract inhibited tumor growth without producing significant side effects. These results suggest that angelica extract may have the potential to be a novel therapeutic agent for treating liver cancer patients. Background: Angelica extract is a furanocoumarin compound found in the fruit of Psoralea corylifolia L. This study aimed to investigate the protective effect of angelica dahurica on inflammation in a lipopolysaccharide (LPS)-stimulated RAW 264.7 cell and LPS-induced in vivo acute lung injury (ALI) model. Materials and Methods: Twenty-four hours after LPS administration, the concentrations of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in the culture supernatant of RAW 264.7 cells were measured. An acute lung injury model was induced by intratracheal instillation of LPS. Six hours after LPS inhalation, bronchoalveolar lavage fluid and lung tissue samples were collected for enzyme-linked immunosorbent assay (ELISA), histological analysis, and Western blot analysis. Results: The results showed that angelica dahurica pretreatment significantly reduced the levels of TNF-α and IL-6 in vitro and in vivo, and significantly reduced the number of inflammatory cells, lung wet/dry weight ratio, and myeloperoxidase activity in the lung tissue of LPS-induced acute lung injury (ALI) mice. Furthermore, Western blot analysis showed that angelicin inhibited the phosphorylation of IκB, NF-κBp65, p38 MAPK, and JNK in LPS-induced ALI. Conclusion: These results suggest that angelicin may have potential advantages in preventing inflammatory diseases by inhibiting the NF-κB and MAPK pathways. Our data indicate that angelicin may be a potential novel drug for clinical prevention of inflammatory responses and diseases.

---

Background: Angelicin is a furanocoumarin compound found in the fruit of Psoralea corylifolia L. This study aimed to investigate the anti-inflammatory protective effects of angelicin on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and an LPS-induced in vivo acute lung injury model. Materials and Methods: The concentrations of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in the culture supernatant of RAW 264.7 cells were measured 24 hours after LPS administration. An acute lung injury model was induced by intratracheal infusion of LPS. Six hours after LPS inhalation, bronchoalveolar lavage fluid and lung tissue samples were collected for enzyme-linked immunosorbent assay (ELISA), histological and Western blot analysis. Results: The results showed that angelica root extract pretreatment significantly reduced the levels of TNF-α and IL-6 in vitro and in vivo, and significantly reduced the number of inflammatory cells, lung wet weight/dry weight ratio and myeloperoxidase activity in the lung tissue of LPS-induced acute lung injury (ALI) mice. In addition, Western blot analysis showed that angelica root extract inhibited the phosphorylation of IκBα, NF-κBp65, p38 MAPK and JNK in LPS-induced ALI. Conclusion: These results suggest that angelica root extract may play a potential role in preventing inflammatory diseases by inhibiting the NF-κB and MAPK pathways. Our data suggest that angelica root extract may be a potential novel drug for the prevention of inflammatory responses and diseases. [3]

C11H6O3

186.16354

186.031

C, 68.89; H, 5.44; N, 9.45; O, 5.40; S, 10.82

523-50-2

29462-18-8; 29462-19-9 (HCl)

10658

White to off-white solid powder

1.4±0.1 g/cm3

362.6±27.0 °C at 760 mmHg

132-134ºC

173.1±23.7 °C

0.0±0.8 mmHg at 25°C

1.667

2.01

0

3

0

14

284

0

O=C1C=CC2=CC=C(OC=C3)C3=C2O1

XDROKJSWHURZGO-UHFFFAOYSA-N

InChI=1S/C11H6O3/c12-10-4-2-7-1-3-9-8(5-6-13-9)11(7)14-10/h1-6H

furo[2,3-h]chromen-2-one

Knoll brand of bentazepam; QM 6008; Angelicin; 523-50-2; ISOPSORALEN; 2H-Furo[2,3-H]chromen-2-one; furo[2,3-h]chromen-2-one; Angecin; 2-Oxo-(2H)-furo(2,3-h)-1-benzopyran; Furo(2,3-h)coumarin; QM-6008; Bentazepam; Thiadipone; Tiadipone; Bentazepam [USAN:INN]; CI-718; Tiadipona; Thiadipone

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: 14~33.3 mg/mL (75.2~179.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (13.43 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (13.43 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.

---

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