Urolithin M5 is a neuraminidase inhibitor isolated from olive leaves. Urolithin M5 has anti-influenza virus activity in vitro and in vivo by inhibiting viral NA activity. Urolithin M5 can improve the survival rate of mice and improve pulmonary edema in a pr8-infected mouse model. Urolithin M5 can be used for anti-influenza research.

Physicochemical Properties

Molecular Formula	C13H8O7
Molecular Weight	276.20
Exact Mass	276.027
CAS #	91485-02-8
PubChem CID	18504424
Appearance	White to off-white solid powder
Density	1.894±0.06 g/cm3 (20 °C, 760 mmHg)
Boiling Point	727.4±60.0 °C (760 mmHg)
LogP	1.474
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	0
Heavy Atom Count	20
Complexity	400
Defined Atom Stereocenter Count	0
SMILES	O=C1C2C(=C(C(=C(C=2)O)O)O)C2C(=C(C(=CC=2)O)O)O1
InChi Key	ZELMDXUEWHBWPN-UHFFFAOYSA-N
InChi Code	InChI=1S/C13H8O7/c14-6-2-1-4-8-5(3-7(15)9(16)11(8)18)13(19)20-12(4)10(6)17/h1-3,14-18H
Chemical Name	3,4,8,9,10-pentahydroxybenzo[c]chromen-6-one
Synonyms	urolithin M5; 91485-02-8; 3,4,8,9,10-Pentahydroxybenzo[c]chromen-6-one; 3,4,8,9,10-Pentahydroxy-6H-dibenzo[b,d]pyran-6-one; 3,4,8,9,10-Pentahydroxyurolithin; Decarboxyellagic Acid;; SCHEMBL2357304; 3,4,8,9,10-pentahydroxydibenzo[b,d]pyran-6-one;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Neuraminidase
ln Vitro	In Vitro Activities of Urolithin M5 [1] The cytotoxicity of urolithin M5 was initially assessed using the MTT assay on MDCK cells. Urolithin M5 had a CC50 value of 227.4 μM (Figure 1a). A plaque reduction assay was performed to find the inhibition of urolithin M5 on amantadine-resistant strain A/WSN/33(H1N1) (WSN), oseltamivir-resistant strain A/California/7/2009(H1N1) (pdm09), A/PR/8/34(H1N1) (PR8) and A/Hong Kong/1/68(H3N2) (HK68). As shown in Figure 1b, urolithin M5 suppressed all four strains in a dose-dependent manner. Notably, 25 μM urolithin M5 inhibited all virus growth, indicating that compared with other phytochemicals from GLY, canaroleosides A, B and C, urolithin M5 was the most effective compound, with an IC50 ranging from 3.74 to 16.51 μM. Urolithin M5 Did Not Act on Attachment Stage [1] To investigate if urolithin M5 exerts an anti-influenza virus activity by interacting with virus attachment, hemagglutination inhibitory assays were performed. In this assay, influenza virus could induce hemagglutinin in chicken erythrocytes by means of forming lattices. This is due to the interaction of the HA1 unit and the sialic acid receptor of the host. Without WSNs, urolithin M5 treatment led to a red spot appearing in the U-bottom reaction well (Figure S5). With WSN, lattice appearance showed in 5–500 μM urolithin M5 or PBS control treatment. PGG, as a positive control, could interrupt hemagglutination and generate erythrocyte red spots. Oseltamivir, as a negative control, did not have an effect on the lattice appearance. These results demonstrated that urolithin M5 had no activity on HA1. Urolithin M5 Inhibited NA Activities [1] The effects of urolithin M5 on NA were tested using the NA substrate MUNANA. The reaction of MUNANA and IAV produced a fluorescent signal compound, 4-methylumbelliferone. Urolithin M5 at 10, 100, 125, 250, 500, and 1000 μM was added to the reaction wells, and ten-fold diluted oseltamivir acid was used as a positive control of this assay (except resistant strain pdm09). As shown in Figure 2, urolithin M5 could inhibit NA activity with an IC50 of 243.2 μM (WSN), 191.5 μM (pdm), 257.1 μM (PR8), and 174.8 μM (HK68).
ln Vivo	Urolithin M5 Improved the Survival Rate of Mice [1] The in vivo effect of urolithin M5 was examined in a mouse model. Its dose was decided according to reports on in vivo doses of the urolithin family. Generally, infected mice lost body weight until day 10, and all died. From the result in Figure 3b, body weights of urolithin M5 and oseltamivir groups rebound at day 9. A total of 200 mg/kg/d urolithin M5 treatment improved the survival rate to 50%, which was significantly higher than the vehicle group (Figure 3a). These results indicated that 200 mg/kg/d urolithin M5 had a protective effect on PR8-infected mice. Urolithin M5 Reduced Lung Index and Lung Viral Load [1] Six mice of each group were selected and sacrificed randomly on the fourth day post-infection to obtain their lung index and lung viral titer. Compared with the vehicle group, urolithin M5 treatment decreased the lung index from 1.445 to 0.8875 (Figure 3c), indicating an improvement in lung edema. Lung viral titer was decreased by 0.52 log from Figure 3d, coinciding with the anti-influenza virus effect of urolithin M5 treatment in a cell model. Urolithin M5 Reduced the Cytokine Expression of Mouse Lung [1] Influenza virus infection could induce inflammatory storm in the lungs showing from the overexpression of cytokines. To determine the inflammatory markers after urolithin M5 treatment, the right part of the mouse lungs was homogenized on day 4 post-inoculation, and the expression levels of NF-κB, TNF-α and IL-6 were evaluated. As shown in Figure 4a, the three cytokines were upregulated in the vehicle group. Oseltamivir and urolithin M5 treatment both decreased the production of cytokines. Urolithin M5 had better performance than oseltamivir in the regulation of NF-κB levels. Urolithin M5 Reduced Lung Pathology [1] The left parts of lung tissues collected on the fourth day post-infection were examined to determine the histopathological changes. As shown in Figure 4b, changes in inflammation were observed after hematoxylin and eosin staining, including interstitial expansion, edema and inflammatory cell infiltration around small vessels. After oseltamivir treatment, exudate around small vessels and bronchus was significantly reduced. After urolithin M5 treatment, fewer bronchi were injured, and fewer inflammatory cells appeared, but inflammatory cell infiltration around small vessels was still observed. These results indicated that urolithin M5 treatment alleviated lung pathology and lesions in influenza virus infection.
Enzyme Assay	Hemagglutination Inhibitory Assay [1] A hemagglutination inhibitory assay was performed with erythrocyte and WSN virus. WSN (4HA units) and two-fold diluted F2-3-4-6 were added to a U-bottom plate and mixed slightly. After incubating for 30 min at 37 °C, 0.05% chicken erythrocytes in PBS were freshly prepared and added to the reaction wells. After incubating for 30 min at room temperature, hemagglutination could be observed. Pentagalloyglucose (PGG) was used as a positive control, and oseltamivir acid was used as a negative control. The reaction well without virus solution was included to confirm the effect of compounds on chicken erythrocytes. Three parallels were performed for confirmation. Neuraminidase Inhibitory Assay [1] To confirm the inhibitory effect of F2-3-4-6 on influenza virus NA, MUNANA and four different viral strains were used to perform an NA inhibitory assay. MES buffer and MUNANA were prepared, respectively. Each reaction well of a black 96-well plate (Costar) contained 30 mM MES buffer, virus solution, F2-3-4-6 dilution and distilled water to make up the volume of 90 μL. After incubating at 37 °C for 30 min, 10 μL 1 mM MUNANA solution was added immediately to each well. After 30 min at 37 °C, the fluorescence intensity (F) of each well was measured by a CLARIOstar multi-mode microplate reader with an excitation wavelength of 322 nm and an emission wavelength of 450 nm. Oseltamivir acid was used as positive control, and three parallels were performed for confirmation. The inhibitory effect of F2-3-4-6 on NA activity was calculated by: NA inhibition (%) = (Fcontrol − Fcompound)/(Fcontrol − Fblank) × 100%
Cell Assay	Cytotoxicity Assay [1] The cytotoxicity of F2-3-4-6 was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Monolayer MDCK cells seeded on a 96-well plate were treated with two-fold diluted F2-3-4-6 in FBS-free MEM. After incubating for 48 h at 37 °C, freshly prepared MTT in PBS was added to each well. After 4 h incubation, the supernatant was removed, and 100 μL of DMSO was added per well to dissolve the formazan crystals. The assay was repeated three times for confirmation. A ClarioStar microplate reader was used to measure the absorbance at 570 nm. CC50 was defined as the concentration that generated a 50% cytotoxic effect. Virus Infection and Titer [1] MDCK cells on 6-well plates were washed with PBS. Virus solution A/WSN/33(H1N1) (WSN), A/PR/8/34(H1N1) (PR8), A/California/7/2009(H1N1) (pdm) or A/Hong Kong/1/68(H3N2) (HK68) was 10-fold diluted and added into MDCK cells at 1000 μL per well. After incubating at 37 °C for 2 h, the monolayer cell was coated with FBS-free MEM containing 2 μg/mL TPCK-treated trypsin (unnecessary for WSN infection) and 1% melted agarose in PBS. After infection for 48 h, the agarose plugs were removed, and the cells were stained with a staining solution (0.25% coomassie blue, 10% acetic acid, 50% methanol). The dilution resulting in 200 plaque-forming units (PFU) was determined and used for infection in method 4.7. Plaque Reduction Assay [1] MDCK cells were seeded on 6-well plates and incubated overnight. Confluent MDCK cells were washed with PBS and infected with 200 PFU of A/WSN/33(H1N1) (WSN), A/PR/8/34(H1N1) (PR8), A/California/7/2009(H1N1) (pdm) or A/Hong Kong/1/68(H3N2) (HK68). F2-3-4-6 was two-fold diluted with freshly prepared FBS-free MEM containing 2 μg/mL TPCK-treated trypsin (unnecessary for WSN infection). After incubating at 37 °C for 2 h, the monolayer cell was coated with a mixture of F2-3-4-6 solution and 1% melted agarose in PBS. After infection for 48 h, the agarose plugs were removed, and the cells were stained with a staining solution (0.25% coomassie blue, 10% acetic acid, 50% methanol). The assay was repeated three times for confirmation. The concentration of F2-3-4-6 that inhibited 50% of virus-induced plague was determined as the IC50 (50% inhibitory concentration).
Animal Protocol	Anti-Influenza Virus Test in Mouse Model [1] Mice were divided randomly into four groups, with 12 mice per group. The F2-3-4-6, oseltamivir, and vehicle groups were intranasally infected with 2 MLD50 of PR8 in a volume of 20 μL. The F2-3-4-6 groups were orally administered 200 mg/kg/day of F2-3-4-6. The oseltamivir group were orally administered oseltamivir at 65 mg/kg/d as a positive control. The control and vehicle groups were treated with 0.5% CMC solution only. Herb treatment was administered once a day for six consecutive days. Body weights were recorded for 16 consecutive days.
References	[1].Urolithin M5 from the Leaves of Canarium album (Lour.) DC. Inhibits Influenza Virus by Targeting Neuraminidase. Molecules. 2022 Sep 5;27(17):5724.
Additional Infomation	3,4,8,9,10-Pentahydroxybenzo[c]chromen-6-one has been reported in Lagerstroemia speciosa, Punica granatum, and other organisms with data available. Ganlanye, the leaves of Canarium album (Lour.) DC., were recorded as a major traditional herb for warm disease treatment. In our study, urolithin M5 was identified as a potent anti-influenza virus agent from this herb. Our research provides scientific evidence for the application of GLY in influenza treatment and improves public confidence to traditional medicine. [1]

Solubility Data

Solubility (In Vitro)

Typically soluble in DMSO (e.g. 10 mM)

Solubility (In Vivo)

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 Formulations (e.g. IP/IV/IM/SC) 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). 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 Formulations 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). 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.6206 mL	18.1028 mL	36.2056 mL
	5 mM	0.7241 mL	3.6206 mL	7.2411 mL
	10 mM	0.3621 mL	1.8103 mL	3.6206 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.