Natural product; secondary metabolite

Osteoclasts are the only cells that can resorb bone and they are produced from monocytes/macrophages in the presence of M-CSF and RANKL and are activated in vivo by an immune response. Usnic acid is a secondary metabolite of lichen and has a unique dibenzofuran skeleton. It has been used for years in cosmetics, fragrances, and traditional medicines. It has a wide range of bioactivities, including anti-inflammatory, anti-bacterial, anti-cancer, anti-viral, and so on. However, the anti-osteoclastogenic activity of usnic acid has not been reported yet. In this study, we investigated whether usnic acid could affect RANKL-mediated osteoclastogenesis. Usnic acid significantly inhibited RANKL-mediated osteoclast formation and function by reducing the transcriptional and translational expression of NFATc1, a master regulator of osteoclastogenesis [1].

Effects of Usnic Acid on Serum Biochemical Marker in LPS-Induced Mice [1]
After 8 days of the first usnic acid injection, the concentrations of TRAP-5b, bone resorption marker, were measured from serum by ELISA. The level of serum TRAP-5b in the LPS group was about two times higher than that in the control group, but the level of serum TRAP-5b in the LPS + usnic acid group was about 30% lower than that of the LPS group.

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Usnic Acid Inhibited Bone Loss in LPS-Induced Mice [1]
The anti-resorptive activity of usnic acid was evaluated with the LPS-induced mouse bone erosion model. Femora were collected from mice and analyzed by a micro-computed tomography (μCT) system. μCT displayed that bone mass of trabecular bone in the femur metaphyseal region was decreased by LPS treatment, whereas the treatment of usnic acid significantly prevented LPS-mediated trabecular bone loss (Figure 6A). The LPS-mediated changes in bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface/total volume (BS/TV), and trabecular separation (Tb.Sp) were significantly prevented by usnic acid.

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Usnic acid prevented lipopolysaccharides (LPS)-induced bone erosion in mice. Taken together, our results suggest that usnic acid might be a potential candidate for the treatment of osteoporosis [1].

Measurements of Serum TRAP by Enzyme-Linked Immunosorbent Assay (ELISA) [1]
Blood was collected from the retro-orbital plexus and centrifuged at 18,000 g for 5 min with LPS treated osteoporosis model mice. Serum was separated and stored at −20 °C. The serum TRAP-5b levels were measured using a Tartrate resistant acid phosphatase 5b ELISA kit. Analyses were performed according to protocols provided by the manufacturers.

Cell Viability Assay The effect of Usnic acid on the viability of BMMs was determined using Cell Counting Kit-8 (CCK-8) method. Briefly, BMCs were seeded in a 96-well plate at a density of 1 × 104 cells/well and cultured with indicated concentration of Usnic acid (0, 0.3, 1, and 3 µM) for 3 days. Cell viability was evaluated using a CCK-8 kit according to the manufacturer’s protocol.

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Bone Pit Formation Assay Bone pit formation assay was performed as described previously. BMMs were differentiated on an Osteo Assay Plate (24 well plate) at a density of 3 × 105 cells/well and stimulated with 10 ng/mL RANKL and 30 ng/mL M-CSF in the presence of Usnic acid (0, 1, and 3 µM). After 4 days, the cells were removed with 5% sodium hypochlorite for 5 min, then the resorption area was observed under a light microscope (magnification, ×50), and after that, measured by ImageJ software.

Lipopolysaccharides (LPS)-Induced Bone Erosion All procedures involving mice were conducted in strict accordance with SCNU IACUC guidelines for the care and use of laboratory animals (Permit No: SCNU IACUC-2016-08). LPS-induced bone erosion was performed as described previously [28]. Five-week-old male ICR mice were divided into 3 groups of 6 mice. One day before injection of LPS (Sigma-Aldrich, St. Louis, MO, USA) and subsequently on every day for up to 8 days till the end of the experimental period, intraperitoneal injections of usnic acid (1 μg/g of body weight) or 10% Kolliphor ER in PBS (control) were administered. LPS (5 μg/μL in 0.1% BSA PBS) was injected intraperitoneally on days 1 and 4. All mice were sacrificed by cervical dislocation, and their femora were scanned with High-resolution micro-CT (SKYSCAN 1272; Bruker, Billerica, MA, USA) and imaged by DataViewer (SKYSCAN). The bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface/total volume (BS/TV), and trabecular separation (Tb.Sp) were measured to assess the trabecular bone microstructure of the femur using the CTAn software provided with the SKYSCAN analysis tool.

Adverse Reactions
Skin Sensitizers - Substances that can induce skin allergic reactions.
Subcutaneous LD50 in mice: 75 mg/kg, CRC Handbook of Antibiotic Compounds, Vol. 1-, Berdy, J., Boca Raton, Florida, CRC Press, 1980, 9(89), 1982.
Intravenous LD50 in mice: 25 mg/kg, Antibiotics, 1(611), 1967.
Oral LD50 in rabbits: >500 mg/kg, Pharmaceutical Research, 5(510), 1955 [PMID:13276269].
Antidotes and Emergency Treatment
/SRP:/Basic Treatment: Maintain a patent airway. Suction if necessary. Watch for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 liters/minute. Monitor for pulmonary edema and treat if necessary…. Monitor for shock and treat if necessary…. Anticipate seizures and treat as necessary… If eyes are contaminated, rinse immediately with water. During transport, continuously rinse each eye with saline… Do not use emetics. If swallowed, rinse mouth; if the patient can swallow, has a strong gag reflex, and does not drool, dilute with 5 ml/kg body weight to 200 ml of water… After cleansing, cover burns with a dry, sterile dressing… /Class A and Class B Poisoning/
/SRP:/ Advanced Treatment: For patients with altered consciousness, severe pulmonary edema, or respiratory arrest, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation using a bag-valve-mask may be effective. Monitor heart rhythm and treat arrhythmias as necessary… Establish intravenous access using 5% glucose solution /SRP: "Keep access open," minimum flow rate. If signs of hypovolemia are present, use lactated Ringer's solution. Be aware of signs of fluid overload. Consider drug treatment for pulmonary edema… For hypotension accompanied by signs of low blood volume, administer intravenous fluids with caution. Be alert for signs of fluid overload… Use diazepam (Valium) to treat epileptic seizures… Use promecaine hydrochloride to assist with eye irrigation… /Toxins A and B/

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Non-human toxicity values
Canine intravenous LD50 40 mg/kg PMID:12453567
Rabbit intravenous LD50 30 mg/kg PMID:12453567
Rats intravenous LD50 30 mg/kg PMID:12453567
Rabbit oral LD50 500 mg/kg

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Non-human toxicity values
Canine intravenous LD50 40 mg/kg
Rabbit intravenous LD50 30 mg/kg
Rats intravenous LD50 30 mg/kg
Rabbit oral LD50 500 mg/kg
For more complete non-human toxicity data for USNIC acids (6 in total), please visit the HSDB record page.

[1]. Effect of Usnic Acid on Osteoclastogenic Activity. J Clin Med. 2018 Oct 12;7(10). pii: E345.

7-Hydroxy-(S)-Usnic acid belongs to the benzofuran class of compounds. It has been reported in lichens such as Dimelaena oreina, Flavoparmelia haysomii, and other organisms with relevant data. See also: (-)-Usnic acid (note moved here). Mechanism of Action: This study investigated the physiological effects of usnic acid on two cultured lichen algae, Trebouxia. Exposure of Trebouxia to sodium usnic acid inhibited its growth and photosynthesis, increased cell membrane permeability, and immobilized zoospores. /Sodium usnic acid/ The mechanism of action of usnic acid is not yet clear. (+)-Usnic acid has been shown to uncouple and oxidatively phosphorylate at a concentration of 1 μM in mouse liver mitochondria. Inhibition of photosynthesis stimulates the accumulation of usnic acid, suggesting that glucose may play a role in regulating phenolic synthases.
Experiments showed that usnic acid is an allelochemical that inhibits bryophyte spore germination, and its inhibitory effect is pH-dependent.
In vitro experiments showed that usnic acid has a slight inhibitory effect on leukotriene biosynthesis in bovine polymorphonuclear leukocytes. Its mechanism is through interaction with specific enzymes, rather than acting as an antioxidant to combat peroxidation, scavenge free radicals, or even act as a source of free radicals.

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Osteoclasts are the only cells capable of absorbing bone. They are produced by monocytes/macrophages in the presence of M-CSF and RANKL and are activated by the immune system in vivo. Lichenic acid is a secondary metabolite of lichens with a unique dibenzofuran skeleton. For many years, it has been used in cosmetics, fragrances, and traditional medicines. Lichenic acid has a wide range of biological activities, including anti-inflammatory, antibacterial, anticancer, and antiviral effects. However, the anti-osteoclastogenic activity of lichenic acid has not been reported. This study investigated whether lichenic acid affects RANKL-mediated osteoclastogenesis. The results showed that lichenic acid significantly inhibited RANKL-mediated osteoclast formation and function by reducing the transcriptional and translational expression of NFATc1, a key regulator of osteoclastogenesis. In addition, lichenic acid also prevented lipopolysaccharide (LPS)-induced bone erosion in mice. In summary, our results suggest that lichenic acid may be a potential candidate drug for the treatment of osteoporosis. [1]

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Therapeutic use
Recent studies compared the anti-inflammatory activity of (+)-lichenic acid and ibuprofen using the rat paw edema test (acute effect) and cotton ball test (chronic effect). The results showed that oral administration of 100 mg/kg of (+)-lichenic acid showed significant efficacy in both tests and was comparable to that of ibuprofen at the same dose. PMID:12453567
/EXPL THER:/ Studies have shown that applying commercially available (+)-lichenic acid to filter paper and then placing the filter paper on virus-infected African green monkey kidney cells (BS-C-1) can inhibit the cytopathic effects of herpes simplex virus type 1 and poliovirus type 1. PMID:12453567
/EXPL THER:/ Oral administration of lichenic acid at doses of 30 and 100 mg/kg showed significant analgesic effects in acetic acid-induced writhing and tail compression tests. Oral administration of usnic acid at doses of 100 and 300 mg/kg showed significant antipyretic activity in a lipopolysaccharide-induced hyperthermia model. PMID:12453567
Usnic acid inhibits the growth of drug-resistant Staphylococcus aureus, Enterococcus, and Mycobacteria. PMID:12061397

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Usnea acid inhibits NFATc1 expression by downregulating RANKL-mediated ERK activation, thereby exerting an anti-osteoclast effect and significantly preventing LPS-induced bone loss in vivo. Therefore, lichenic acid may be used as a novel structural scaffold for the treatment of osteoporosis and other bone diseases. [1]

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Therapeutic Uses
Recent studies compared the anti-inflammatory activity of (+)-lichenic acid and ibuprofen using rat paw edema test (acute effect) and cotton ball test (chronic effect). The results showed that oral administration of 100 mg/kg dose of (+)-lichenic acid showed significant efficacy in both tests and was comparable to the efficacy of the same dose of ibuprofen.
/EXPL THER:/ Studies have shown that coating commercially available (+)-lichenic acid onto filter paper and then placing it on virus-infected African green monkey kidney cells (BS-C-1) can inhibit the cytopathic effects of herpes simplex virus type 1 (HSV-1) and poliovirus type 1 (PPV-1).
/EXPL THER:/ Oral administration of lichenic acid at doses of 30 and 100 mg/kg significantly reduced the cytopathic effect. Analgesic effects were assessed by acetic acid-induced writhing and tail pressure tests. Oral administration of 100 and 300 mg/kg of urenic acid showed significant antipyretic activity as assessed by a lipopolysaccharide-induced hyperthermia test.
Usnic acid inhibits the growth of multidrug-resistant Staphylococcus aureus, Enterococcus, and Mycobacteria.
For more complete data on the therapeutic uses of urenic acid (8 types), please visit the HSDB record page.

C18H16O7

344.31

344.089

C, 62.79; H, 4.68; O, 32.53

125-46-2

(+)-Usnic acid;7562-61-0

5646

White to yellow solid powder

1.5±0.1 g/cm3

594.8±50.0 °C at 760 mmHg

204 °C

219.1±23.6 °C

0.0±1.7 mmHg at 25°C

1.643

1.4

2

7

2

25

708

0

O=C(C1(C)C(OC2=C(C(C)=O)C(O)=C(C)C(O)=C12)=C3)C(C(C)=O)C3=O

CUCUKLJLRRAKFN-UHFFFAOYSA-N

InChI=1S/C18H16O7/c1-6-14(22)12(8(3)20)16-13(15(6)23)18(4)10(25-16)5-9(21)11(7(2)19)17(18)24/h5,11,22-23H,1-4H3

2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3-dione

NSC-8517; 125-46-2; Usno; 1,3(2H,9bH)-Dibenzofurandione, 2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-; usnic-acid; L-Usnic acid; NSC 8517; NSC5890; NSC 8517; Usnic acid

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 : ~3.33 mg/mL (~9.67 mM)

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.

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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).

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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)

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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).

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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

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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.

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