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Apiin

Alias: apiin; 26544-34-3; Apigenin-7-apioglucoside; Apioside; UNII-6QU3EZE37U; 6QU3EZE37U; CHEBI:15932; EINECS 247-780-0;
Cat No.:V4467 Purity: ≥98%
Apiin is a naturally occurring flavonoid isolated from celery, it shows antiinflammatory activity mediated through inhibition of NO (nitric oxide synthesis) and inhibition of iNOS expression.
Apiin
Apiin Chemical Structure CAS No.: 26544-34-3
Product category: NO Synthase
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

Apiin is a naturally occurring flavonoid isolated from celery, it shows antiinflammatory activity mediated through inhibition of NO (nitric oxide synthesis) and inhibition of iNOS expression. Flavonoids are natural compounds that are widely distributed in the plant kingdom, and are reported to affect the inflammatory process and to possess anti-inflammatory as well as immunomodulatory activity in-vitro and in-vivo.

Biological Activity I Assay Protocols (From Reference)
Targets
NO production (IC50 = 0.08 mg/mL);iNOS (IC50 = 0.049 mg/ mL)
ln Vitro
The extract of A. graveolens var. dulce contained Apiin as the major constituent (1.12%, w/w, of the extract). The extract and Apiin showed significant inhibitory activity on nitrite (NO) production in-vitro (IC50 0.073 and 0.08 mg mL(-1) for the extract and apiin, respectively) and iNOS expression (IC50 0.095 and 0.049 mg mL(-1) for the extract and apiin, respectively) in LPS-activated J774.A1 cells. Our results clearly indicated the inhibitory activity of the extract and apiin in-vitro on iNOS expression and nitrite production when added before LPS stimulation in the medium of J774.A1 cells. [1]
Compound 1 was used as biological marker of the extract for quantitative HPLC analysis by a direct calibration method. We found that the whole extract was composed of a high ratio (1.12% w/w) of Apiin (1). It is well known that in inflammatory disease NO production is elevated by the constitutive and the inducible nitric oxide synthase, and NO produced by iNOS is an important inflammatory mediator. Therefore, we investigated the invitro activity of the extract and compound 1 on inducible nitric oxide synthase expression (iNOS) and on NO production by LPS-stimulated J744.A1 macrophages. Cytotoxicity was evaluated using cell cultures (J774.A1 (murine monocyte/macrophage) and HEK-293 (human epithelial kidney)). NO release in the cellular medium of J774.A1 macrophages incubated with compound 1 (0.005–0.05 mg mL−1 ) or the extract (0.01–0.1 mg mL−1 ) 1 h before LPS stimulation was evaluated 24 h after LPS (6 × 103UmL−1 ) challenge. Results were expressed as % of inhibition calculated vs controls (Aquino et al 2002). As shown in Figures 2 and 3, extract (0.01, 0.05 and 0.1 mg mL−1 ) and Apiin (1; 0.01 and 0.05 mgmL−1 ) added 1 h before and simultaneously with LPS inhibited NO release significantly and in a concentration related manner; so that the IC50 value was calculated as 0.073 mgmL−1 of extract and 0.08 mgmL−1 of apiin. To establish whether the inhibitory effect of compound 1 and of the extract on NO release was related to the modulation of iNOS induction, iNOS expression was evaluated by Western blot analysis on cell lysates obtained by J774.A1 incubated with 1 (0.005–0.05mgmL−1 ) or the extract (0.01–0.1mg mL−1 ), 1 h before and simultaneously with LPS. Compound 1 (IC500.049 mgmL−1 ) and the extract (IC50 0.095 mgmL−1 ) showed a significant and concentration-dependent inhibition of iNOS expression (P < 0.1, compound 1 0.05 and 0.01 mg mL−1 ; P<0.01, extract 0.1 and 0.05mg mL−1 ) (Figures 4 and 5). To verify the effects on cell viability, the extract (0.01– 0.1 mgmL−1 ) and compound 1 (apiin) (0.005–0.05 mgmL−1 ) were tested on two different cell lines, J774.A1 (murine macrophage cells) and HEK-293 (human epithelial kidney cells) using the MTT test. Our results indicated that they did not affect cell viability (data not shown). [1]
ln Vivo
The croton-oil ear test on mice showed that the extract exerted anti-inflammatory activity in-vivo (ID50 730 microg cm(-2)), with a potency seven-times lower than that of indometacin (ID50 93 microg cm(-2)), the non-steroidal anti-inflammatory drug used as reference. The anti-inflammatory properties of the extract demonstrated in-vivo might have been due to reduction of iNOS enzyme expression [1].
As to the in-vivo topical anti-inflammatory activity of the extract, tested using the croton oil ear test in mice, the anti-oedematous effect of the extract, at doses of 100, 300 or 900mgcm−2 , is reported in Table 2. The dose–activity relationship of the extract was investigated in comparison with indometacin (ID50, dose inducing 50% oedema inhibition=93mgcm−2 ). The extract provoked a significant and dose-dependent oedema inhibition with potency seven-times lower than that of indometacin (ID50 730mgcm−2 ) [1].
Cell Assay
Cytotoxic activity [1]
Potential cytotoxic activity of the extract (0.01–0.1 mg mL−1 ) and compound 1 (0.005–0.05 mg mL−1 ) in PBS solutions was evaluated in cell cultures (J774.A1 and HEK-293 cell lines) by MTT assay as previously described (Mosmann 1983; Picerno et al 2005). The optical density (OD) of each well was measured with a microplate spectrophotometer equipped with a 620 nm filter. The viability of each cell line in response to treatment with tested compounds and 6-MP was calculated as: % dead cells = 100− (OD treated/OD control) × 100. Data on cell viability were expressed as percentages of viability vs negative controls (PBS-treated cells).
Analysis of nitrite [1]
Monolayers of J774.A1 cells were routinely harvested by gentle scraping with a Teflon cell scraper, diluted in fresh medium and cultured to confluency at 37°C. Before each experiment cells were harvested, plated to a seeding density of 1.5 × 106 in P60 well plates. After 2 h of cell adhesion, the extract (0.01–0.1 mg mL−1 ) or 1 (0.005–0.05 mg mL−1 ) in PBS solution was added to the culture medium 1 h before and simultaneously to LPS (6 × 103 U mL−1 /24 h). Nitrite accumulation, indicator of nitric oxide (NO) release, was measured in the culture medium by the Griess reaction (Green et al 1982) 24 h after LPS challenge, according to Picerno et al (2005). The amount of nitrite in the samples was calculated using a sodium nitrite standard curve freshly prepared in culture medium. Results were expressed as percentages of inhibition calculated vs NO production of cells treated with LPS alone.
Western blot analysis for iNOS [1]
After 24 h of incubation with LPS, medium was removed, cells were lysed and Western blot analysis was performed according to Picerno et al (2005).
Animal Protocol
Topical anti-inflammatory activity [1]
Topical anti-inflammatory activity was evaluated as inhibition of the croton-oil-induced ear oedema in mice (Tubaro et al 1985). All experiments complied with the Italian D.L. n. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC). Male CD-1 mice (28–32 g) were anaesthetized with ketamine hydrochloride (145 mg kg−1 , i.p.). Cutaneous inflammation was induced on the inner surface of the right ear (surface: approximately 1 cm−2 ) of anaesthetized mice by application of 80 mg croton oil dissolved in 42% aqueous ethanol (v/v), used as vehicle for extract and its control. Control mice received only the irritant solution, whereas the other mice received the irritant and the test substance. At the maximum oedematous response, 6 h later, mice were killed and a plug (6-mm diameter) was removed from the treated (right) and the untreated (left) ears. The oedematous response was measured as the weight difference between the two plugs. Anti-inflammatory activity was expressed as percent reduction of the oedema in treated mice compared with control mice. The non-steroidal antiinflammatory drug (NSAID) indometacin was used as the reference drug.
References

[1]. An extract of Apium graveolens var. dulce leaves: structure of the major constituent, apiin, and its anti-inflammatory properties. J Pharm Pharmacol. 2007 Jun;59(6):891-7.

Additional Infomation
Apiin is a beta-D-glucoside having a beta-D-apiosyl residue at the 2-position and a 5,4'-dihydroxyflavon-7-yl moiety at the anomeric position. It has a role as an EC 3.2.1.18 (exo-alpha-sialidase) inhibitor and a plant metabolite. It is a beta-D-glucoside, a dihydroxyflavone and a glycosyloxyflavone. It is functionally related to an apigenin. It is a conjugate acid of an Apiin(1-).
Apiin has been reported in Crotalaria micans, Ageratina calophylla, and other organisms with data available.
See also: Chamomile (part of); Chamaemelum nobile flower (part of).
Flavonoids, natural compounds widely distributed in the plant kingdom, are reported to affect the inflammatory process and to possess anti-inflammatory as well as immunomodulatory activity in-vitro and in-vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of the ethanol/water (1:1) extract of the leaves of Apium graveolens var. dulce (celery) on iNOS expression and NO production in the J774.A1 macrophage cell line stimulated for 24 h with Escherichia coli lipopolysaccharide (LPS) were evaluated. The extract of A. graveolens var. dulce contained Apiin as the major constituent (1.12%, w/w, of the extract). The extract and Apiin showed significant inhibitory activity on nitrite (NO) production in-vitro (IC50 0.073 and 0.08 mg mL(-1) for the extract and apiin, respectively) and iNOS expression (IC50 0.095 and 0.049 mg mL(-1) for the extract and apiin, respectively) in LPS-activated J774.A1 cells. The croton-oil ear test on mice showed that the extract exerted anti-inflammatory activity in-vivo (ID50 730 microg cm(-2)), with a potency seven-times lower than that of indometacin (ID50 93 microg cm(-2)), the non-steroidal anti-inflammatory drug used as reference. Our results clearly indicated the inhibitory activity of the extract and apiin in-vitro on iNOS expression and nitrite production when added before LPS stimulation in the medium of J774.A1 cells. The anti-inflammatory properties of the extract demonstrated in-vivo might have been due to reduction of iNOS enzyme expression.[1]
Flavones are a class of natural products with a large number of derivatives in plants and they are common components in the human diet. They are reported to possess a wide range of biochemical and pharmacological effects including antioxidant, anti-inflammatory, anticancer, antimicrobic and immunomodulatory activity (Gryglewski et al 1987; Middleton & Kandaswami 1992; Cooks & Samman 1996). As it has been reported, their mechanisms of action could be explained with the inhibition of different enzymes such as prostaglandin synthase, lipoxygenase and cyclooxygenase, involved in the inflammatory process and tumorigenesis, and with the induction of detoxifying enzymes such as glutathione S-transferase (Cooks & Samman 1996; Comalada et al 2006; Horinaka et al 2006; Vargo et al 2006). Despite the large use of A. graveolens var. dulce in traditional remedies, there are not enough data in the literature on its leaf components as anti-inflammatory agents and on their mechanism of action. In-vitro cyclooxygenase and topoisomerase inhibitory activity has been reported for compounds of different chemical structures (sedanolide, senkyunolide-N and -J, L-tryptophan, chromenone and indole derivatives), isolated from the seeds of A. graveolens (Momin & Nair 2002). Crude ethanol extracts of A. graveolens showed invivo anti-inflammatory activity in rats on carrageenaninduced paw oedema (Atta & Alkofahi 1998) and on cotton pellet granuloma (Al-Hindawi et al 1989). No significant antiexudative effect was observed in xylene-induced ear oedema in mice with the same extract (Al-Hindawi et al 1989). Previously water extract of celery, a rich source of apigenin, was shown to enhance prostaglandin E2 (PGE2) production in the absence of LPS, and ethyl acetate extract of celery enhanced PGE2 production in the presence of LPS in RAW264.7. Wu & Huang (2001) reported that the flavone aglycone, apigenin, inhibited the production of PGE2. It is also well known that in inflammatory disease NO production is elevated by the constitutive and the inducible nitric oxide synthase, and NO produced by iNOS is another important inflammatory mediator. The results of this study indicated that polar extract of A. graveolens contained Apiin as the major constituent and a high content of polyphenols that may all have contributed to the biological activity of celery. Moreover, Apiin and celery extract were able to inhibit significantly and in a concentration-related manner in-vitro NO release and iNOS expression when added 1 h before LPS stimulation, with IC50 values of 0.049 mgmL−1 (86.8 mM) and 0.08 mgmL−1 (141.8 mM), respectively. Different results were obtained by Kim et al (1999) using different experimental conditions. They did not observe inhibition of NO release and iNOS expression when flavone glycosides, such as Apiin, were added simultaneously (from 1 to 100 mM) with LPS in the macrophage cell line RAW264.7, whereas they reported the inhibition of NO production for flavones, such as apigenin (IC50 23 mM), under the same experimental conditions. The activity of the celery extract was confirmed by our results obtained in-vivo that firstly indicated the topical antiinflammatory capability of the extract of celery leaves to ameliorate inflammation or other conditions in which enhanced expression of iNOS could be observed. These results were in agreement with recent researches on flavonoids as anti-inflammatory and antioxidative agents (Chu et al 2002; Ninfali & Bacchiocca 2003), as well as with studies on their mechanisms of action (Cooks & Samman 1996; Comalada et al 2006; Horinaka et al 2006; Vargo et al 2006).[1]
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C26H28O14
Molecular Weight
564.4921
Exact Mass
564.147
Elemental Analysis
C, 55.32; H, 5.00; O, 39.68
CAS #
26544-34-3
PubChem CID
5280746
Appearance
White to light yellow solid powder
Density
1.7±0.1 g/cm3
Boiling Point
942.2±65.0 °C at 760 mmHg
Melting Point
230ºC (dec.)
Flash Point
316.7±27.8 °C
Vapour Pressure
0.0±0.3 mmHg at 25°C
Index of Refraction
1.744
LogP
0.74
Hydrogen Bond Donor Count
8
Hydrogen Bond Acceptor Count
14
Rotatable Bond Count
7
Heavy Atom Count
40
Complexity
923
Defined Atom Stereocenter Count
8
SMILES
C1[C@@]([C@H]([C@@H](O1)O[C@@H]2[C@H]([C@@H]([C@H](O[C@H]2OC3=CC(=C4C(=C3)OC(=CC4=O)C5=CC=C(C=C5)O)O)CO)O)O)O)(CO)O
InChi Key
NSVHIOLUPMJKID-RQUVOORVSA-N
InChi Code
InChI=1S/C26H30O14/c27-9-17-20(31)21(32)22(39-25-23(33)26(34,10-28)11-36-25)24(38-17)37-14-7-16(30)15-5-6-18(40(35)19(15)8-14)12-1-3-13(29)4-2-12/h1-4,6-8,17,20-25,27-34H,5,9-11H2/t17-,20-,21+,22-,23+,24-,25+,26?/m1/s1
Chemical Name
7-((2-O-beta-D-Apiofuranosyl-beta-D-glucopyranosyl)oxy)-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyranone
Synonyms
apiin; 26544-34-3; Apigenin-7-apioglucoside; Apioside; UNII-6QU3EZE37U; 6QU3EZE37U; CHEBI:15932; EINECS 247-780-0;
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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO : ~100 mg/mL (~177.15 mM)
Solubility (In Vivo)
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.43 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 (4.43 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.

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 1.7715 mL 8.8576 mL 17.7151 mL
5 mM 0.3543 mL 1.7715 mL 3.5430 mL
10 mM 0.1772 mL 0.8858 mL 1.7715 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.

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