NO production (IC50 = 0.08 mg/mL);iNOS (IC50 = 0.049 mg/ mL)

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]

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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]

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].

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).

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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.

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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).

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.

[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.

Apiin is a β-D-glucoside with a β-D-apiosyl residue at the 2-position and a 5,4'-dihydroxyflavone-7-yl moiety at the anomeric position. It is an EC 3.2.1.18 (exo-α-sialidase) inhibitor and a plant metabolite. It is a β-D-glucoside, dihydroxyflavone, and glycosyloxyflavone. It is functionally related to apigenin. It is the conjugate acid of Apiin (1-). Apiin has been reported in Crotalaria micans, Ageratina calophylla, and other organisms with relevant data. See also: Chamomile (partial); Chamaemelum nobile flower (partial). Flavonoids are naturally occurring compounds widely distributed in the plant kingdom, and have been reported to influence inflammatory processes, exhibiting anti-inflammatory and immunomodulatory activities both in vitro and in vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, this study evaluated the effects of an ethanol/water (1:1) extract of celery (Apium graveolens var. dulce) leaves on iNOS expression and NO production in the J774.A1 macrophage cell line stimulated with Escherichia coli lipopolysaccharide (LPS) for 24 hours. The main component of the celery extract was apigenin (accounting for 1.12% of the extract by weight). Both the extract and apigenin showed significant inhibitory activity in vitro on nitrite (NO) production (IC50 values of 0.073 and 0.08 mg mL⁻¹ for the extract and apigenin, respectively) and iNOS expression (IC50 values of 0.095 and 0.049 mg mL⁻¹ for the extract and apigenin, respectively). The mouse croton oil ear test showed that the extract had anti-inflammatory activity in vivo (ID50 value of 730 μg cm⁻²), which was 7 times less potent than the reference nonsteroidal anti-inflammatory drug indomethacin (ID50 value of 93 μg cm⁻²). Our results clearly show that the addition of the extract and apigenin before LPS stimulation of J774.A1 cells effectively inhibited iNOS expression and nitrite production. The anti-inflammatory properties of the extract in vivo may be attributed to the reduction of iNOS enzyme expression. [1]

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Flavonoids are a class of natural products with a large number of derivatives in plants and are also common components of the human diet. They have been reported to have a wide range of biochemical and pharmacological effects, including antioxidant, anti-inflammatory, anticancer, antibacterial and immunomodulatory activities (Gryglewski et al., 1987; Middleton and Kandaswami, 1992; Cooks and Samman, 1996). It has been reported that its mechanism of action may be related to the inhibition of multiple enzymes involved in inflammatory processes and tumorigenesis (such as prostaglandin synthase, lipoxygenase, and cyclooxygenase) as well as the induction of detoxification enzymes (such as glutathione S-transferase) (Cooks & Samman 1996; Comalada et al 2006; Horinaka et al 2006; Vargo et al 2006). Although stevia (A. graveolens var. dulce) is widely used in conventional medicine, data on its leaf components as anti-inflammatory agents and their mechanisms of action remain insufficient in the literature. Compounds with different chemical structures isolated from caraway (A. graveolens) seeds (such as apigenin lactone, senkyunolide-N and -J, L-tryptophan, tryptophanone, and indole derivatives) have been reported to have in vitro cyclooxygenase and topoisomerase inhibitory activities (Momin & Nair 2002). Crude ethanol extract of parsley showed in vivo anti-inflammatory activity in carrageenan-induced rat paw edema models (Atta & Alkofahi 1998) and cotton pellet granuloma models (Al-Hindawi et al 1989). However, no significant anti-exudative effect was observed in xylene-induced mouse ear edema models (Al-Hindawi et al 1989). Previous studies have shown that celery water extract (a rich source of apigenin) enhances prostaglandin E2 (PGE2) production in the absence of lipopolysaccharide (LPS); acetic acid extract of celery also enhances PGE2 production in RAW264.7 cells in the presence of LPS. Wu and Huang (2001) reported that the flavonoid aglycone apigenin inhibits PGE2 production. It is well known that in inflammatory diseases, both constitutive and inducible nitric oxide synthase (iNOS) increase nitric oxide (NO) production, and NO produced by iNOS is another important inflammatory mediator. The results of this study indicate that the main component of celery polar extract is apigenin, which is also rich in polyphenols. These components may collectively promote the bioactivity of celery. Furthermore, the addition of apigenin and celery extract one hour before LPS stimulation significantly and in a concentration-dependent manner inhibited NO release and iNOS expression in vitro, with IC50 values of 0.049 mgmL−1 (86.8 mM) and 0.08 mgmL−1 (141.8 mM), respectively. Kim et al. (1999) obtained different results under different experimental conditions. They found that in the macrophage line RAW264.7, when flavonoid glycosides (such as apigenin) were added simultaneously with LPS (concentration range of 1 to 100 mM), no inhibition of NO release and iNOS expression was observed; however, under the same experimental conditions, they reported the inhibitory effect of flavonoids (such as apigenin) on NO production (IC50 value of 23 mM). Our in vivo experiments confirmed the activity of celery extract, demonstrating for the first time that celery leaf extract has local anti-inflammatory capabilities and can improve inflammation or other conditions with observable enhancement of iNOS expression. These results are consistent with recent studies on flavonoids as anti-inflammatory and antioxidant agents (Chu et al 2002; Ninfali & Bacchiocca 2003) and on their mechanisms of action (Cooks & Samman 1996; Comalada et al 2006; Horinaka et al 2006; Vargo et al 2006). [1]

C26H28O14

564.4921

564.147

C, 55.32; H, 5.00; O, 39.68

26544-34-3

5280746

White to light yellow solid powder

1.7±0.1 g/cm3

942.2±65.0 °C at 760 mmHg

230ºC (dec.)

316.7±27.8 °C

0.0±0.3 mmHg at 25°C

1.744

0.74

8

14

7

40

923

8

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

NSVHIOLUPMJKID-RQUVOORVSA-N

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

7-((2-O-beta-D-Apiofuranosyl-beta-D-glucopyranosyl)oxy)-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyranone

apiin; 26544-34-3; Apigenin-7-apioglucoside; Apioside; UNII-6QU3EZE37U; 6QU3EZE37U; CHEBI:15932; EINECS 247-780-0;

2934.99.9001

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.

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

DMSO : ~100 mg/mL (~177.15 mM)

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.

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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.

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