1. Aim or purpose
Plants belonging to Galium genus (Rubiaceae) are known in ethnobotanical field for the treatment of a variety of pathological conditions and for producing several classes of secondary metabolites such as iridoid glycosides, saponins, triterpenes, anthraquinones, and flavonoids (Mocan et al., 2016). Iridoids are a large class of natural products, exhibiting a wide range of pharmacological activities including anti-inflammatory and anticancer. Nowadays there is a growing interest to discover new angiomodulators as angiogenesis inhibition might be a promising approach for anticancer therapies (Kadioglu et al., 2013). Galium tunetanum Lam. is a perennial herb, native to Tunisia, Algeria, Marocco, Spain, and Sicily. Despite the claimed health benefits that the folk medicine attributes to this species, there is still a lack of scientific data to support this information. In the present study, a phytochemical investigation of G. tunetanum leaves was performed along with the assessment of its anti-angiogenic properties.
2. Materials and methods
G. tunetanum leaves were preliminary extracted with solvents of increasing polarity n-hexane, chloroform, chloroform-methanol (9:1), and methanol by exhaustive maceration. The methanol extract was then partitioned between n-butanol and water to afford a n-butanol residue which was submitted to Sephadex LH-20 column chromatography. Nine major fractions were collected and subjected to RP-HPLC analysis. The chloroform-methanol extract was subjected to Isolera Biotage column chromatography to obtained five major fractions submitted to RP-HPLC analysis. The structural determination of the isolated compounds was performed by 1D and 2D NMR spectroscopic data, as well as ESI-MS experiments. The anti-angiogenic effects of the isolated compounds were tested on new blood vessels formation using the chick embryo chorioallantoic membrane (CAM) as in vivo model (Certo et al., 2017). Fertilized eggs of Gallus gallus, after four days of incubation, were treated with pure compounds (0.5-2.0 μg/egg). Retinoic acid (2 μg/egg) was used as anti-angiogenic reference compound. After treatment, the eggs were reincubated for 24 h, then they were observed by a steromicroscope equipped with a digital camera and photographed. The anti-angiogenic effects on the CAMs were quantified by counting the number of blood vessel branch points in a standardized area and expressed as % of inhibition respect to control.
3. Results
The phytochemical analyses of G. tunetanum extracts led to the isolation of thirteen compounds from the chloroform-methanol and the methanol extracts: six iridoid glycosides, one non-glycoside iridoid, two p-coumaroyl iridoid derivatives, two phenolic acids and two flavonoid glycosides.
The isolated iridoids were subjected to the CAM assay and results showed that among them asperuloside, geniposidic acid and iridoid V1 reduced microvessel formation on morphological observations of the CAMs. The anti-angiogenic effects, expressed as percentages of inhibition versus control, were 67% (asperuloside), 59% (geniposidic acid), and 54% (iridoid V1), respectively. In addition, the active compounds were able to inhibit CAM angiogenesis in a dose-dependent manner as compared to the standard retinoic acid.
4. Conclusions
The results obtained suggest the potential use of G. tunetanum extracts as new sources of anti-angiogenic compounds.

References
Certo, G., Costa, R., D’Angelo, V., et al. Anti-angiogenic activity and phytochemical screening of fruit fractions from Vitex agnus castus. Nat. Prod. Res. 2017, 31, 2850-2856.
Kadioglu, O., Seo, E.J., Efferth, T. Targeting angiogenesis by phytochemicals. Med. Aromat. Plants. 2013, 2, 2-8.
Mocan, A., Crisan, G., Vlase, L. et al. Phytochemical investigations on four Galium species (Rubiaceae) from Romania. Farmacia 2016, 64, 95-99.

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