Anti-angiogenic activity of iridoids from Galium tunetanum

ABSTRACT

The phytochemical study of Galium tunetanum Lam., Rubiaceae, leaves led to the isolation of 13 compounds from the chloroform–methanol and the methanol extracts, including six iridoid glycosides, one non-glycoside iridoid, two p-coumaroyl iridoid glycosides, two phenolic acids, and two flavonoid glycosides. The structural determination of the isolated compounds was performed by mono- and bidimensional NMR spectroscopic data, as well as ESI-MS experiments. All compounds were isolated from this species for the first time. The anti-angiogenic effects of the isolated iridoids were also reported on new blood vessels formation using the chick embryo chorioallantoic membrane as in vivo model. Results showed that among the isolated iridoids tested at the dose of 2 µg/egg, asperuloside (1), geniposidic acid (2), and iridoid V1 (3) reduced microvessel formation of the chorioallantoic membrane on morphological observations using a stereomicroscope. The anti-angiogenic effects of the active compounds, expressed as percentages of inhibition versus control, were 67% (1), 59% (2), and 54% (3), respectively. In addition, the active compounds were able to inhibit angiogenesis in the chorioallantoic membrane assay, in a dose-dependent manner (0.5–2 µg/egg) as compared to the standard retinoic acid.

Keywords:
Anti-angiogenic activity; Asperuloside; Chick chorioallantoic membrane; Geniposidic acid; Iridoid V1

Introduction

Plants belonging to Galium genus, Rubiaceae, comprising approximately 1300 species, are known in ethnobotanical field for the treatment of a variety of pathological conditions, such as psoriasis, skin infections (Oumeish, 1999), hepatitis (Bolivar et al., 2011), kidney disorders, and as sedative, diuretic, and to treat the epilepsy and hysteria (Shah et al., 2006). G. tunetanum Lam. is a perennial herb, native to Tunisia, Algeria, Marocco, Spain, and Sicily (Casimiro et al., 2012). To the best of our knowledge, in the literature there is only one report about the antioxidant activity of the methanol extract of its leaves (Gaamoune et al., 2014) but no phytochemical studies have been carried out so far.

Galium genus is well-known for producing several classes of secondary metabolites such as iridoid glycosides, saponins, triterpenes, anthraquinones, and flavonoid glycosides (Mocan et al., 2016). Iridoids are a large class of natural products, exhibiting a wide range of pharmacological activities such as anti-inflammatory, anticancer, cardioprotective, and neuroprotective. Interestingly, the iridoid glycoside geniposide was found to have a potent anti-angiogenic activity in the chick embryo chorioallantoic membrane (CAM) assay (Koo et al., 2004). Angiogenesis is the growth of new blood vessels to ensure wound healing, reproduction, and developments of cells. This physiological process plays an important role in the expansion of veins and blood capillaries and in the nutrition of tumor cells. Thus, angiogenesis inhibition might be a promising approach for anticancer therapies.

In the course of our investigation on plants belonging to the African flora (Beladjila et al., 2017), the chemical study of G. tunetanum leaves was performed, and the isolation and structural characterization of 13 compounds, including nine iridoid glycosides (1–9), two phenolic acids (10–11), and two flavonoid glycosides (12–13) was herein reported. The anti-angiogenic effect of iridoids 1–8 on new blood vessels formation, using the CAM assay as in vivo model, was also explored.

Materials and methods

One and two-dimensional NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K (Bruker BioSpin, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe, acquiring the spectra in methanol-d₄. Pulse sequences and phase cycling were used for DQF-COSY, TOCSY, HSQC, and HMBC, experiments. NMR data were processed using XWinNMR software (De Leo et al., 2017). ESI-MS were obtained from an LCQ Advantage ThermoFinnigan spectrometer (ThermoFinnigan, USA), equipped with Xcalibur software. Column chromatographies (CC) were performed over Sephadex LH-20 (40–70 µm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) and Isolera^® Biotage^® purification system (flash Silica gel 60 SNAP 340 g cartridge, flow rate 90 ml/min) (Milella et al., 2016). Reverse phase – high performance liquid chromatography (RP-HPLC) separations were conducted on a Shimadzu LC-8A series pumping system equipped with a Shimadzu RID-10A refractive index detector and Shimadzu injector on a C₁₈ µ-Bondapak column (30 cm × 7.8 mm, 10 µm Waters, flow rate 2 ml/min, Milford, MA, USA). Thin Layer Chromatography (TLC) analyses were carried out using precoated Kieselgel 60 F₂₅₄ (0.20 mm thickness) plates (Merck, Darmstadt, Germany); compounds were detected by cerium disulfate/sulfuric acid (Sigma–Aldrich, Milan, Italy). All the solvents used for the extraction and separation processes and retinoic acid used for the CAM assay as antiangiogenic reference compound were purchased from Sigma-Aldrich (Milan, Italy).

Galium tunetanum Lam., Rubiaceae, leaves were collected and identified by authors Smain Amira and Fatima Benchikh in Djilma, 45 km away from Jijel, Northeast Algeria, in June 2013. A voucher specimen has been deposited at the Herbarium Horti Botanici Pisani, Pisa, Italy (n. 8486 Galium tunetanum/1, Nuove Acquisizioni).

Briefly, dried leaves of the plant (1 kg) were extracted with solvents of increasing polarity: n-hexane, chloroform, chloroform–methanol (9:1), and methanol by exhaustive maceration to give 4.0, 13.3, 11.9, and 48 g of the respective residues. The methanol extract was partitioned between n-butanol and water to afford a n-butanol residue (10.8 g), that was submitted to Sephadex LH-20 column chromatography (5 × 75 cm, flow rate 1 ml/min) using methanol as eluent and collecting nine major fractions (A–I) grouped by TLC. Part of the fraction B (1.5 g) was subjected to RP-HPLC with methanol–water (3:7) as eluent, to give compounds 2 (0.7 mg, t_R 7 min) and 7 (1.4 mg, t_R 14 min). Fractions E (273.3 mg), F (707.3 mg), G (724.0 mg), and I (818.2 mg) were submitted to RP-HPLC using methanol–water (35:65) as eluent, to give compounds 3 (5.0 mg, t_R 14 min) and 8 (1.7 mg, t_R 55 min) from fraction E; compounds 10 (1.5 mg, t_R 9 min) and 9 (0.5 mg, t_R 22 min) from fraction F; compounds 11 (6.0 mg, t_R 6 min) and 12 (1.3 mg, t_R 32 min) from fraction G; compound 13 (2.6 mg, t_R 39 min) from fraction I, respectively. The remaining fractions B (874.2 mg) and C (922.3 mg) were subjected to RP-HPLC with methanol–water (1:4) as eluent, to give compound 6 (1.3 mg, t_R 5 min) from fraction B and compound 2 (1.3 mg, t_R 8 min) from fraction C, respectively. Part of the chloroform–methanol residue (5.6 g) was subjected to Isolera Biotage column chromatography (340 g silica SNAP cartridge, flow rate 90 ml/min), eluting with chloroform followed by increasing concentrations of methanol in chloroform (between 1% and 100%). Fractions of 27 ml were collected, analyzed by TLC and grouped into five major fractions (A–E). Fractions B (331.4 mg) and C (1481.8 mg) were subjected to RP-HPLC with methanol–water (3:7) as eluent, to give compounds 5 (1.3 mg, t_R 6 min) and 7 (3 mg, t _R 15 min) from fraction B; compound 1 (23.6 mg, t_R 8 min) from fraction C, respectively. Fraction E (509.7 mg) was submitted to RP-HPLC with methanol–water (1:4) as eluent, to give compound 4 (6.6 mg, t_R 7 min).

The CAM assay was performed following the method of Germanò et al. (2015) modified (Certo et al., 2017). Fertilized eggs of Gallus gallus were previously maintained in a humidified incubator at 37 ºC and, after four days of incubation, a small window was created on the broad side of the eggs to apply different doses of pure compounds (0.5–2 µg/egg) directly on the CAM surface, previously suspended in albumen. Retinoic acid (2 µg/egg) was used as antiangiogenic reference compound. After treatment, the eggs were reincubated for 24 h, then they were observed by means of a steromicroscope (Zeiss Stemi 2000-c) equipped with a digital camera (Axiocam MRc 5 Zeiss) and photographed. The antiangiogenic effects on the CAM were quantified by counting the number of blood vessel branch points in a standardized area using a Zeiss software for micromorphometric analysis and expressed as % of inhibition respect to control. Each experiment was repeated three times. The significance of the differences was assessed on the basis of the t-test, considering the differences for p < 0.05, and finally calculated with respect to the lot of control eggs treated only with albumen.

Results and discussion

The phytochemical study of chloroform–methanol and methanol extracts of G. tunetanum leaves afforded the isolation of thirteen compounds 1–13. Their structural determination was performed by 1D and 2D NMR spectroscopic techniques, mass spectrometry analyses, and comparison of these data with those reported in the literature. Isolated compounds included six iridoid glycosides identified as asperuloside (1) (Otsuka et al., 1991), geniposidic acid (2) (Güvenalp et al., 2006), iridoid V1 (3) (Mitova et al., 1999), deacetylasperuloside (4) (Otsuka et al., 1991), monotropein (6) (Tzakou et al., 2007), and daphylloside (7) (Demirezer et al., 2006); one non-glycoside iridoid macedonine (5) (Mitova et al., 1996); two p-coumaroyl iridoid derivatives, 10-O-p-coumaroyl-10-deacetyldaphylloside (8) (Ahn and Kim, 2012) and 10-O-p-coumaroyl-10-deacetylasperuloside (9) (Bai and Hu, 2006); two phenolic acids characterized as p-hydroxyhydrocinnamic acid (10) and chlorogenic acid (11) (Owen et al., 2003); and two flavonoid glycosides rutin (12) and apigenin-7-O-glucoside (13) (Agrawal, 1989).

Isolated iridoids, except 9 that was obtained in too small quantity, were subjected to CAM assay in order to evaluate their anti-angiogenic effects.

The anti-angiogenic effects of isolated iridoids (2 µg/egg) in the CAM assay showed that compounds 1, 2, and 3 were able to reduce CAM microvessel formation with inhibitions of 67%, 59%, and 54%, respectively. Besides, compounds 4–8 demonstrated the following inhibition values: 43%, 31%, 23%, 19%, and 16%. Noteworthy, 1 has a higher anti-angiogenic activity in respect to the standard retinoic acid (62%). Representative microscopic images of the CAM after treatment with the active compounds 1–3 are reported in Fig. 1. Control eggs showed the presence of a clear vascular net-work with large vessels converging toward the embryo (Fig. 1A). Conversely, a visible reduction of blood vessel branch points is evidenced in the CAM treated with 1, 2, and 3 (Fig. 1B–D). In addition, these active compounds demonstrated to inhibit CAM angiogenesis in a dose-dependent manner (0.5–2 µg/egg) (Fig. 2).

Fig. 1
Chick embryo chorioallantoic membrane (CAM) treated with at a dose of 2 µg/egg. (A) Control; (B) asperuloside (1); (C) geniposidic acid (2); (D) iridoid V1 (3).

Fig. 2
Dose-dependent anti-angiogenic activity of asperuloside (1), geniposidic acid (2), and iridoid V1 (3) in the chick embryo chorioallantoic membrane (CAM) assay. Retinoic acid was used as a positive control. CAMs were treated with compounds at doses of 0.5–2 µg/egg. Each group contained at least 10 eggs. Each value represents the mean ± SD of three experiments.

It is known that inhibition of angiogenesis has been recognized to be advantageous for the prevention of inflammation and neoplastic growth. For this reason nowadays there is a growing interest to discover new inhibitors of angiogenesis from natural sources. The CAM model offers advantages that include the comparative ease of culture, low cost, and easy observation of the neovascularisation (Koutsaviti et al., 2017). Among the isolated iridoids tested, asperuloside (1), geniposidic acid (2), and iridoid V1 (3) exhibited high inhibitory activity on CAM angiogenesis. These results are in accordance with the study of Koo et al. (2004) where the iridoid geniposide is considered largely responsible for the anti-angiogenic activity of Gardenia jasminoides fruits ethanol extract. In summary, the results obtained may be the starting point for considering G. tunetanum a new source of anti-angiogenic compounds.

Ethical disclosures

Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data. The authors declare that no patient data appear in this article.

Right to privacy and informed consent. The authors declare that no patient data appear in this article.

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Publication Dates

• Publication in this collection
  May-Jun 2018

History

• Received
  29 Jan 2018
• Accepted
  29 Mar 2018