Simultaneous isolation of gallotannins and a related phenolic from Mangifera indica kernels and assessment of their anti-Trichomonas vaginalis activities

Mohamed A. A. Orabi1*, Soad A. L. Bayoumi2, Hanaa M. Sayed2, Enas A. M. Huseein3, Amgad I.M. Khedr4, Tsutomu Hatano5 1Department of Pharmacognosy, Faculty of Pharmacy, Al Azhar University, Assiut 71524, Egypt. 2Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt. 3Department of Parasitology, Faculty of Medicine, Assiut University, Assiut 71526, Egypt. 4Department of Pharmacognosy, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt. 5Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 8530, Japan.


General experimental procedures
The one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectra were recorded on a Varian INOVA AS 600 instrument (Agilent, Santa Clara, CA, USA; 0.151 GHz for 13 C and 0.6 GHz for 1 H). The chemical shift values were shown in δ (ppm) relative to the solvent signals [(CD 3 ) 2 CO-d 6 (δ H 2.04; δ C 29.8) on the tetramethylsilane (TMS) scale. Reversedphase-high performance liquid chromatography (RP-HPLC) was done on a YMC-Pack ODS-A A-303 (product of YMC, Japan) column (4.6 i.d. × 250 mm) using acetonitrile -water (2:8, v/v) with 0.1% acetic acid. The flow rate was set at 1 ml/min and the oven temperature at 40°C. The eluates were monitored by a Ultraviolet (UV) detector at λ max = 280 nm. Preparative RP-HPLC was carried out on a YMC-Pack ODS-A, A-324 column (10 i.d. × 300 mm) using the mobile phase acetonitrile -water (2:8, v/v) with 0.1% acetic acid. The flow rate of 2 ml/min at column oven temperature 40°C, and UV detection (λ max = 280 nm) were applied. The gels, Dia-ion HP-20 and MCI-gel CHP-20P (products of Mitsubishi Chemical, Japan), were used for the chromatographic experiments.

Plant material
Fresh seed kernels of M. indica variety Tymor were collected from mature ripe fruits which were purchased from a private farm in Assiut. The plant species was identified by Prof. Dr. Ayman Kotb, Horticulture Department, Faculty of Agriculture, Assiut University. An authentic sample (No. 2012MT) was kept in the Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt.

Culture of T. vaginalis
Fresh T. vaginalis was isolated from a female patient visiting the Patient Treatment Center, Gynecology and Obstetrics Hospital, Faculty of Medicine, Assiut University. After explaining the aim of the study, informative consent was signed by the patient. This study was endorsed by the Research Ethics Committee, Faculty of Medicine, Assiut University, Egypt. The live trophozoites were grown in trypticase-yeast extract-maltose (TYM) medium (Innocente et al., 2014) in 15-ml screw-stoppered glass tubes at 37°C. The medium was complemented with 1-ml heat-inactivated fetal calf serum, crystalline penicillin (1,000,000 IU/ml), and streptomycin sulfate (100,000 μg/ml), and the pH was adjusted at 6.0. The isolate was regularly transferred to fresh TYM medium every 48 hours, and maintained in the Parasitology Department, Faculty of Medicine, Assiut University. The growth of T. vaginalis was monitored until it reached the logarithmic growth phase. The trophozoites exhibiting normal viability and morphology were harvested, isolated from the medium by centrifugation, and regrown in fresh TYM medium for the investigations; 10 5 /ml cells were used for the evaluation of the anti-trichomonal effect of the isolated compounds. The organism count number was monitored using hemocytometer slides.

The trichomonacidal assay
The anti-T. vaginalis activity of the pure compounds (2-8) was determined in vitro based on the reported method (Ibrahim, 2013). The test compounds were diluted by DMSO as solubilizing vehicle at a concentration not more than 0.01% in the experiment medium. A series of two-fold dilutions for all compounds tested were obtained and used at final concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 μg/ml. A 50 μl of 1 × 10 5 trophozoite/ml was inoculated in 150 μl of the test compound in a sterile 96-well culture plate at 37°C (Innocente et al., 2014). Three controls were considered: negative control with parasites only; vehicle control (DMSO); and the positive control [metronidazole (MTZ) (Sanofi-Aventis, Egypt). MTZ was dissolved in distilled H 2 O, diluted by the incubation medium giving four concentrations, 3.12, 1.56, 0.75, and 0.0037 μg/ml, and used as the reference standard. The 96-well plate was incubated for 72 hours at 37°C, 5% CO 2 . The number of living organisms was determined by counting the parasites by a hemocytometer using the trypan blue dye (0.2%) exclusion method. Control cultures were treated by the same procedure as that of the test cultures. All assays were carried out in triplicate and repeated three times. For evaluation of the activity of the compound at different contact times, samples from the cultures were taken after 24-, 48-, and 72-hours incubations. The mortality percentage of the parasites was determined from the equation {100 × [100-a/b]} where "a" is the living organisms count in test wells and "b" is the living organisms count in the control wells (Ali, 2007).

Statistical analysis
The IC 50 values were computed from a non-linear regression curve using the GraphPad Prism 5.0 software.
The easy and fast isolation procedures described here for gallotannins could be useful for the rapid purification of such compounds from other gallotannins-rich plants including other morphological parts of M. indica.

Anti-T. vaginalis activities of the isolated compounds
Secondary metabolites from natural sources have played a unique role in the discovery of anti-infectious compounds. Studies on the inhibitory effects of plant extracts on the growth of T. vaginalis trophozoites evidenced that extracts from the volatile oils producing plants Mentha piperita, Salvia officinalis, and Tanacetum parthenium demonstrate anti-T. vaginalis activity identical to MTZ (Ezz Eldin and Badawy, 2015;Sharafi et al., 2013). A commercial garlic-based product (Tomex ® ) has been also shown to significantly reduce the multiplication and motility of the T. vaginalis trophozoites (Ali, 2007). In another study, the hydrolyzable tannins containing ethyl acetate fraction of a Eucalyptus extract exhibited MTZ-like activity on the growth of the Trichomonas trophozoites (Hassani et al., 2013). Meanwhile, limited research on the effects of pure compounds from natural sources, such as berberine alkaloid (de Brum Vieira et al., 2015;Sharafi et al., 2013) and piperazinyl derivatives of betulinic acid (Innocente et al., 2014), have been reported.
Herein, the effect of a group of gallotannins and ethyl gallate from kernels of M. indica on the viability of T. vaginalis clinical isolates was estimated (see experimental section). Although the common chemical nature of the investigated compounds, galloyl esters of a glucose core (Fig. 1), their antiprotozoal activities were varied based on the molecular structure (Fig. 3). All the examined compounds (2-8) exhibited a remarkable dosedependent and time-dependent decrease in the percentage of living trophozoites (Fig. 3). After 24 hours of incubation, ethyl gallate, a tannin-related compound, showed the highest inhibitory effect on the T. vaginalis trophozoites viability (IC 50 1.3 μg/ml, Fig. 4). The gallotannin with an unacylated O-3 position of the glucose core, 1,2,4,6-tetra-O-galloyl-β-D-glucose (7), exerted a potent effect (IC 50 2.4 μg/ml), while the inhibitory effect of ~1:1 mixture of 5 and 6 on the T. vaginalis trophozoites was relatively low (IC 50 36.1 μg/ml). The other gallotannins (3, 4 and 8) exhibited noticeable anti-T. vaginalis activities with comparable potencies (IC 50 3.9 -9.9 μg/ml). Due to the presence of some differences in the activity of the examined gallotannins, a structural activity relationship can't be generated because of the limited number of the investigated compounds.
The broad-spectrum antiprotozoal activities of the mango leaf and stem bark extracts (Núñez Sellés et al., 2002) against Entamoeba histolytica (Tona et al., 1998), Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp (Grabensteiner et al., 2008), Plasmodium falciparum (Awe et al., 1998;Ruiz et al., 2011;Zirihi et al., 2005), and Giardia lamblia (Amaral et al., 2006) agree with our herein obtained results. In addition, the reported leishmanicidal activity of several tannins against amastigotes of Leishmania donovani (Kolodziej et al., 2001) also supports our results. Likewise, the antibacterial activity of gallotannins and/ or extracts of mango kernels on different bacterial species have been also shown in several reports (El-Gied et al., 2012;Engels et al., 2010;Ka buki et al., 2000;Rajan et al., 2011;Rakholiya et al., 2015;Shabani and Sayadi, 2014;Subbiya et al., 2013). The antimicrobial properties of the kernel gallotannins were ascribed to its ability to intermingle with proteins, hinder the enzyme activity (Rajan et al., 2011), and/or its ability to make a complex with metal ions such as iron (Engels et al., 2009). Altogether, the anti-T. vaginalis activity of the isolated gallotannins may be attributed to either or all of the aforementioned mechanisms. T. vaginalis uses the iron-containing proteins lactoferrin and hemoglobin (Sehgal et al., 2012 delivered by the menstruation blood (Figueroa-Angulo et al., 2012). A study on the effect of iron deficiency in the host on T. vaginalis demonstrated changes in the parasite propagation, cytotoxicity, and immune evasion (Alvarez-Sánchez et al., 2007). Therefore, iron deficiency by complexation with gallotannins may be the cause of the trophozoite cellular damage and the parasite survival inhabitation at the experimental conditions.

CONCLUSION
We are reporting here on the accumulation of gallotannins (galloylglucoses), gallic acid, and gallate derivatives in mango kernels. The procedure as described here is an easy and fast isolation for gallotannins that could be useful for the preparation of these compounds either as a crude fraction or single pure sample from the kernels and from gallotannins-rich plant extracts including other morphological parts of M. indica. The present study demonstrated, to the first time, that the mango kernels along with its isolated gallotannins and ethyl gallate could be used for further studies on the development of novel preventive or therapeutic agents for the treatment of trichomoniasis. However, it is still required to achieve animal lab-work and more mechanistic studies to approve our in vitro finding.

CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.