# Plant Iridoids Affect Intraocular Pressure and Vascular Flow in the Rabbit Eye
## 2 Materials and Methods  
The approval for the study was obtained from the local ethics committee (No. 21/2015 dated 18 March 2015). The iridoids used in the experiment where purchased from Sigma Aldrich, St. Louis, MO, USA. The purity was above 98%, according to HPLC (High-Performance Liquid Chromatography). For the evaluation of the effect of iridoids on intraocular pressure and blood flow in the blood vessels of the iris, 14 young, sexually mature _New Zealand white_ rabbits, aged between 6 months and 1 year, were used: 7 males and 7 females. The average female mass was 4.5 kg, whereas for male rabbits, it was 4.1 kg. Each compound was tested in 7 rabbits, of mixed sex. The time interval between the experiments (use of new compounds) was 3-4 days. In all experiments, control was the untreated eye. During the experiment, the rabbits were kept in individual cages at 21-23 \({}^{\circ}\)C. The animals were fed a full-meal blend feed for rabbits (a special mix for the time of biological testing LSK, Agropol S.J., Motycz, Poland), ad libitum, with unlimited access to drinking water. Before the experiment itself, the animals were under 4-week quarantine. Their behavior and physical condition were observed during that time. The measurement of data throughout the experiment was carried out by one person. This course of action was aimed at domestication, as well as stress reduction, as stress could interfere with the results of the experiment. The experiment was performed within two months (1 day of experimentation, followed by a 3-4 day interval). A solution of iridoids was administered in an aqueous solution of artificial tears containing 0.15% sodium hyaluronate (vehicle). The iridoids that were administered topically included verbenalin (VERB, 1), loganin (LOGA, 2), aucubin (AUCU, 3), catalpol (CATAL, 4), harapagoside (HARP, 5), oleuppein (OLEU, 6), geniopticroside (GENCJ), 7), and secologanin (SECO, 8), as presented on Figure 1. In addition, a 0.5% timolol solution was also administered as a positive control. Experimental drops where prepared by dissolving the corresponding iridoids in Kropia eyedrops, S-lab, Mirkow, Poland (0.3% hyaluronic acid, without preservatives). A 0.15% solution was obtained by dilution in distilled water. In the first part of the study, each  
Figure 1: Chemical structures of investigated iridoids: (**1**) verbenalin (VERB), (**2**) loganin (LOGA), (**3**) aucubin (AUCU), (**4**) catalpol (CATAL), (**5**) harpagoside (HARP), (**6**) oleupropein (OLEU), (**7**) geniopticroside (GENCJ) and (**8**) secologanin (SECO). animal was administered 0.5% iridoids in a 0.9% NaCl (saline) fraction intraconjunctivally, in a volume of 1 drop corresponding to the volume of 50 \(\upmu\)L to the right eye (study group), and to the left eye, saline in the volume of 1 drop (50 \(\upmu\)L) was administered as a placebo (control group). Then, the intraocular pressure was measured in both eyes at six time points: before (time 0) and 1, 2, 3, and 6 h after iridoid and vehicle administration. An induction Icare VET rebound tonometer (Icare Finland Oy, Vantaa, Finland) was used for the measurement of intraocular pressure, without anesthesia, before measurement. We repeated the experiment with the 0.1% iridoids in the same way. In the second part of the study, each animal was administered iridoids intraconjunctivally, in a volume of 1 drop (50 \(\upmu\)L) to the right eye, and to the left eye, saline was once again administered in the volume of 1 drop (50 \(\upmu\)L) as the control. Blood flow in the iris was measured before (time 0) and 1, 2, 3, and 6 h after administration. The measurement was completed using a laser Doppler flow meter--Laser Blood Flow Monitor MBD3 (Moor Instruments, Axminster, GB). Following local anesthesia of the cornea (Alcaine, Alcon Laboratories, Inc., Fort Worth, TX, USA), the laser probe was placed in direct contact with the cornea (approximately 2 mm from the corneal limbus). The laser beam was directed perpendicularly to the surface of the iris.