**4.1 Saponin mediated dispersal of lipid deposits**

The potential for saponin mediated dispersal of lipids was assessed in both isolated deposits and intact human Bruch's membrane. Bruch's from four donor eyes (ages 50 and 82 years) was homogenised in Tris-buffer and spun to obtain pellets containing the deposits. Pellets were resuspended in Tris buffer containing ginsengderived saponins in the range 0-1.2 mg/ml and incubated for 12 hours at 37o C. Samples were then spun and any lipids released into the supernatants extracted with chloroform: methanol (2:1 v/v). Lipids were then separated on Silica Gel thinlayer chromatography (TLC) plates using solvent system #1, chloroform: methanol: acetic acid: water (50:30:8:3 v/v) and solvent system #2, heptane: diethyl ether: acetic acid (70:30:2 v/v). Lipid spots were visualised by staining with amido-black 10B stain and following densitometry, levels quantified with reference to standard curves. Saponins were observed to rapidly release various lipid classes from the deposits in a dose dependent manner (**Figure 5**).

In addition, 14 Bruch's preparations were obtained from 4 human donors (age range 64-75 years) and mounted in Ussing chambers. All chambers were perfused with Tris buffer to remove loosely attached debris and then half the chambers were incubated with Tris buffer and the other half with Tris buffer containing 4.6 mg/ml saponins for 12 hours at 37o C. Chambers were rinsed in Tris buffer and the content of the various lipid classes present in Bruch's membrane quantified as detailed above. The content of lipids in the samples incubated with saponins was significantly reduced compared to controls (**Figure 6**). Thus, saponin molecules are able to solubilise and disperse the lipid deposits in Bruch's membrane.

#### **4.2 Saponin-mediated release of deposited proteins**

Soluble proteins that are either damaged due to chemical modification or denatured tend to unfold exposing the hydrophobic regions to the aqueous environment. This leads to aggregation and deposition within Bruch's membrane. The possibility that saponins could interact with these 'amphipathic' structures to de-segregate and solubilise them was also assessed. Bruch's membrane from four human donors (age range 49-77 years) was mounted in 8 Ussing chambers and perfused with Tris buffer. The perfusate was collected every 5 hours and the protein content determined. As indicated in **Figure 7**, perfusion with Tris buffer resulted in slow release of loosely adherent proteins up to the fourth perfusion period (20 hours). In four chambers, the perfusion fluid was then switched to one containing 167 μg/ml of saponin Rb1. This resulted in the rapid and copious release of further, presumably trapped proteins from the membrane (**Figure 7**). The possibility that trapped MMP enzymes may also have been released is assessed below.

#### **Figure 5.**

*Saponin-mediated release of lipids from deposits extracted from Bruch's membrane. Extracted deposits were incubated with saponins in the range 0-1.2 mg/ml for 12 hours, spun, and lipids present in the supernatant quantified. Saponins released cholesterol, cholesterol esters, phospholipids, and triglycerides in a dose-dependent manner. Data is given as Mean ± SD.*

#### **Figure 6.**

*Saponin-mediated release of lipids from intact human Bruch's membrane. The level of the major lipid classes was reduced following saponin treatment. Data is given as Mean ± SD.*

*Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

#### **Figure 7.**

*Solubilisation and release of trapped proteins from Bruch's membrane. On perfusion with saline, loosely adherent proteins are released slowly. When there was no further release (after period 4), the perfusion medium was switched to one containing Rb1, resulting in the copious release of trapped proteins. TBS-Tris buffered saline; Rb1-Ginsenoside Rb1. Data as Mean ± SD.*

#### **Figure 8.**

*Saponin-mediated release of trapped MMP enzymes. (A) Perfusion with Tris buffer did not release any MMP species from Bruch's membrane. (B) Saponin perfusion released trapped MMP species and in particular activated forms of MMPs 2&9. FCS-foetal calf serum standard. P- pro-MMPs; A-activated MMPs. From reference [43].*

### **4.3 Saponin-mediated release of trapped MMP enzymes**

MMPs are detected by the technique of gelatine zymography. This is standard electrophoresis but with the gel containing 1% gelatine, a substrate for MMPs. Samples are electrophoresed with the MMPs migrating according to molecular weight, the smaller ones running fastest. Following electrophoresis, the gel is incubated in Tris-buffer (containing calcium) for a period of 18-24 hours to allow the MMP enzymes to digest the gelatine in their locality. The gel is then stained with Coomassie Blue and after de-staining, regions containing gelatine stain blue but where the gelatine has been hydrolysed (by MMP enzymes), the region is colourless. Reversing the grey-scale of the gel image shows the MMP regions as dark bands and the identity of the MMP is confirmed from its molecular weight.

To assess the likely effect of saponins on release of trapped MMP enzymes, Bruch's membrane from donors aged 49-71 years was mounted in Ussing chambers and perfused for a period of 12 hours to remove plasma-derived and loosely bound MMP species. Half the chambers were then perfused with Tris buffer for three periods of 3 hours each and the remaining half with saponins at a level of 4.6 mg/ml. After each period, the perfusate was collected and examined by zymography. Incubation with Tris did not show the release of any MMP species (**Figure 8A**). However, perfusion with saponins showed a trace release of MMPs in the first period followed by greater release in the two subsequent periods (**Figure 8B**). The release profiles showed the presence of activated MMP9 and MMP2. If this release occurred in vivo, it would kick start the MMP degradation machinery, rejuvenating the membrane.
