B i t t e r G o u r d

V. Isolation & Synthesis of Charantin

One procedure to identify and isolate charantin is affinity chromatography on gel which showed that it has many arginine and glutamate amino acids. The compound isolated from the seeds of the bitter gourd, charantin, was found to be 9.7 kDa . This process starts off with the crushing of bitter gourd seeds and centrifuging it to obtain a crude extract and was treated with buffers and until the turbidity reading at 280nm was less than 0.05. Some of the extract was applied to an Affi-Blue gel and fractions of the 6mL were collected at a constant rate of 1 mL/min. This was then assayed for translation-inhibitory action and the adsorbed protein that did that was dialysed against 4^oC distilled water for 36 hours. This was then dissolved again in a buffer and treated to get another constant flow of 0.4 mL/min and the purified substance obtained was named charantin ^[12] . Mass spectroscopy was also used to determine the composition of charantin in helping to figure out its structure based on how many peaks and where and the number of carbons in the spectrum ^[11] .

This is an example of a Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of charantin compared to other molecular weight markers.

Charantin is in column 3 as compared to the other weight markers. The top one in column 1 is phosphorylase B, with a mass of 94 kDa. The very bottom of column 2 is oxidized insulin B chain with a mass of 3.5 kDa. As you can see, charantin is in between those 2 weights and was measured to be 9.7 kDa ^[12] .

There is a fairly new technique for the extraction of charantin from Momordica charantia called Pressurized liquid extraction (PLE) and is done by using ethyl alcohol as the solvent. By changing the solvents, solvent flow rate, and temperature, scientists were able to achieve a more efficient way to isolate charantin from the actual fruit of the plant at an increased rate in time and a decrease to how much solvent they lost ^[5] . Before liquid chromatography could be performed, the charantin needed to be purified so that there were no other contaminants like chlorophyll and other sugars. The most efficient way to extract the charantin depended on the temperature and the composition of the solvents.

This is a picture of the device needed for the Pressurized Liquid Extraction taken from this journal [5] .

So pressurized hot water is heated upto 400^oC so that it is above boiling but below critical heat (where it could just decompose and destroy most the chemical structures), and it's kept in this subcritical area so that the water dielectric constant decreases which makes it possible for the compounds to be soluble. This type of PLE requires low solvent volume and a short extraction time because the solvents are heated to the critical region where they have high extraction properties.

First, a crude extract needs to be taken from the bitter gourd seeds and 5 mL of methanol-water was added to it. The mixture was centrifuged for 15 minutes. The precipitate was taken and with 5 mL methanol-water, it was centrifuged again. It was centrifuged a third time with 3 mL of hexane. After this, the mixture was dissolved in a 200 microliter chloroform-methanol mixture. This mixture was filtered through nylon membrane filter and analyzed. The flow rate of methanol-water on the extract was 1 mL/min. This was pressurized to 10 MPa and extracts were collected every 10 minutes. The percent recovery was almost above 90% with this new extraction technique ^[5] . Charantin is a mixture of two compounds called sitosteryl glucoside and stigmasteryl glucoside.

VI. References