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Hendrik Mueller-Werkmeister, student
Georg Feuerriegel, student
Jelena Krasilnikova, professor, ph.d. of medicine, associate professor
Riga Stradins University, Latvia
This research work is related to the European COST program, action CM0804:
Chemical Biology with Natural Products:
“The main objective of the Action is to advance the use of natural products as tools for
chemical biology. Applying modern techniques and advancing them, natural products will
prove to be instrumental in discovering target proteins and biological pathways that are of
relevance to diseases. This in turn, should facilitate and speed up subsequent drug discovery
efforts in the pharmaceutical industry.”
Lignin is a complex dendritic network polymer of phenyl propene basic units is one of the most important substances in the secondary cell wall of plant cells. It is responsible for the strength and density of wood. Next to cellulose it is the second most abundant renewable carbon source on earth.
It is difficult to use lignin in its natural form in any analytical methodology therefore the lignin used in this research has been fractioned with Isopropanol.
For broad effects of the influences of lignin on the activity of digestive enzymes, two initial experiments with artifical body media have been made:
1) ASSESSMENT OF LIGNIN (JP I, JP II) INFLUENCE ON SALIVA ACTIVITY
Amylases are enzymes, produced in the human body by salivary glands and the pancreas , that catalyzes the breakdown of starch into monosaccharides. Salivary ?(1 → 4)-amylase hydrolyses randomly ?(1 → 4) glucoside bonds of polysaccharides in the oral cavity.
The amylase activity is characterized with amyloclastic force, that is the volume of the 0,1% starch solution in milliliters that is hydrolysed by lml of saliva at 38°C dyring 30 minutes.
The aim of this research work is the investigation of the influences of Lignin in different concentrations on human saliva activity, especially on the enzyme amylase.
For the experimental determination of the influences of Lignin on amylase activity five sets of testtubes are used, the saliva is donated by a sober student (without nutrition, chewing gum, nicotin etc.) to get clean results. All sets consist of a test and a control row. Within the first set the natural activity of amylase of the used saliva is investigated, in the second set one drop of Lignin JP II (concentration c = 1600 mg/L) is added, in the third set Lignin JP I (concentration c = 800 mg/L), in the fourth set diluted Lignin JP I (concentration c = 400 mg/L) and in the fifth set diluted Lignin JP I (concentration c = 200 mg/L). The Lignin, that is used in the experiments is extracted from black alder wood.
Calculation of the native amyloclastic force of the donated saliva after 30 minutes at 38°C: Testtube number 5 showed a reddish-brown colour after 30 minutes at 38°C and 1 drop of 1% iodine solution, the saliva quantity in it is 1/320 ml. This number is used for the calculation of the amyloclastic force. D 30/38o. = (l*2)/(l/320) = 640
Second test row with l drop of Lignin II (c = 1600 mg/L), testtube number 8 showed reddish-brown colour, the resulting amyloclastic force is D 30/38o. = (l*2)/(l/2560) = 5120
Third test row with 1 drop of Lignin I (c = 800 mg/L) and fourth test row with 1 drop of Lignin I (c = 400 mg/L), testtube number 7 showed a reddish-brown colour, the resulting amyloclastic force is D 30/38o. = (l*2)/(l/1280) = 2560
Fifth test row with 1 drop of Lignin I (c = 200 mg/L), testtube number 8 showed reddish-brown colour, the resulting amyloclastic force is D 30/38o. = (l*2)/(l/2560) = 5120
A strong influence of Lignin on amylase activity can be concluded from the observations made during the experiments. Even in very low concentrations the amyloclastic force increases clearly (by factor 4 – 8).
2) PROPANOL FRACTION OF LIGNIN (JP III, JP IV) IN GASTRIC JUICE
Pepsin is an endopeptidase (enzyme) resident in the gastric cavity. It is secreted as inactive zymogene called Pepsinogen and activated by autocatalysis.
Autocatalysis is triggered by an acidic stomach medium caused by the secretion of HCL during the cephalic and gastric stages of digestion. Pepsin catalyzes the hydrolysis of peptide bonds adjacent to aromatic and branched-chain amino acids and methionine.
Casein is a phosphoprotein found in milk. It consists of many proline peptides and makes up to 80% of the proteins in cow milk. Casein is found as major component in cheese but also used as food additive in sports.
For the experimental determination of the influence of Lignin on the activity on the enzyme Pepsin, six sets of test tubes are used. Casein, is used as a protein source in equal amounts of 1 g in each test row. Every set consists of a test and a control row. Lignin is used in five groups in two different concentrations. JP-3 (concentration c = 800 mg / L) is added to the Sets 2 – 4, JP-4 (concentration c = 1.600 mg / L) is used in the sets 5 and 6. The first part of the experiment is the simulation of a acidic gastric medium (HCL is added) to show the effects of Pepsin on the Casein resin and, by a visual observation, determine the changes of Casein in the different test rows. In the second part, qualitative verification of the pepsin activity is given by the biurete reaction, a chemical test for detecting the presence of proteins (violet colour) or pink (light violet) in the presence of short-chain polypeptides (evidence on Pepsin activity).
Control Group II showed, that the greatest effects of lignin on pepsin are present in low amounts. Higher amounts (for JP-3) and concentrations (for JP-4) seemed to act as an inhibiting factor on the enzyme activity.
The biurete reaction has proved the results from the visual observation. As stated in the theory, the colour reflects the presence of polypeptide bonds. Depending on the intensity, the test gives qualitiativ verification of proteins (violet – dark violet) or short-chain polypeptides if the colour is pink (or light violet).
As seen in the test rows, the effects of Lignin on Pepsin are present. Interestingly, the higher the amounts and concentrations (Evidence given by visual observation test), the lower the effects are, thus, in these regions, an inhibiting role of Lignin is assumed. The greatest effects are present with the lower concentrated JP-3 (c = 800 mg / L) and an amount of only 1 drop (1 / 20 ml), which has been proven by both tests. Therefore an accurately dosage has to be worked out to assure the effective usage of Lignin as a medical drug. The current approximation is 160 mg, which reflects 2 mg of Lignin per kg of body weight. It can help in disease treatments affecting the effects of pepsin, such as Pepsin deficiency or Hydrochloric acid deficiency to elevate the activity of the enzyme available in the patients.
These first experiments have clearly demonstrated the positive effects of lignin on the activity of the enzymes amylase and pepsin. Further investigation will drift more into evaluating practical purposes and prove the genereal suspected effects on all gastric enzymes.
Lignin is an interesting product due to the fact, that it is easily obtained from the black alder tree by simple fractioning of its juice. Therefore it can be developed into a very cost effective drug for many gastrointestinal, and related, disorders.