Summer 2001 Issue — PRODIET™ F 200
A promising anti-stress casein hydrolysate

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INGREDIA is a "still young" but experimenting French company (nearly 10 years old), but although a market leader, specialized in functional dairy ingredients for the food industries.

INGREDIA has recently launched a new range of health ingredients for functional foods and dietary supplements. PRODIET(TM) F 200, a unique milk protein hydrolysate, is the main product of this new range, with its anti-stress properties, which have been scientifically proved. According to the Leatherhead Food research Association, 1 stress is one of the 3 top health concerns in Europe. People asked which problem they were most concerned about placed stress in the top three in United Kingdom and Germany, and placed first in France.

In the USA, 48% of consumers are concerned about stress. 2 Noise, social and professional pressures, lifestyle, overwork, and pollution is often mentioned as sources of stress and irritability.

Anxiety and sleeping disorders are frequent outcomes of stress. Absenteeism, loss of productivity, social problems and accidents lead to a significant economic cost for society, and to increasing psychotropic drugs misuse. Moreover, the most common psychotrops used by consumers occurs further side effects, such as dependency, tolerance or memory loss.

The milk protein hydrolysate PRODIET (TM) F 200 contains a bioactive peptide with anti-stress properties.

Many biological activities have been connected with different milk peptides, including opioïd or anti-opioïd, anti-hypertensive, anti-thrombotic, immuno-modulating or calcium transport activities. Nevertheless, only a few of them have resulted in a commercial food product.

PRODIET (TM) F 200 comes as a result of a 10-year research, including a close collaboration between INGREDIA and public (CNRS, INRA, Université de nancy) and private research laboratories. Observing the calm state of a baby after drinking milk, researchers of the "Laboratoire des Biosciences de l'Aliment" of nancy (France) aimed to evaluate a possible relationship between milk digestion and potential calming properties of peptides issued from this digestion. Some American researchers have recently confirmed that milk infant formula delivery quiets effectively human newborns.(3) Reproducing and optimizing proteolytic digestion in the digestive tract, INGREDIA and the Université de Nancy developed a milk casein hydrolysate. They tested firstly its potential anxiolytic effect on rats, and subsequently checked it in vitro on biological receptors. In this way they showed the anxiolytic-like activity of a bioactive peptide. Researchers then purified this decapitate, identified it and tested the activity of the same peptide chemically synthesized, and finally modeled the spatial structure with two-dimensional NMR spectroscopy. (4,5)

INGREDIA researchers and their contributors designed a food-grade process to manufacture an industrial casein hydrolysate and evaluated the anxiolytic-like activity of this industrial food grade ingredient in vivo. Pre-clinical studies were firstly conducted on rats, before doing clinical trials on humans. INGREDIA mainly wanted to market a food grade ingredient with clearly proved anti-stress properties with side effects.

Firstly, PRODIET(TM) F 200 activity and efficacy after oral absorption in male Wistar rats has been checked and quantified. These results have been always obtained in comparison with a placebo and with a chemical anxiolytic (benodiazepine family) usually used in those types of tests as a well-known model control. Secondly, the researchers checked already in rats if there were any side effects that would make PRODIET(TM) F 200 comparable to a chemical anxiolytic, for example memory loss, tolerance and dependency.

Finally, with human clinical trials, INGREDIA had tested the anti-stress efficacy of PRODIET(TM) F 200 after oral absorption in healthy subjects, evaluated during a stress test. These studies were made in collaboration with well-known medical structures, measuring psychometric and haemodynamic parameters and determining quantities of saliva or plasmatic cortisol and plasmatic ACTH (biological stress indicators).

Pre-clinical studies: efficacy without side effects

All the pre-clinical studies on rats were double blind conducted, versus placebo, and versus diazepam as an experimental model control. First of all, the Conditioned Defensive Burying (CDB) model, (6.7) (see Figure 1) was used to check and quantify the bioactive peptide and the PRODIET(TM) F 200 efficacy. In the CDB model, based on the natural tendency of a rat to bury with sawdust any aversive object causing anxiety, the rats were put in a box containing an electrical probe, manually started for a single electrical shock. A global anxiety score was associated with any result obtained with each of the products tested. After oral absorption of PRODIET(TM) F 200, a strong decrease in the global anxiety score of the rats was observed unlike that obtained in placebo group (see Figure 2). PRODIET(TM) F 200 shows a real efficacy, similar to that of the controlled group.

Behavioral side effects were then checked using appropriate models. Firstly, the Irwin test showed that PRODIET(TM) F 200 neither does nor result in any consecutive effect concerning behavior, neurology or toxicology in Wistar rats. Different models were used to measure other potential side effects: tolerance, addictive effect and memory loss.

Firstly, in the Conditioned Place Preference test, a possible dependency effect was investigated, in measuring the time spent by the rat in a "non preferred" compartment, in order to obtain the wished (and potentially dependency inducer) product.

Secondly, in the Social Memory test, the potential amnesia effect (memory loss) was evaluated. The studied variable was the modification in the investigation duration of a young rat by an adult one during the two sessions of a two steps exposure (30 minutes interval).

Finally, the Conditioned defensive burying test was used for testing a potential tolerance effect, to investigate the necessity of progressively increase the dose in order to obtain the same effect.

As a result, none of these side effects were observed after the bioactive peptide administration to the rats, unlike the results obtained with the chemical model (dependency, social memory loss and tolerance).

INGREDIA was therefore able to conclude that PRODIET(TM) F 200 had an excellent biological activity and efficacy with no side effects. INGREDIA then obtained the approval of the National Ethics Committee to engage clinical studies on humans, with the collaboration of private or public Clinical Investigation centers. The new objective was to investigate the activity of PRODIET(TM) F 200, and to evaluate its optimal dosage, in humans.

Fig 1. Conditioned Defensive Burying Model CBD.

Main clinical studies

Though PRODIET(TM) F 200 is a food ingredient, it was important of INGREDIA to work following Good Clinical Practice guidelines, in agreement with official ethical committees, in order to obtain undeniable results. It must be underlined that PRODIET(TM) F 200 is not a drug but a food ingredient; it could consequently only be tested on healthy volunteers, not on pre-selected people with a natural tendency to stress. A first double blind pilot study was made on 24 healthy volunteers, with a 15 days of twice-daily oral absorption of PRODIET(TM) F 200, to check the PRODIET(TM) F 200 efficacy in moderate stress conditions. Cognitive functions were evaluated through psychometric tests (Stroop test, 8.9 mental rapidity tests and concentration tests). Anxiety questionnaires enabled subjects to be classified according to their anxiety level, when in a stable disposition, and according to the anxiety induced by a stressful situation state depending on the stress situation (respectively Catell 10 and Spielberger 11 scales). Haemo-dynamical parameters were followed and saliva cortisol dosages were done.

This study showed that volunteers with a high anxiety levels as a stable disposition have a smaller increase in their global anxiety-state score in the PRODIET(TM) F 200 group compared to the placebo group.

In the double blind randomized second study, the aim was to determine the PRODIET(TM) F 200 efficacy in greater stress conditions, after a little number of ingestions and at high dosage. It aimed also to confirm its efficacy with incontestable evidence. The 42 healthy volunteers had to ingest PRODIET(TM) F 200 twice daily the previous day and once more in the morning of the test. The parameters were systolic and diastolic blood pressures, heart rate, plasmatic cortisol and ACTH (adenocorticotropic hormone) dosages. ACTH is secreted by the anterior pituitary gland, and controls the secretion of the cortisol by the adrenal cortex. Both hormones are recognized biological indicators of stress. 12 There were two types of stress situations. The first was due to a Stroop test (mental conflict situation) made on computer, with a rapid rhythm and an alarm, which sounded when mistakes were made. The second type was caused by a Cold pressor 13 test: the volunteer had to put his hand past the wrist in slushy ice water for 5 minutes (causing a sharp pain, sometimes resulting in faintness).

The results of this trial are convincing: During the Stroop test, when measuring the deviation percentages of systolic and diastolic blood pressures, there was a significantly lower increase in the PRODIET(TM) F 200 group than in the placebo group.

During the Cold presser test, the heart rate remained statically stable in the PRODIET(TM) F 200 group, while it increased significantly in the placebo group. The blood ACTH level tended to increase during the stress tests for the control group while it tended to decrease in the PRODIET(TM) F 200 group.

The main result of this study is about the plasmatic cortisol level. Initial cortisol levels of both groups should not be compared, because of the important statistical variation existing among people. This variation would perhaps be reduced if the sample were bigger. What should on the other hand be observed inside each group is the sharp statistical decrease of the plasmatic cortisol concentration in the PRODIET(TM) F 200, unlike the placebo one, between the beginning and the end of the tests. Indeed, there is usually a natural biological decrease of the cortisol level from the morning till the night in humans, and the absolute values differ among people. Despite that proven fact, the plasmatic cortisol level remained statistically stable in the placebo group due to the stress tests, while it decreased significantly in PRODIET(TM) F 200 group (see Figure 3).

This study has clearly established the efficacy of the PRODIET(TM) F 200 against stress effects.

Fig 2. Preclinical Studies

Fig 3. Blood Hormonal Dosage: Cortisol

Other health ingredients

PRODIET(TM) F 200 offers a good stability towards pH, temperature and ferments. An international patent covers the hydrolysate PRODIET(TM) F 200. Looking to the future, a number of applications are possible. PRODIET(TM) F 200 is already sold in North America, in dietary supplements, in tablet and capsule form. PRODIET(TM) F 200 may also be incorporated in food products such as chocolate and beverages as it is a free-flowing, water-soluble powder.

Well-being and stress are very important human concerns. INGREDIA's health team goes on "working hard" on health ingredients. There are a large number of potential discoveries for providing lots of interesting bioactive health properties.

Catherine Lefranc
Veterinary Doctor
Doctor of the "Université de Compiègne"

INGREDIA - Health Products Research and Development
Department. 51 - 53 avenue Lobbedez - BP946 -62033
Arras - France

Notes:

Cortisol: Is it a stress indicator? During the stress tests, blood Cortisol level remains stable in the placebo group. While it decreases significantly in the PRODIET(TM) F 200 GROUP.

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