Drug – bio-affecting and body treating compositions – Topical sun or radiation screening – or tanning preparations
Reexamination Certificate
2001-05-25
2002-05-28
Dodson, Shelley A. (Department: 1616)
Drug, bio-affecting and body treating compositions
Topical sun or radiation screening, or tanning preparations
C424S060000, C424S400000, C424S401000, C424S725000, C424S771000
Reexamination Certificate
active
06395261
ABSTRACT:
The invention relates to a walnut seed cake extract. It also relates to a cosmetic composition comprising said extract. Its subject is also various applications of the extract and therefore of the cosmetic composition.
In the remainder of the description and in the claims, “walnut seed cake” designates the residue from the pressing of walnut seed after extraction of the oil which it contains, commonly called “walnut oil”.
Walnut trees belonging to the Juglandaceae family are widely distributed and cultivated in temperate countries. This includes in particular
Juglans regia
which is found in Europe or
Juglans regia
which is found in America, which are the subject of a number of applications.
There has thus been described, in the document RO-A-103 270, a cosmetic composition used for washing hair and comprising, in combination, a salicylate and an alcoholic extract of walnut tree of the
Juglans regia
type. However, nothing is indicated regarding the part of the tree from which the extract is obtained.
There has also been described, in the document EP-A-0306853, a cosmetic composition exhibiting germicidal properties, containing extracts of barks of roots of trees of the Juglandaceae family.
Likewise, certain parts of the walnut have been identified as having advantageous properties.
This is for example the case for the walnut hull constituting the outer fleshy part of the walnut, which contains a naphthoquinone compound (juglone) used as hair dye.
On the other hand, the residue from the pressing of the walnut seed constituting the cake has never been upgraded and has consequently not received any particular application.
The Applicant has sought to isolate a walnut seed cake extract and has observed that it exhibits a number of advantageous properties.
The invention therefore relates to a seed cake extract of walnuts produced by a walnut tree of the genus
Juglans
which can be obtained by a step of aqueous extraction of the cake followed by a concentration step.
Advantageously, the concentration is carried out by reverse osmosis.
The preparation of an aqueous extract can be accomplished by any of the techniques known to persons of skill in the art, including maceration, percolation, digestion, microwave, and ultrasonic waves. The temperature can vary depending on the process. Indeed, the extraction can be conducted at ambient or higher temperature, although high temperatures and long exposure times will result in partial destruction of active ingredients. The period of contact may vary depending on which extraction process is used. If the extraction is conducted using microwaves and the weight of seed cake is small in comparison to the power of the microwave, the period of contact can be as short as a few seconds. There is an inverse relationship between time and temperature. The ratio of walnut seed cake to solvent (water) is a balance between the amount of solvent needed for efficient extraction and the amount of solvent that must be subsequently removed. Typical reasonable ratios fall between 1/99 and 20/80 (by weight). Ratios falling outside this range will work, but they sacrifice process efficiency. We have found that 5:95 is a good ratio. The optimal extraction process is maceration, carried out at a temperature between 3 and 10° C., advantageously at about 4° C. A temperature less than 3° C. may cause the formation of ice; a temperature greater than 10° C. risks causing a decline in the activity of the extract. When the extraction process is maceration at 3 to 10° C. for less than 40 hours, exposure time determines only the efficiency of the extraction, not the nature of the extract. We have found that about 20 hours is optimal.
The extract obtained is provided in the form of a concentrated aqueous solution.
To obtain the extract in powdered form, the concentration step is followed by a drying or freeze-drying step.
Advantageously, the walnuts from which the cake extract is derived are produced by a walnut tree (Jugans regia).
As already stated, the walnut cake extract of the invention has a number of advantageous properties, such that it can be used for various applications, in particular in cosmetic compositions.
1/Protective Activity of the Walnut Seed Cake Extract Toward the Intracellular Oxidation Caused by an Oxidative Stress of the UVB Type
The experimental approach used for this evaluation is based on the measurement of the intracellular oxidation level, after exposure to LVB radiation, of human keratinocytes cultured in the absence and in the presence of the extract of the invention.
Methods
Normal human keratinocytes isolated from foreskins obtained during surgical operations are cultured in “serum-free” KGM medium at 37° C., in a humid air-CO
2
(95-5%) atmosphere.
After cytotoxicity studies, three concentrations of extract were selected for evaluation: 10 &mgr;g/ml, 100 &mgr;g/ml, 250 &mgr;g/ml.
Principle of the Test
The principle of the test is based on the measurement of the degree of intracellular oxidation with the aid of a specific probe: DCFH-DA (2′,7′-trichlorodihydrofluorescein diacetate).
DCFH-DA, a nonfluorescent probe, penetrates by passive diffusion into the cells. After cleavage of the acetate groups by intracellular esterases, DCFH accumulates in the cytosol. The intracellular oxidation of DCFH by various Reactive Oxygen Species (ROS) leads to the formation of a fluorescent product.
The measurement of the fluorescence intensities makes it possible to evaluate the degree of oxidation of the cells subjected to an oxidative stress.
After having been detached from their support, the suspensions of keratinocytes are inoculated into 24-well plates. The cells are incubated at 37° C. for 24 hours with the medium containing the extract to be studied.
After incubation with the various concentrations of extract to be tested, the cultures are rinsed with a PBS solution and then exposed to UVB radiation, through a PBS solution.
After irradiation of the cells, the PBS solution is replaced with a solution of DCFH-DA. After incubation of the fluorescent probe, the cellular lawns are abundantly rinsed with PBS and the cells are then reincubated at 37° C. for 24 hours with fresh culture medium.
At the end of the assay, the suspended keratinocytes are transferred into thermostatted vessels and the fluorescence intensities (Abs=502 nm, Em=520 nm) are measured and expressed in percentage relative to the nonirradiated control cultures.
The results are expressed in the following table:
Degree of oxidation
Protective activity
Control
no UV
100
—
25 mJ/cm
2
144
—
50 mJ/cm
2
176
—
10 &mgr;g/ml
no UV
100
—
25 mJ/cm
2
116
39
50 mJ/cm
2
125
65
100 &mgr;g/ml
no UV
100
—
25 mJ/cm
2
107
74
50 mJ/cm
2
115
79
250 &mgr;g/ml
no UV
100
—
25 mJ/cm
2
98
104
50 mJ/cm
2
106
92
Under the conditions of this study, the extract of the invention protects in a dose-dependent manner the cell against oxidative stress induced by UVB irradiation. The extract is therefore capable of regulating the level of reactive oxygen species (ROS) which form. The extract possesses the capacity to protect the cell from oxidative stress, that is to say to block the formation of ROS and/or to inhibit their reactivity by a “trapping” process.
In a first application, the extract of the invention may therefore be used to protect skin cells from oxidative stress induced by UVB irradiation.
2/Activity for Protecting Cells Against Apoptosis Induced by UVB Radiation
ROSs capable of reacting directly with DNA can introduce into this constituent multiple chemical modifications. These modifications, which have the consequence of disrupting the genetic program of the cell, are as a whole corrected by so-called repair enzymes.
However, when this repair is not complete, the cell enters into a program of programmed cell death or apoptosis leading to their elimination.
Evaluation of the Activity for Protecting Cells Against Apoptosis Induced by UVB Radiation
This protective power against apoptosis induced by TVB radiation was evaluated on monolayer keratinocyte cultures by determining th
Dodson Shelley A.
Gattefosse S.A.
Heslin Rothenberg Farley & & Mesiti P.C.
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