# Patent

## Title
Plant extracts with anti-radical type action

## Abstract

The use of plant extracts, especially plant extracts with an anti-radical-type action, and a cosmetic or dermo-pharmaceutical composition containing extracts of this type. The use of at least one plant extract whose botanical genus belongs to the group formed by the following: Clidemia, Inga, Sabicea, Astrocaryum, Siparuna, Eperua, Byrsonima, Priva, Coutoubea and Goupia genera; as an active substance which has especially anti-radical-type activities, for preparing a cosmetic or dermopharmaceutical product for local, external use for the skin, the mucous membranes and/or the epithet or appendage structures.

## Background
The invention relates to the cosmetics field, particularly to the use of plant extracts in dermo-cosmetics, and its object is the use of certain medicinal herbs from French Guyana for the preparation of cosmetic or dermo-pharmaceutical products for the skin, mucous membrane and/or the epithelium or body appendages (hair, nails etc).
The Creole and Palikur peoples of French Guyana use numerous local plants in their traditional medicine. These plants and their therapeutic use are described particularly in the work entitled: “Pharmacopées traditionelles en Guyane: Créoles. Palikur, Wayapi” by Grenand P., Moretti C. and Jacquemin H., published by Orstom in 1987.
The authors of this invention have however discovered that extracts of certain plants of the above-mentioned type have various biological and biochemical actions linked with very high cutaneous tolerance, which enables them to be used directly in compositions for cosmetic use and particularly in dermo-cosmetics.
The plants chosen by the inventors therefore enable extracts to be obtained, all of which unexpectedly and surprisingly have a stronger anti-radical-type action but also an inhibiting action on tyrosinase (de-pigmenting), on tyrosinase activators (pigmenting), an anti-UVA and anti-UVB action, an action protecting catalase from UVA, and an anti-elastase, anti-collagenase, anti-gylcation and/or lypolytic action (for slimming).
The main object of the invention is therefore to use at least one extract of a plant of a botanical genus belonging to the group formed by Clidemia, Inga, Sabicea, Astrocaryum, Siparuna, Eperua, Byrsonima, Priva, Coutoubea or Goupia as an active ingredient, particularly with an anti-radical action, in the preparation of a cosmetic or dermo-pharmaceutical product for external, topical use on the skin, mucous membrane and/or epithelium or body appendages.
The following particular species may advantageously be selected from the above-mentioned genera:

    
    
        Clidemia hirta, Clidemia dentata 
        Inga bourgoni, Inga pezizifera, Inga alata, Inga alba, Inga capitata, Inga meissneriana 
        Sabicea cinerea, Sabicea glabrescens, Sabicera velutina, Sabicea villosa, Sabicea hirsuta 
        Eperua falcata 
        Byrsonima verbascifolia, Byrsonima crassifolia, Byrsonima chrysophylla, Byrsonima coriacea 
        Astrocaryum vulgare, Astrocaryum murumuru, Astrocaryum chambira, Astrocaryum jauari, Astrocaryum macrocalyx 
        Priva lappulacea 
        Coutoubea spicata, Coutoubea ramosa 
        Siparuna guianensis, Siparuna emarginata 
        Goupia glabra 
    
    


In a preferred embodiment of the invention however, giving extracts with a qualitatively and quantitatively optimised action particularly in respect of the anti-radical-type action, the active ingredient comprises at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia, Priva lappulacea, Coutoubea spicata and Goupia glabra. 
Owing to their many forms of action these extracts are advantageously used alone or combined together or with other active compounds in products designed mainly to control ageing skin, hyper-pigmentation of the skin, patches of pigment, loss of skin elasticity, wrinkles, irritation and inflammation (treatment of sensitive skin types), irritation caused by environmental pollution and/or sunshine and/or for slimming formulae.
The parts of the plants used to prepare and obtain the above-mentioned extracts are chosen from roots, cortices (roots, stalks and/or trunk of plants) leaves and foliated stalks, fruit, grains and/or blossoms.
When the parts of the appropriate plants have been gathered and dried they are subjected to an extraction process. The solvent used may advantageously be selected from the group formed by water, alcohols, ketones, esters, types of ether, chlorine-containing solvents or polyhydric alcohols, or mixtures of at least two of the above-mentioned miscible solvents.
In a special embodiment of the present invention the extract or extracts is/are obtained by an extraction process based on wave radiation, for example microwaves or ultrasound.
The extracts involved in the invention may alternatively be obtained by extraction of supercritical CO2, alone or mixed with a secondary solvent.
In a special embodiment of the present invention the plant extract(s) comprise(s) one or more separated fractions, purified particularly by chromatography and based on one or more extracts of said plants obtained by one of the afore-mentioned extraction processes.
To illustrate the invention, though without restricting its scope, various processes which can be carried out to prepare the above-mentioned plant extracts will now be described.
In the following examples the parts of the plants used are only given as an indication. The extracts involved in the invention can be obtained from any accessible parts of the aforementioned plants.


EXAMPLE 1
Cortices of Epurea falcata trunk are crushed then mashed finely by a blade-type pulping machine.
Three liters of distilled water are put into a reactor with an agitator then the following operations are successively carried out, comprising:

    
    
        adding 300 g of pulped cortex to the reactor
        extracting with agitation for an hour while boiling
        cooling to ambient temperature
        removing the insoluble materials by centrifuging or filtering
        filtering the materials floating on the surface to a porosity of about 0.45 μm
        collecting the filtrate
        extracting the residue with 2 liters of distilled water under the same operating conditions and proceeding as described above
        after determining the content of dry material relative to the aqueous extracts obtained, adding substance of the malto dextrin type to obtain 2⁄3 added substance to 1⁄3 extracted dry material
        de-watering the solution obtained, by spraying or another method known to a person skilled in the art (dehydrating, freeze drying—lyophilisation, drying in a drying chamber etc).
    
    


The total yield of dry extract from this process is 15% by weight relative to the cortices.
EXAMPLE 2
Cortices of Inga bourgoni trunk are crushed then mashed finely by a blade-type pulping machine.
Three liters of 50% ethyl alcohol (ethanol) are put into a reactor with an agitator then the following operations are successively carried out, comprising:

    
    
        adding 300 g of pulped cortex to the reactor
        extracting with agitation for an hour and with (reflux) heating
        cooling to ambient temperature
        removing the insoluble materials by filtering
        filtering the materials floating on the surface to a porosity of about 0.45 μm
        collecting the filtrate
        extracting the residue with 1.5 liter of 50% ethanol under the same operating conditions then proceeding as described above
        evaporating the alcohol phase of both filtrates under vacuum
        after centrifuging and determining the content of dry material, if necessary making the addition to the aqueous phases obtained in the same proportions as mentioned in Example 1
        if necessary de-watering (dehydrating) the solution obtained, by conventional methods.
    
    


The total yield of dry extract from this process is 13.8% by weight relative to the cortices.
EXAMPLE 3
Cortices of the roots of Byrsonima verbascifolia are crushed then mashed finely by a blade-type pulping machine.
Three liters of 80% methyl alcohol (methanol) are put into a reactor with an agitator then the following operations are successively carried out, comprising:

    
    
        adding 300 g of pulped cortex to the reactor
        extracting with agitation for an hour and with (reflux) heating
        cooling to ambient temperature
        filtering off the insoluble materials
        filtering the materials floating on the surface to a porosity of about 0.45 μm
        collecting the filtrate
        extracting the residue with 1.5 liter of 80% methanol under the same operating conditions then proceeding as described above
        evaporating the methanol phase of both filtrates under vacuum
        after centrifuging and determining the content of dry material, making the addition to the aqueous phase obtained in the same proportions as described in Example 1
        if necessary de-watering the solution by conventional methods.
    
    


The total yield of extract obtained by this method is 17.4% by weight relative to the cortices.
EXAMPLE 4
Roots of Astrocaryum vulgare are crushed then finely pulped.
Three liters of absolute ethanol are put into a reactor with an agitator then the following operations are successively carried out, comprising:

    
    
        adding 300 g of crushed Astrocaryum vulgare roots to the reactor
        extracting with agitation for an hour and with (reflux) heating
        cooling to ambient temperature
        filtering off the insoluble materials
        filtering the materials floating on the surface to a porosity of about 0.45 μm
        collecting the filtrate corresponding to Extract 1
        extracting the residue with 3 liters of ethanol under the same operating conditions then proceeding as described above to obtain Extract 2
        evaporating the ethane phase of both filtrates under vacuum at 40° C.
        removing the traces of solvent by drying the extracts in a ventilated drying oven at 40-50° C.
    
    


7.15 g E1 extract and 1.47 g E2 extract are thus obtained and are finally mixed, representing a total yield of extract of 2.87% by weight relative to the roots.
The following table contains a list of all the plants and extracts with which the inventors worked, as an indication.

















% yield (2




Plant parts

successive


Plant
extracted
Type of extract
extractions)
Appearance



















Clidemia hirta
Leaves
Aqueous extract
20.9
Brown powder


Inga bourgoni
Cortices
Aqueous extract
9.7
Wine-red powder




Ethanol E 50%
13.8
Wine-red powder




Ethanol extract
11.7
Wine-red powder


Sabicea cinerea
Foliated
Aqueous extract
16.4
Brown powder



stalks
Ethanol E 50%
25.5
Brown powder




Ethanol extract
18.7
Green paste


Astrocaryum
Roots
Aqueous extract
5.3
Brown powder


vulgare

Ethanol E 50%
5.6
Brown powder




Ethanol extract
2.8
Brown-orange






paste


Siparuna
Leaves
Aqueous extract
16.1
Brown powder


guianensis

Methanol E 80%
21.9
Brown powder




Ethanol extract
22.6
Green paste


Eperua falcata
Cortices
Aqueous extract
15
Brown powder




Methanol E 80%
17.6
Brown powder




Ethanol extract
16.7
Brown paste


Byrsonima
Root
Aqueous extract
10.8
Brown powder


verbascifolia
cortices
Methanol E.80%
17.4
Brown powder




Ethanol extract
15.7
Brown paste


Priva
Foliated
Aqueous extract
24.0
Brown powder


lappulacea
stalks
Methanol E 80%
14.5
Brown powder




Ethanol extract
13.9
Brown paste


Goupia glabra
Leaves
Aqueous extract
30.0
Brown-orange




Methanol E 80%
30.4
powder






Yellowish powder


Coutoubea
Stalks + ears
Aqueous extract
18.5
Brown powder


spicata

Methanol E 80%
18.8
Brown powder




Ethanol extract
18.0
Brown-green paste









It was possible for the biological and biochemical properties and actions of the plant extracts studied, which may be used directly in products of cosmetic importance, to be determined and measured by tests known to a person skilled in the art. The principles of the tests will be briefly discussed below and the results will be set out in the corresponding tables.
1) Anti-radical-type Action
The oxidative anti-stress properties were evaluated by “in tubo” and “in vitro” tests.
The group of in tubo tests includes both the initial radical-type forms of oxygen and the reactive forms introduced in vivo: radical hydroxyl (HO0 and anion superoxide (O{overscore (20 )}).
1) “Chemical” Tests in Tubo
a) Anti-DPPH Test
DPPH (diphenylpicryl hydrazyl) is a free, stable, violet-coloured radical which, in its leuco derivative, is modified by substances which capture free radicals (neutralising effect, also described as a “scavenger effect”).
The result is given as percent inhibition of DPPH0 in radical form relative to the control material without extract.
b) Anti-HO0 Test with Salicylic Acid (Fenton Reaction)
The HO0 (formed by H2O2 with Fe++ and EDTA present) hydroxylate the salicylic acid, which then forms a reddish compound.
The optical density at 490 nm corresponds to the hydroxylated salicylic acid content.
An anti-radical substance reacts with the HO0 radicals and reduces the formation of this red compound.
The results are given as percent inhibition of the hydroxylation content (average of 2 tests).
c) Anti-HO0 Test with Deoxyribose (Fenton Reaction)
The HO0 (formed by H2O2 with Fe++ and EDTA present) oxidise the deoxyribose to aldehyde derivatives which are then metered by thiobarbiturate acid.
The thiobarbiturate acid is condensed with the aldehydes to form a pink compound (DO at 532 nm).
The Fenton reaction is also carried out without EDTA in order to measure the ability to complex iron; the deoxyribose is then oxidised by HO0.
The results are given as percent inhibition (an average of 2 tests).
2) “Biochemical” in Tubo Tests=Anti-anion Superoxide O{overscore (20)} Tests
Xanthine oxidase is an enzyme which is introduced during oxidative stress and catabolises the purine bases (adenine and guanine) to uric acid and O{overscore (20)}. O{overscore (20)} is dismutated spontaneously (or by SOD=superoxide dismutase) to H2O2 and O2.
a) Anti-O{overscore (20)} Action: Method with Luminol
O{overscore (20)} can be recognised in terms of luminescence by Luminol.
An enzyme system (hypoxanthine/xanthine oxidase) forms the superoxide anions O{overscore (20)} which react with Luminol to form an intermediate compound. When stabilised this compound emits luminescence which is picked up by a photomultiplier.
A substance with an anti-radical action absorbs or destroys the anion O{overscore (20)}, thereby reducing the formation of luminescence.
b) Anti-O{overscore (20)} and H2O2 Action: Method with Luminol in the Presence of Microperoxidase
An enzyme system (hypoxanthine/xanthine oxidase) forms the superoxide anions O{overscore (20)} which are gradually dismutated to O{overscore (20)} and H2O2.
H2O2 and O{overscore (20)} react with the microperoxidase (M) to form O21 (singlet oxygen) which degrades Luminol (Lum), forming a luminescent compound, the light emission from which is picked up by a photomultiplier.
An anti-radical substance absorbs either H2O2 or O{overscore (20)} or O21, thereby reducing the formation of luminescence.
c) Anti-O{overscore (20)} Action: Method with NBT (Tetrazole Salt)
An enzyme system (hypoxanthine/xanthine oxidase) forms superoxide anions O{overscore (20)} which react with the NBT to form a red composition, formazan.
An anti-radical substance absorbs or destroys O{overscore (20)}, thereby reducing the formation of formazan.
The results of these three tests a), b) and c) are given as percent inhibition (average of 2 tests).
The results of above-mentioned tests 1) and 2) are set out in the following table:

















ARL
Fenton test (% 1)
HX + XOD test
















(%1)
Salicylic

(%1)















Plant
Type of extract
DPPH
acid
Deoxyribose
LUM
LUM + M
NTB

















Clidemia
Aqueous extract
91
40
70
99
51
53


hirta


Inga bourgoni
Aqueous extract
94

40
100
98
64



Ethanol E 50%
94

27
100
98
71



Ethanol extract
95

74
100
99
83


Sabicea
Aqueous extract
90
47
39
99
89
41


cinerea
Ethanol E 50%
94
39
50
100
99
62



Ethanol extract
93
51

100
98
50


Astrocaryum
Aqueous extract
76


98
27
23


vulgare
Ethanol E 50%
93


100
98
74



Ethanol extract
93
48
59
100
100
81


Siparuna
Aqueous extract
92


99
67
47


guianensis
Methanol E 80%
94

47
100
99
71



Ethanol extract
38
49

99
96
54


Eperua
Aqueous extract
91


100
85
49


falcata
Methanol E 80%
91


99
94
61



Ethanol extract
94

64
100
100
89


Byrsonima
Aqueous extract
95

80
100
100
83


verbascifolia
Methanol E 80%
95

84
100
100
75



Ethanol extract
94

75
100
100
80


Priva
Aqueous extract
95
46

100
70
35


lappulacea
Methanol E 80%
100
51
45
100
97
45



Ethanol extract
93
37

100
96
44


Goupia glabra
Aqueous extract
96
49
32
100
94
34



Methanol E 80%
89
61
31
100
90


Coutoubea
Aqueous extract
92
79

99
41


spicata
Methanol E 80%
93
81

100
89



Ethanol extract
92
72

99
50





Lum: Luminol; 


Lurn + M: Luminol + microperoxidase 






II) Anti-UVA Cell Protection on Human Fibroblasts in “in vitro” Survival
A) Metering of the Salted-out MDA (Morlière P. and Colleagues: “UV-A Lipid Peroxydation in Cultured Human Fibroblasts”, Biochim. Biophys. Acta 1084, 3: 261-269,1991)
1) The Theory
The UV-A penetrate into the dermis, where they set up an oxidative stress indicated particularly by lipoperoxidation of the cytoplasmic membranes.
Lipoperoxides are fragmented into malonal dialdehyde, which will crosslink/connect many biological molecules, for example proteins (enzyme inhibition) and nuclein bases (mutagenesis).
2) The Procedure
The fibroblasts are injected into a culture medium defined by calf's foetus serum.
The plant extract (in the culture medium defined by 2% serum) is added 72 hours after injection.
After 48 hours' incubation at 37° C. and CO2=5% the culture medium is replaced by a saline solution and the fibroblasts are irradiated with a dose of UV-A (15 J/cm2).
When irradiation is over the MDA (malonal dialdehyde) rate in the saline solution floating on the surface is metered and the protein rate in the fibroblasts is measured.
MDA is metered by the reaction on thiobarbiturate acid, and proteins by the so-called Bradford method.
B) Protecting the Action of the Catalase Enzyme from UVA
1) The Theory
UVA penetrates into the dermis where it sets up an oxidative stress, causing a lessening of the action of many enzymes including catalase.
Catalase is an enzyme which enables the H2O2 produced spontaneously by oxidation of substrates such as glucose or hypoxanthine to be removed.
H2O2 has to be removed quickly, otherwise it forms very toxic hydroxyl radicals (HO0) if iron is present.
2) The Procedure
(by L H Johansson and L A H Borg in “A spectrophotometric method for determination of catalase activity in small tissue samples”, Anal. Biochem. 174, 331-336, 1988).
Fibroblasts are injected into a culture medium defined by calf's foetus serum.
The plant extract (dissolved in a defined culture medium containing 2% serum at a concentration of 0.01 to 0.05% by wt/vol) is added 72 hours after injection.
After 48 hours' incubation at 37° C. and CO2=5% the culture medium is replaced by a saline solution and the fibroblasts are irradiated with a dose of UV-A (5 J/cm2).
When irradiation is over the rate of the active catalase is metered by a spectrophotometric method and its activity is related to the protein rate measured in the fibroblasts.
The action of the plant extract is given as a percent increase in catalase activity relative to the irradiated control material in the absence of any plant extract (two tests on average).
III) Evidence of the Anti-UVB Cell Protection Effect on Human Keratin Cells in “in vitro” Survival
1) The Theory
UV-B causes slight inflammation (erythema, oedema) by activating an enzyme, phospholipase A2.
This enzyme removes the arachidonic acid from the phospholipids of the plasmic membrane. Arachidonic acid is the precursor of prostaglandins, including E2 prostaglandins (=PGE2), which are formed by cyclooxygenase.
2) The Procedure
A431 keratin cells are injected into a culture medium defined by calf's foetus serum. After 72 hours' incubation at 37° C. and CO2=5% the culture medium is replaced by a saline solution containing the plant extract to be tested. The keratin cells are immediately irradiated with a dose of UV-B (30 mJ/cm2) and incubated for 24 hours at 37° C., then the PGE2 and LDH rates in the culture medium floating on the surface are measured.
The number of adhering keratin cells is determined by a particle counter (after trypsination) and the PGE2 rate by an ELISA test.
The LDH (lactate dehydrogenase) rate is also determined by an enzyme reaction. LDH is a cytoplasmic enzyme which marks cell disorders.
The activity of the plant extract is tested and given as the percent inhibition of the two markers relative to the values reached with the control materials (2 tests on average, each in duplicate).
IV) “Anti-protease” Activity
The proteases which have been separated from the neutrophil polymorphonuclear substance during a slight inflammation or by fibroblasts exposed to UV-A radiation cause decomposition of the proteins which structure the extracellular matrix of the dermis.
Two types of protease were tested by enzyme reactions in tubo.
1) Anti-elastase test (Bieth J., “Elastase: structure, function and pathological role”, Front Matrix Biol. 6: 1-82, Karger, 1978).
PNN segregate an elastase which is active on elastins (serine protease), proteoglycans and collagens.
The in tubo tests are carried out on an elastase from the pancreas (serine protease) using two types of substrate: a synthetic, chromogenic one (SS test) and a natural substrate, namely elastin combined with Congo red (Rc test).
The incubations take 30 minutes at ambient temperature or 2 hours at 37° C. and the colouring effects are in each case measured at 410 and 520 nm.
The inhibition proof mass which is tested as a comparison is α-1 antitrypsin.
2) Anti-collagenase in tubo test (Van Wart and colleagues, “A continuous spectrophotometric assay for Clostridium histolyticum collagenase”, Anal. Biochem., 113, 356-365, 1981).
Collagenase is segregated by PNN during a slight inflammation and by the “old” or irradiated fibroblasts.
The tests are carried out with a clostridium hystoliticum collagenase and a synthetic chromogenic substrate, FALGPA.
Incubation takes 30 minutes at ambient temperature and the optical density is measured at 324 nm.
The results are given as percent inhibition of the enzyme relative to the control material without activator.
The inhibition control material tested as a comparison is cysteine.
The results of test series II) III) and IV) are listed in the following table:



















Catalase %








increase in




UV-A
activity/

Elastase




(%1)
radiated control
UV-B (%)
(%)
Collagenase















Plant
Type of extract
MDA
Material
PGE2
LDH
Rc
SS
(%)


















Clidemia
Aqueous extract
23


67
92
67
93


hirta


Inga bourgoni
Aqueous extract
71

78
92
72
34
100



Ethanol E 50%
70

58
85
91
38
100



Ethanol extract



100
100
51


Sabicea
Aqueous extract
74

95
100

68
83


cinerea
Ethanol E 50%
86

40
75

60
100



Ethanol extract
78
81
87
100

47
27


Astrocaryum
Aqueous extract
91

78
97

9
15


vulgare
Ethanol E 50%
82

68
79
29
56
100



Ethanol extract
75

79
83
100
75
91


Siparuna
Aqueous extract
79

60
86

29
36


guianensis
Methanol E 80%
72
55
50
75
78
62
99



Ethanol extract






21


Eperua
Aqueous extract
79

100
100


36


falcata
Methanol E 80%
66

99
100


85



Ethanol extract
59

88
93

49
72


Byrsonima
Aqueous extract
63
23
70
92
100
68
100


verbascifolia
Methanol E 80%
66
26
64
89
101
86
100



Ethanol extract
42
24
50
73
100
68
100


Priva
Aqueous extract
26

21
27


lappulacea
Methanol E 80%



Ethanol extract





70
99


Goupia glabra
Aqueous extract



Methanol E 80%


Coutoubea
Aqueous extract


spicata
Methanol E 80%



Ethanol extract






47





MDA: malonal dialdehyde, 


PGE2: prostaglandin E2, 


LDH: lactate dehydrogenase, 


%1: percent inhibition relative to control material. 






V) Inhibition of Melanogenesis
1) Inhibition of Tyrosinase “in Tubo”
Tyrosinase is the key enzyme in the synthesis of the melanin in the melanocytes of the human skin.
This enzyme catalyses the first two stages in the conversion of tyrosine to melanin, i.e. oxidation of melanin to DOPA (dihydroxyphenylalanine) and subsequently to dopachrome.
Dopachrome is a coloured compound which is quantified at 475 nm by spectrophotometry.
The activity of the enzyme used in tubo (obtained from fungi) is determined as 20 seconds by the kinetics of the reaction.
The proof mass substance is hydroquinone (C150=0.025% wt/vol).
The results are expressed as percent inhibition relative to the control material.
2) Inhibition of Melanogenesis on B16 Melanocytes in the “in vitro” Culture
Inhibition of melanogenesis is assessed by spectrophotometric dosing of the melanin produced by melanocytes (B16 line), incubated “in vitro” in the presence of the plant extract to be tested.
The B16 melanocytes are cultivated in a defined growth medium and incubated for 3 days at 37° C., CO2=5%. The plant extracts are dissolved in a medium which activates melanogenesis and put into contact with the B16 for 3 days at 37° C.
The rate of salted-out melanin in the medium floating on the surface is quantified by spectrophotometry at 475 nm.
The rate of intra-cellular melanin is quantified by spectrophotometry at 475 nm in the homogenised B16 cells, which have been dissolved in a mixture of NaOH 1N+10% (v/v) DMSO.
The protein rate in the homogenised B16 cells is quantified by the so-called Bradford method.
The comparative substances are hydroquinone and arbutin.
VI. Evidence of Activity Inhibiting Non-enzymatic in Tubo Glycation (“Anti-glycation Activity)
(DEYL and colleagues: “Increased glycation and pigmentation of collagen in aged and young parabiotic rats and mice”, Mechanisms of ageing and development, 55, 39-47, 1990/MONNIER and colleagues: “Accelerated age-related browning of human collagen in diabetes mellitus”, Proc. Natl. Acad. Sci., USA: 81 583-587, 1984)
Non-enzymatic glycation of proteins is a crucial process in the ageing of human tissue.
This reaction, discovered by Maillard, explains the reticulation of extracellular matrices and base membranes which are mainly responsible for pathologies observed in diabetics.
In addition, Schiff's bases catalyse the production of reactive forms of oxygen, which worsen the effects of non-enzymatic glycation.
The in tubo tests are carried out on I-type collagen incubated for 21 days at 45° C. in the presence of 1% (p/v) glucose.
The rate of the Schiff's bases is assessed by fluorimetry at 430 nm (excitation at 350 nm) in the substances floating on the surface (fs) and in the centrifuging deposits (fgc). The result is given as percent inhibition relative to the control material without plant extract.
VII) Activation of Lipolysis on Human Adipocytes in “in vitro” Survival
Lipolysis is removal of triglycerides (or TG) stored in the adipocytes by an enzyme, triglyceride lipase (or TGL), causing the TG to be split into free fatty acid and glycerol, which are eliminated in the bloodstream.
The fatty acids can then be absorbed by muscle cells to produce energy.
The in vitro test is carried out as described below:
Activation of lipolysis is metered by spectrophotometric metering of the rate of glycerin salted out by adipocytes, incubated “in vitro” with the substance to be tested present.
The adipocytes are liberated by enzymatic digestion of subcutaneous human tissue by the so-called Rodbell method (Rodbell M., “Metabolism of isolated fat cells”, The journal of biological chemistry, vol. 239, no. 2, pages 375 to 380, 1964).
The plant extracts are dissolved in a defined medium which is put into contact with the adipocytes for 90 minutes at 37° C.
The rate of salted-out glycerin is quantified by spectrophotometry in a medium which floats on the surface by the method of Carpéné C. and colleagues (J. Pharmacol., vol. 12, no. 2, pages 219-224, 1981).
The rate of liberated glycerin is given relative to the rate of all lipids metered by turbidimetry. The result is given as a percent increase relative to the control material without plant extract.
The comparative substances are theophylline and isoprenaline.
The results of test series V), VI) and VII) are summarised in the following table:


















Anti-glycation
B16 Melanin
















Type of
Tyrosinase
(%)


Mel.
Lipolysis
















Plant
Extract
C150
CA50
fs
fgc
Dose
Index
extra
% increase



















Clidemia
Aqueous E.
0.31

37
50






hirta


Inga
Aqueous E.
0.060

100
76
0.05
3.9
68


bourgoni
Ethanol E 50%
0.070



0.03
3.5
80



Ethanol E
0.010



0.02
1.4
10


Sabicea
Aqueous E.


21
50


cinerea
Ethanol E 50%
0.090



0.01
1.6
49



Ethanol E.
0.060



0.03
1.8
10


Astrocaryum
Aqueous E.


vulgare
Ethanol E 50%
0.63



Ethanol E.
0.23


Siparuna
Aqueous E.


guianensis
Methanol E 80%



Ethanol E.


Eperua
Aqueous E.


97
46


falcata
Methanol E 80%
0.38






30



Ethanol E.
0.080



0.01
1.5
93


Byrsonima
Aqueous E.
0.090



0.02
1.7
72


verb.
Methanol E 80%
0.050


0.02
2.5
91



Ethanol E.
0.010


0.03
2.5
58


Priva
Aqueous E.
0.35


lappulacea
Methanol E 80%
0.13



Ethanol E.
0.060


Goupia
Aqueous E.

0.037


glabra
Methanol E 80%


Coutoubea
Aqueous E.
0.64


spicata
Methanol E 80%
0.39



Ethanol E.
0.27





In the above table: 


C150: concentration of active substances by weight/volume giving 50% inhibition 


CA50: concentration of active substances by weight/volume giving 50% activation 


Dose: concentration of active substances tested by weight/volume 


Index (B16 melanin): protein rate/rate of intracellular melanin 


Mel. extra: rate of extracellular melanin (salted out in the substance floating on the surface) as percent inhibition. 






In evaluating the results of the above test tables the inventors worked out various preferred embodiments of the invention.
To obtain a cosmetic or dermo-pharmaceutical composition giving an increased anti-elastase action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia and Priva lappulacea. 
To obtain a cosmetic or dermo-pharmaceutical composition giving an increased anti-collagenase action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia and Priva lappulacea. 
To obtain a cosmetic or dermo-pharmaceutical composition giving an increased de-pigmenting action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Eperua falcata, Byrsonima verbascifolia, Priva lappulacea, Astrocaryum vulgare and Coutoubea spicata. 
To obtain a cosmetic or dermo-pharmaceutical composition giving an important anti-UVB action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia and Priva lappulacea. 
To obtain a cosmetic or dermo-pharmaceutical composition giving an important anti-UVA action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia and Priva lappulacea. 
To obtain a cosmetic or dermo-pharmaceutical composition giving an action protecting catalase from UVA as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Sabicea cinerea, Siparuna guianensis and Byrsonima verbascifolia. 
To obtain a cosmetic or dermo-pharmaceutical composition giving a strong anti-glycation action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea and Eperua falcata. 
To obtain a cosmetic or dermo-pharmaceutical composition giving an important pigmenting action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by an extract of the Goupia glabra plant.
To obtain a cosmetic or dermo-pharmaceutical composition giving a considerable slimming (lipolytic) action as well as an anti-radical-type action, the active ingredient contained in said composition and passing on these properties is advantageously formed by an extract of the Eperua falcata plant.
However the active ingredient is preferably formed by an extract or a mixture of extracts obtained from one or more plants selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia and Priva lappulacea. 
The object of the present invention also concerns a cosmetic or dermo-pharmaceutical composition for topical, external use on the skin, mucous membrane and/or the epithelium or body appendages, characterised in that it contains a plant extract as the active ingredient giving an anti-radical-type action in particular, alone or with at least one other added ingredient, the botanical genus of the plant belonging to the group formed by the Clidemia, Inga, Sabicea, Astrocaryum, Siparuna, Eperua, Byrsonima, Priva, Couloubea and Groupia genera, and that a plant in the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata, Byrsonima verbascifolia, Priva lappulacea, Coutoubea spicata and Goupia glabra is preferably selected.
According to the other additional properties relating to the anti-radical-type action which the cosmetic or dermo-pharmaceutical composition may need to provide, the group of plants which may be selected from the above-mentioned ones will contain various representatives according to the action of each of them disclosed in the tables showing the results of the tests described above.
Thus in a first embodiment of the invention the cosmetic or dermo-pharmaceutical composition may contain an active ingredient with an anti-UVA, anti-UVB and anti-collagenase action, which is formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna quianensis, Eperua falcata and Byrsonima verbascifolia. 
In a second embodiment of the invention the cosmetic or dermo-pharmaceutical composition may contain an active ingredient with an anti-UVA, anti-UVB, anti-collagenase and anti-elastane action, which is formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Eperua falcata and Byrsonima verbascifolia. 
In a third embodiment of the invention the cosmetic or dermo-pharmaceutical composition may contain an active ingredient with an anti-UVA, anti-UVB, anti-collagenase and anti-glycation action, which is formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea and Eperua falcata. 
In a fourth embodiment of the invention the cosmetic or dermo-pharmaceutical composition may contain an active ingredient with an anti-UVA, anti-UVB, anti-collagenase, anti-elastane and de-pigmenting action, which is formed by at least one extract of a plant selected from the group formed by Clidemia hirta, Inga bourgoni, Sabicea cinerea, Eperua falcata and Byrsonima verbascifolia. 
In a fifth embodiment of the invention the cosmetic or dermo-pharmaceutical composition may contain an active ingredient with a pigmenting action, which is formed by an extract from the Goupia glabra plant.
In a sixth embodiment of the invention the cosmetic or dermo-pharmaceutical composition may contain an active ingredient with a slimming action, which is formed by an extract from the Eperua falcata plant.
The cosmetic composition advantageously contains as its active ingredient—alone or combined with other active ingredients—between 0.001% and 20% by weight, preferably between 0.1% and 3% by weight, of an extract or mixture of extracts from a plant or plants defined and obtained in the above-mentioned manner.
The extract or mixture of extracts forming an active ingredient may be used in any galenic form possible in cosmetics, particularly in a galenic form chosen from oil in water emulsions, water in oil emulsions, face lotions, cosmetic milk, gels, hydrogels, cremes, pomades, soaps, pellets, spraying materials, hair lotion and shampoos.
The extract or mixture of extracts may also be added to one or more cosmetic vectors, particularly one or more vectors selected from the group formed by liposomes, macrocapsules, microcapsules, nanocapsules, macro-particles, microparticles and nanoparticles.
Various products or cosmetic preparations containing a plant extract as described above will now be described, as examples to which the practical use of the invention is not limited.
Example 1
A cosmetic product in the form of a skin bleaching cream may for example be of the following composition by weight:















Fraction A:










Glycerin stearate and Ceteareth-20
15.0% 



Paraffin oil
3.0%



Ascorbyl palmitate
3.0%



Dimethicon
3.0%



Cetyl alcohol
0.5%



PEG 30 glycerol isostearate
2.0%



Fraction B:



Water
72.2% 



Methyl parabene
0.2%



Imidazolinidyl urea
0.3%



Ethanol extract of Inga bourgoni
0.5%



Fraction C



Perfume
0.3%










The procedure for preparing this cream consists of melting the Fraction A constituents with agitation at 75° C., preparing Fraction B at 75° C. then pouring Fraction B into Fraction A with turbine agitation, cooling with planetary agitation then adding to Fraction C.
Example 2
A cosmetic product in the form of an anti-blotch emulsion for the hands, to treat cutaneous pigment marks, may for example be of the following composition by weight.
In accordance with the invention the product could alternatively have multiple actions, particularly anti-radical, anti-elastase and anti-collagenase.















Fraction A:










Glycerin stearate and PEG 100 stearate
6.0%



Olein alcohol
1.5%



Glycerin stearate
2.0%



Steareth 2
2.0%



Karite butter
3.0%



Dimethicon
4.0%



Caprylic capric triglyceride
8.0%



Propyl parabene
0.1%



Tocopherol acetate
0.1%



Fraction B:



Water
60.8% 



Elastab 388 (Laboratoires Serobiologiques)
2.5%



Byrsonima verbascifolia extract (80% methanol)
1.5%



Ethanol Astrocaryum vulgare extract
1.5%



Propylene glycol
5.0%



Fraction C:



Polyacrylamide, isoparaffin and Laureth 7
2.0%










The procedure for making this emulsion consists of preparing Fractions A and B separately at 75° C., adding Fraction A to Fraction B with turbine agitation at 75° C., cooling to 50° C. then adding Fraction C and lastly cooling the final mixture to ambient temperature.
Example 3
A cosmetic product in the form of an anti-wrinkle day cream and a multiple-action anti-ageing cream may for example be of the following composition by weight:















Fraction A:










Glycerin stearate
14.0% 



Octyl dodecanol
16.0% 



Dibutyl adipate
6.0%



Ceteareth 12
1.5%



Ceteareth 20
1.5%



Fraction B:



Propylene glycol
5.0



Aqueous extract of Inga bourgoni
3.25% 



Extract of Sabicea cinerea (50% ethanol)
1.0%



Ethanol extract of Eperua falcata
0.75% 



Elastab 4112 (Laboratoires Serobiologiques)
0.4%



Water
50.6% 



Fraction C:



Perfume
0.3%










The procedure for making the cream comprises preparing Fractions A and B separately at 80° C. with agitation, adding Fraction A to Fraction B with turbine agitation, cooling the mixture to 45° C. then adding Fraction C, and lastly bringing the final mixture back to ambient temperature.
The invention is not of course restricted to the embodiments described and illustrated in the accompanying drawings. Modifications are possible, particularly in the composition of the various elements or through replacement by industrial equivalents, without thereby going beyond the scope of the invention.

## Claims

1. A composition for topical application to skin, mucous membrane, epithelium, body appendages or combinations thereof, characterized in that said composition contains a first active ingredient, alone or combined with a second active ingredient, wherein said first active ingredient is an extract of Eperua, and wherein said first and second active ingredients provide anti-UV, anti-elastase, anti-collagenase, anti-glycation, tyrosinase activation, anti-tyrosinase activation, lypolytic and/or anti-radical action.

2. The composition according to claim 1, wherein said first active ingredient is an extract of Eperua falcata bark.

3. The composition according to claim 1, wherein the total amount of active ingredients are in an amount between 0.001% and 20% by weight of said composition.

4. The composition according to claim 1, wherein the extract is in a galenic form selected from the group consisting of oil in water emulsions, water in oil emulsions, face lotions, cosmetic milk, gels, hydrogels, crèmes, pomades, soaps, pellets, spraying materials, hair lotion and shampoos.

5. The composition according to claim 1, wherein said first and/or second active ingredients are added to one or more cosmetic vectors selected from the group consisting of liposomes, macrocapsules, microcapsules, nanocapsules, macroparticles, microparticles and nano-particles.

6. The composition according to claim 1, wherein said composition has anti-UVA, anti-UVB and anti-radical actions.

7. A composition for topical application to skin, mucous membrane, epithelium, body appendages or combinations thereof, characterized in that said composition comprises an extract of Eperua falcata, wherein the extract provides anti-UV, anti-elastase, anti-collagenase, anti-glycation, tyrosinase activation, anti-tyrosinase activation, lypolytic and/or anti-radical action.

8. The composition according to claim 7, wherein said extract is prepared from Eperua falcata cortices or bark.

9. The composition according to claim 7, wherein said composition contains said Eperua falcata extract in an amount between 0.001% and 20% by weight of said composition.

10. The composition according to claim 7, wherein the extract is in a galenic form selected from the group consisting of oil in water emulsions, water in oil emulsions, face lotions, cosmetic milk, gels, hydrogels, crèmes, pomades, soaps, pellets, spraying materials, hair lotion and shampoos.

11. The composition according to claim 7, wherein said extract is added to one or more cosmetic vectors selected from the group consisting of liposomes, macrocapsules, microcapsules, nanocapsules, macroparticles, microparticles and nano-particles.

12. The composition according to claim 7, wherein said active ingredients are present in said composition in an amount between 0.001% and 20% by weight of said composition.

13. A method for treating skin, comprising applying the composition according to claim 1 on said skin.

14. A method for treating skin, comprising applying the composition according to claim 7 on said skin.

15. A cosmetic composition, comprising Eperua extract in galenic form selected from the group consisting of oil in water emulsions, water in oil emulsions, face lotions, cosmetic milk, gels, hydrogels, crèmes, pomades, soaps, pellets, spraying materials, hair lotion and shampoos, wherein the extract provides anti-UV, anti-elastase, anti-collagenase, anti-glycation, tyrosinase activation, anti-tyrosinase activation, lypolytic and/or anti-radical action.

16. The cosmetic composition according to claim 15, further comprising an extract selected form the group consisting of Clidemia hirta, Inga bourgoni, Sabicea cinerea, Astrocaryum vulgare, Siparuna guianensis, Byrsonima verbascifilia, Priva lappulacea, Coutoubea spicata and Goupia glabra.