OA16367A - Resveratrol extraction from Gnetum africanum. - Google Patents

Abstract

The invention relates to extracts and compositions from Gnetum africanum. It also concerns plant extracts enriched in resveratrol and methods for obtaining the same. Described herein are processed plant materials which comprise about 20 µg resveratrol per gram of dried plant material and plant extracts which comprise at least about 0.002% w/w resveratrol. Also described are resveratrol glycosides and compositions comprising same. The invention further encompasses food products, nutraceutical products, pharmaceutical compositions which comprise processed plant materials, plant extracts and/or resveratrol glycosides.

Description

The invention relates to the field of natural products. More particularly, it concerns extracts and compositions from Gnetum africanum. It also concerns plant extracts enriched in resveratrol and methods for obtaining the same.

BACKGROUND OF THE INVENTION ' !

Resveratrol is a compound having the chemical formula C₁₄Hi₂O₃ and it is represented by the following structure:

Resveratrol is produced naturally by several plants and can be synthesized chemically. Plant extracts obtained so far only contain low concentrations of resveratrol. For instance, resveratrol is found in the skin of red grapes (at a concentration of about 10 pg/g) and it is a constituent of red wine (at a concentration of about 13 pg/ml).

Resveratrol has attracted considérable attention since anti-cancer, anti-inflammatory, bloodsugar-lowering and numerous bénéficiai cardiovascular effects hâve been attributed to it. However, experiments hâve shown that resveratrol alone, even at a relatively high dose, does not yield significant clinical effects in man. Therefore it is becoming accepted that the bénéficiai effects of resveratrol rely on the presence of one or more unknown compounds. Thus, there is a need for compositions and extracts enriched with high concentrations of resveratrol derived from plants.

Gnetum africanum is a popular vegetable commonly found in tropical Africa. It is collected in the wild (rather than cultivated) and it is added to foods and made into fusions. Although traditional African medicrne includes a few medical uses for Gnetum africanum, there is no clear scientifîc evidence of any therapeutic efficacy for that plant. Furthermore, the conditions under which it would be possible to obtain biologically active extracts or products derived from that plant hâve not been properly documented.

Therefore, there is also a need for plant extracts, particularly extracts from Gnetum

I africanum comprising resveratrol, and methods for extracting resveratrol. There is also a ¹ need for pharmaceutical compositions comprising such plant extracts and resveratrol, as well as nutraceuticals and cosmetics comprising same.

The présent invention addresses these needs and other needs as will be apparent from review ofthe disclosure, figures and description ofthe features ofthe invention hereinafter.

BRIEF SUMMARY OF THE INVENTION

The présent inventors hâve found a new végétal source from which it is possible to obtain plant material and extracts more concentrated in resveratrol than any previously known 10 végétal source which provides only about 10-15 pg resveratrol per gram of plant material.

Accordingly, one aspect of the invention relates to processed plant material comprising about 20 pg resveratrol per gram of dried plant material. In one embodiment the plant material is derived from the plant Gnetum africanum, preferably the leaves or the stem.

Another aspect of the invention concerns a plant extract which comprises at least about 15 0.002% w/w resveratrol. In one embodiment the plant material is derived from the plant

Gnetum africanum, preferably the leaves or the stem.

The invention further relates to purification methods for obtaining a plant extract concentrated in resveratrol. In one embodiment the method comprises:

, providing plant material from Gnetum africanum-, submitting said plant material to à solvent extraction; and obtaining a plant extract concentrated in resveratrol, said plant extract comprising at least about 0.002% w/w resveratrol.

The invention also concerns resveratrol glycosides and compositions comprising same. In one embodiment the resveratrol glycçside consists of a compound identifiable under 25 détection conditions of Table 1 by the presence of a peak having a rétention time of 9.1 min on a HPLC-APCI-MSD chromatogram. In another embodiment the resveratrol glycoside consists of a compound identifiable under détection conditions of Table 1 by the presence of a peak having a rétention time of 9.5 min on a HPLC-APCI-MSD chromatogram.

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I The invention further relates to food products and nutraceutical products which comprise at

J 30 least one of a processed plant material, a plant extract and a resveratrol glycoside as j defined herein.

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The invention also relates to cosmetic compositions which comprise at least one of a processed plant material, a plant extract and a resveratrol glycoside as defined herein in combination with a suîtable cosmetic carrier, diluent or excipient.

The invention also relates to antioxidant compositions which comprise at least one of a processed plant material, a plant extract and à resveratrol glycoside as defined herein in combination in combination with a suîtable carrier, diluent or excipient.

The invention also relates to pharmaceutical compositions which comprise at least one of a processed plant material, a plant extract and a resveratrol glycoside as defined herein in combination in combination with a pharmaceutically acceptable carrier, diluent or excipient.

Another related aspect of the invention concerns the use of a processed plant material, a plant extract and/or a resveratrol glycoside as defined herein for the manufacture of pharmaceutical compositions and drugs. Oné particular example is a pharmaceutical t σ composition comprising a plant extract from.Gnefuzn africanum. In embodiments, the extract comprises at least about 0.002% w/w resveratrol and/or one or more resveratrol glycosides.

Accordingly, in some aspects the processed plant material, a plant extract and/or a resveratrol glycoside according to the invention are . used for the manufacture of médicaments, and more particularly médicaments useful in the prévention and/or treatment of skin diseases, cardiovascular diseases, inflammatory diseases, blood lipid abnormalities and pain.

An advantage of the présent invention is that it provides a new natural source of resveratrol. It also provides plant extracts rich in resveratrol and other components. The invention further provides compositions comprising extracts from Gnetum africanum which may hâve numerous health, nutrition al and cosmetic bénefits.

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Additional aspects, advantages and features ‘of the présent invention will become more 25 apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES ' !' | t ' ¹ ! j;l . r '

Figure 1 is a line graph showing a HP.LC-DAD profile of extract # E-01 (leaves from the Equatorial forest) obtained at the monitoring wavelength of 325 nm. Resveratrol is the major 30 peak.

Figure 2 is a line graph showing an ion chromatogram of HPLC-APCI-MSD profile of ethanolic extract of extract # E-47 (leaves from the Equatorial forest). Two overlaid lines are represented, one of 229 [M+H]⁺ and the other· of 391 [M+ glyc]⁺. In addition to resveratrol (peak #3), two glycosides (peak’#1 and peak #2) are visible.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

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The présent inventors hâve found a new natural source of resveratrol. More particularly the inventors hâve found that the plant Gnetum africanum can be a valuable source of resveratrol and they hâve obtained extracts from that plant containing resveratrol at much higher concentrations than that of any other plant extract produced so far.

As used herein, the term “resveratrol” refers to the compound having the chemical formula Ci₄H₁₂O₃ and the structure illustrated hereinbefore. Resveratrol is also defined by the CAS Registry Number 501-36-0 and known under other names, including, but not limited to, frans-resveratrol, frans-3,5,4'-Trihydroxystilbene; 3,4',5-Stilbenetriol; (E)-5-(p.. i

HydroxystyryQresorcinol; and (E)-5-(4-hydroxystyryl)benzene-1,3-diol.

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Plant Extracts

One aspect of the présent invention relates to harvested plant material comprising about 20 pg, 30 pg, 40 pg, 50 pg, 75 pg, 100 pg oc more resveratrol per gram of dried plant material. In various embodiments, the plant material ip. dried and comprises from at least about 25 pg/g, 50 pg/g, 75 pg/g, about 100 pg/g, about 250 pg/g, about 500 pg/g, about 1000 pg/g, 20 about 1500 pg/g, about 2000 pg/g, about 2500 pg/g, about 3000 pg/g, about 4000 pg/g, about 5000 pg/g, about 10 000 pg/g, about 15 000 pg/g, or at least about 16 500 pg/g, resveratrol.

As used herein, the term “processed plant material” refers to one or more tissues of a plant which has been harvested for its known properties (e.g. resveratrol content) and 25 prepared (e.g. washed, dried, ground, treated, refined, transformed, packaged, etc.) with the purpose of large scale commercialization. For[the purpose of clarification, infusions and other similar concoctions of Gnetum prepared according to African traditional medicine are not considered a “process plant material” according to the invention, and as such, are exclude from the scope ofthe invention.

Another aspect concerns a plant extract comprising at least about 0.002% w/w, 0.003% w/w, 0.004% w/w, 0.005% w/w, 0.075% w/W, 0.01% w/w, or more resveratrol. In various embodiments, the plant material is dried ànd comprises from at least about 0.0025% w/w,

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0.075% w/w, 0.01% w/w, 0.02% w/w, 0.05% w/w, 0.1% w/w, 0.5% w/w, 1 % w/w or more resveratrol.

As used herein, the term “plant extract’ refers to a concentrated préparation of a plant material obtained by isolating or purifying desired active constituées with one or more

J ' extraction techniques. Examples of extraction techniques include, but are not limited to, solvent extraction (e.g. éthanol, methanol, ethyl'acetate), chromatography, and the like. For the purpose of clarification, infusions and other similar concoctions of Gnetum prepared according to African traditional medicine are not considered a plant extract” according to the invention, and as such, are exclude from frie, scope of the invention.

’’ , Preferably the plant material and the plant extract dérivé from the plant Gnetum africanum. As used herein, the term dérivés from or “derived from or refers to a product, compound or composition which is(are) obtained from, or can be traced back, to a given definite source. A particular example according to the invention includes resveratrol extracted or isolated from Gnetum africanum.

As used herein, the terrns “Gnetum africanum”, “G. africanum” or “Gnetum refers to the plant which commonly grows in tropical Africa. The plant is commonly called “mfumbwa (kikongo), “longongia (lingala) and “banva/e (Azande). Many different varieties are known, i

including the Asusutan, the Oron, the Ikom, theWelw and the Koko variety. Various sources of Gnetum africanum can be used accordifig to the invention, including Gnetum from the Mayombe forest the équatorial forests of Congo. In preferred embodiments, the plant extracts according to the invention are obtained from the Gnetum africanum plants growing in the équatorial forests of Congo. Preferably;, the leaves, stems and/or roots are used for obtaining a plant extract according to ^;t^!he invention but it is conceivable that other plant tissues such as flowers, fruits, tubers, corms, etc. may be also used. The invention further t

encompasses the plant Gnetum bucholzianum, and processed materials, extracts and resveratrol glycoside therefrom.

In preferred embodiments, it is the stem or leaves of Gnetum africanum, most preferably its stem, which is used for obtaining the plant material, plant extract(s), and resveratrol or a resveratrol glycoside according to the invention.

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Those skilled in the art know how to identify useful sources of a plant material or plant extract and how to measure leveis ’of resveratrol in different types of extracts or compositions. Exemplary methods for resveratrol quantification are provided herein in the Examples section. Similarly, Example 3 provides detailed analyses of the content of various biochemicals (e.g. lipids, proteihs, sugars, ^etc.j in selected parts of Gnetum africanum.

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i Those skilled in the art can thus refer to ^those analyses for comparison in assessing j whether a plant material or plant extract is derived from Gnetum africanum.

i j The présent înventors hâve further identified new resveratrol glycosides referred to herein ij | as “resveratrol glycoside-1” and resveratrol glycoside-2' As described hereinafter at ; 5 Example 1, and more particularly in Table 1, the resveratrol glycosides are identifiable as i

distinct peaks by HPLC-MSD. More particularly, on a HPLC-APCI-MSD chromatogram and under détection conditions indicated in Table 1, resveratrol glycoside-1 is identifiable by the presence of a peak having a rétention time bf 9.1 min, whereas resveratrol glycoside-2 is identifiable as a peak having a rétention time of_;9.5 min. For comparison, resveratrol has a 10 rétention time of 10.1 min. Table 1 further provides mass spectrométrie fragmentation pattern for identification of resveratrol glycoside-1 and -2,

Accordingly, an additional aspect of the invention concerns resveratrol glycoside-1 and resveratrol glycoside-2 in an isolated or purified form. The resveratrol glycoside-1 and -2 are isolated or purified from a plant, preferably from Gnetum africanum. The invention further encompasses compositions (e.g. pharmaceutical compositions) comprising at least one of | resveratrol glycoside-1 and resveratrol glycoside-2, j Another aspect of the invention further concerns a plant extract or a processed plant ! material, preferably from Gnetum africanum, the extract comprising resveratrol glycoside-1, ^J, resveratrol glycoside-2, or both, The plant extract may further comprise lipids (e.g. omega-6, ί 20 omega-3, saturated and trans fats), minerais (e.g. Mg, Mn, Se, P, etc.), soluble and | insoluble fibers, sugars (e.g. glucose), vitamins (e.g, vitamin E) and amino acids and î proteins. Table 4 herein after provides a non limitative list of constituents which can be found in Gnetum africanum.

s.; . i Compositions and uses ri J ·

Another aspect of the invention concerns compositions and products comprising a plant material, a plant extract and/or a resveratrol glycoside as defined herein.

j Typically the composition and products pfi,.the invention includes one or more active j ingrédients which are derived from Gnetum africanum, including but not limited to resveratrol, resveratrol glycolside-1, and resveratrol glycoside-2. In a preferred embodiment, 30 the composition comprises at least 0.002% w/w resveratrol derived from Gnetum africanum.

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In various embodiments, the composition may comprise from at least 0.0025% w/w, 0.003% w/w, 0,004% w/w, 0.005% w/w, 0,075% w/w, 0.01% w/w, 0.02% w/w, 0,05% w/w, 0.1% w/w, | 0.5% w/w, 1 % w/w or more resveratrol,

Those skilled in the art will readily appreciate that plant materials, plant extracts and/or

J 35 resveratrol glycosides according to the invention possess numerous bénéficiai properties.

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Accordingly, another related aspect of the invention concerns food products, food additives, nutraceuticals, cosmetics, pharpiaceuticals and antioxidant compositions comprising a plant material, a plant extract and/or a resveratrol component, including the glycosides, as defined herein.

Examples of food products or food additives encompassed by the invention to which addition of a plant material, a plant extract and/or a resveratrol and/or resveratrol glycoside as defined herein may be bénéficiai, include but are not limited to dairy products such as yogurt, cheese and spreads etc., nutritional bars, herbal teas, various drinks, including fruit drinks and energy drinks.

Examples of nutraceuticals encompassed by the invention to which addition of a plant material, a plant extract and/or a resveratrol and/or a resveratrol glycoside as defined herein may be bénéficiai, include but are not limited to vitamins, nutritional suppléments, sleep aids, memory aids.

Examples of cosmetic compositions encompassed by the invention to which addition of a 15 plant material, a plant extract and/or a resveratrol and/or a resveratrol glycoside as defined herein may be bénéficiai, include but aie'.not limited to facial creams, lotions, skin tonies, and the like.

Examples of pharmaceutiçal compositions encompassed by the invention to which addition of a plant material, a plant extract and/or a resveratrol and/or resveratrol glycoside as 20 defined herein may be bénéficiai, include but are not limited to various types of cardiovascular drugs, lipid regulating drugs, anti-inflammatory drugs, analgésies, musclerelaxants.

Antioxidant compositions encompassed by the invention to which addition of a plant material, a plant extract and/or a resveratrol 'glycoside as defined herein may bénéficiai, 25 include but are not limited to antioxidant compositions useful for prévention of treatment of cardiovascular diseases and inflammatory diseases.

The invention further contemplâtes methods, of use and methods of treatment, comprising administration of a plant material, a plant éxtract, a resveratrol, a resveratrol glycoside

.. t . f , ? , * ·, Vand/or composition as defined herein. More particularly, methods of treatment according to 30 the invention comprise the administration to a subject in need thereof of an effective amount of such composition or extract, for cosmetic and/or for therapeutic purposes. Methods envisioned by the présent invention include, but are not limited to, methods related to the skin (e.g. rejuvenating the skin, removing wrinkles, skin toners, skin softeners etc.), methods for preventing or treating a cardiovascular disease, methods for addressing blood lipids

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abnormalities and for controlling lipid levels in the blood (e.g, cholestérol, triglycérides, etc.),

I ' ' i methods for treating inflammatory diseases or conditions, and methods for pain réduction.

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I Related aspect concerne the use of a plant material, a plant extract, a resveratrol, a i resveratrol glycoside and/or a composition as defined herein for the manufacture of a médicament, nutraceutical or cosmetic product for use in these methods.

The plant material(s), plant extract(s), '.resveratrol, resveratrol glycoside(s) and/or composition(s) of the invention may be administered using different routes, for instance by the oral, intravenous (iv), intramuscular (im), depo-im, subcutaneous (sc), depo-sc, sublingually, intranasal, intrathecal, topical or rectal routes.

The compositions and extracts of the invention may be formulated under different suitable forms, such as dry powdered compositions, tablets or capsules, liquid solutions, suspensions, cream, ointment, lotion, paste, etc. The compositions and extracts according to the invention may also be marketed as a concentrate to be diluted by an end user.

The plant materials, plant extracts, resveratrol, resveratrol glycosides and/or compositions according to the invention may be prepared by various methods including but not limited to extracting, blending, grinding, homogenizing, suspending, dissolving, emulsifying, dispersing, and/or mixing selected extracts or ingrédients derived therefrom, with selected excipient(s), carrier(s), adjuvant(s), diluent(s), or stabilizers. For preparing the composition of the invention, methods well known in the art may be used.

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The compositions according to the invention' may further comprise compounds and agents commonly used in food products or food additives, nutraceuticals, cosmetic compositions, pharmaceutical compositiôns and/or antioxidant compositions.

In addition, the plant materials, plant extracts, resveratrol, resveratrol glycosides and/or compositions of the invention may also contain additional ingrédients, including but not limited to, métal chelators, meta! scavengers, coating agents, preservîng agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, colorants (e.g. tracer dyes), flavours, odorants, salts, buffers, surfactants, solvents, coating agents and/or antioxidants.

The plant materials, plant extracts, resveratrol,' resveratrol glycosides and/or compositions according to the invention may be packaged in different forms, which may include but are not limited to, sealed containers (e.g. a plastic or glass bottles, ampuls or vials), pouches (e.g. a bag or sachet), jars, tubes, blistef packs, boxes, etc.

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I Those skilled in the art will readily appreciatethat plant materials, plant extracts, resveratrol, | resveratrol glycosides and/or compositions ofthe invention can be readily obtaîned by using

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-816367 various, processing, and/or purification methods and techniques. For instance, as exemplified hereinafter, samples comprising high levels of resveratrol can be obtained by using an éthanol extraction (see Example 1), a methanol extraction (Example 2), or an ethyl acetate extraction (Example 3) of plant materials from Gnetum africanum.

According to one particular aspect, the invention relates to a method for the préparation of a plant extract enriched or concentrated in resveratrol. In one embodiment the method comprises the steps of : , providing plant material from Gnetum.africanum;

submitting the plant material to a solvent,extraction; and obtaining a plant extract concentrated’in resveratrol, the plant extract comprising at least about 0.002% w/w resveratrol.

In another embodiment the method comprises the steps of :

grinding plant material (preferably previously dried) from Gnetum africanum;

extracting the ground plant material with one or more suitable solvents for obtaining a solvent extract;

recovering and drying the solvent extract to obtain the plant extract.

The methods may further comprise thé steps'^:iof ultrasonicating the plant material in the presence of one or more suitable solvents at the solvent extraction step. In one 20 embodiment, obtaining or recovering the resveratrol-concentrated plant extract comprises a centrifugation step. It may be preferableto carry out two rounds of (1) removing the supematant and (2) re-suèpending the pellet after each centrifugation step, and the supematants from the rounds of centrifugation aie pooled and dried.

Those skilled in the art will recognize, or be able to ascertain using no more than routine 25 expérimentation, numerous équivalents to the spécifie procedures, embodiments, claims, and examples described herein. Such équivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES

EXAMPLE 1: Identification and quantification of resveratrol by HPLC-MS in selected ί ; i¹ J f ^lj '

African plants following an éthanol extraction

EXPERIMENTAL ' i . J

Plant Material: The following samples of plant materials were received in dried unprocessed form: M02, M03, M21, M23, EÙ1, E08, E4ê* E47, The samples are leaves collected from

Gnetum africanum growing either in Mayombe forest (M) or the équatorial forest (E) of Congo. The leaves were left to dry before extraction and analysis.

Plant Extraction: Plants were ground with a coffee blender, and stored as a powder at -80°C until used. Each plant material was weighed (1g) into a 50 ml centrifuge tube and 20 mL 80% éthanol was pipetted into each tube for the first extraction. The sealed tubes were put

I into an ultrasonic processor for 30 min at room température (Yong-Jin Chao et al, (2006), Ultrasonication-assisted extraction of resveratrol from grapes, Journal of Food Engineering 77: 725-730). The supematants.were separated from the pellet after centrifugation at 3000 x g for 10 min. Ten mL of 80% éthanol was'added to the pellet and ultrasonication was repeated followed by centrifugation as above. Two supernatants were pooled into a rT : , weighted centrifuge tube and' evaporated to dryness by a Speedvac™ at room température. The dried extracts were weighed and the perdent yields were calculated.

HPLÇ-MSD Analysis: The HPLC-APCI-MS method for the analyses was modified from Saleem et al. (Saleem A, Brendan RW, Harris CH, Muhammad A, Tayamo C, Sit S, Arnason JT. A validated method of phytochemical analysis of Flor-Essence™ - A multiherb product. 2008. Phytochem Analysis Volume 20, Issue 5, pages 395-401). Briefly, an Agilent 1100™ Sériés HPLC-APCI-MSD system was used consisting of an autosampler, a quaternary pump, a column thermostat and a diode array detector. The mass spectrométrie system, connected with HPLC system, was equipped with an atmospheric pressure chemical ionization (APCI) source, and a detector (range 50-1500 amu). Ail solvents were HPLC grade (Fisher Scientific, CA, USA) unless otherwise specified. For analytical scale i

HPLC-MS analysis the solvents were sonicated for 5 min and the column (YMC-ODS AMC18™, 100 x 2.Omm, particle size 3.0 micron, Waters Inc., CA, USA) was equilibrated for 15 min with the starting conditions prior to, analysis. The elution solvent system consisted of A = acetonitrile and B = trifluoroacetic acid 0,05% (aqueous), pH 3.5. The optimal gradient elution conditions were 5-100% TFA over 20 min. The column was subsequently washed by increasing B up to 100% over 5 min and kept at isocratic condition for 5 min. The column was brought back to initial conditions in 5 min and equilibrated for 3 min. The total run time was 30 min. The column was operated at a flow of 0.25 ml/min and its température was maintained at 48 °C with maximum pressure limit of 200 bars.

The extracts were reconstituted in 1 mg/ml in MeOH and filtered through 0.22 pm PTFE membranes and 10 pL of each extract were înjected into the HPLC system though an autosampler. The rest of the filtrate was stored at 4°C as a reference sample. The UV spectra were stored at monitoring wavelengths of 280 nm. The mass spectrometer was operated in scan mode using positive polarity with mass détection range of 100-800 amu.

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The optimized conditions were, fragmenter voltage 80, gain 1.0 EMV, nitrogen gas température 150 “C/350 °C in négative ionization mode, vaporizer 420 ’C, drying gas flow 5,0 l/min, nebulizer pressure 60 psig, capillary voltage 2100 V/4000V in négative ionization mode and corona at 25 μΑ/5 μΑ in negativé ion mode. The quantification of the identified 5 markers in the extracts was performed by calibràting with known concentration of standards on the basis of area under the peaks using diode array détection.

RESULTS

Plant Extraction: The extraction yields, using the ultrasonication-assisted extraction method, ranged from 8.7% in M03 to 20.2% in E01 (Table 3).

LC-MS Analysis

Identification of resveratrol: The confirmation of presence of resveratrol was achieved by HPLC-DAD and LC-MSD analyses. The identification of resveratrol in plant extract chromatograms (Figure 1) was confirmed by performing a UV spectral match of the peak eluting at similar rétention times with that of standard. Table 1 indicates the UV 15 spectrométrie characteristics of resveratrol obtained. The confirmation of the identification was further established by comparing the mass spectrométrie fragmentation pattern of the standard resveratrol with the unknown peaks of the extracts eluting at the same time (Figure 2). The two glycosides (1 and 2) wére tentatively identified by MS (Table 2) eluting earlier than resveratrol. The chemical structure of these two resveratrol dérivatives was not 20 established.

Table 1. The identification of resveratrol and its dérivatives by HPLC-MSD

	Compound	Rt (min)	UV (280 nm)	MSD [Μ+ΗΓ
1	Resveratrol glycoside-1	9.1	210 sh, 300, 310	229 [M+H]+,
			... sh	391 [M+ glycf
2	Resveratrol glycoside-2	9.5	:210sh, 300, 310	229 [M+H]+,
			'rsh	391 [M+ glycf
3	Resveratrol	10.1	, 210 sh, 300, 310	229 [M+H] +
*			sh

confirmée! by a reference standard ^{1 b} b^,; j

Table 2. Resveratrol and its dérivatives in plant extracts determined by HPLC-MSD . i '

COMPOUND (see Figure 2)
Plant extract	Glycoside 1	Glycoside 2	Resveratrol
E01
E08
E21
E46	A
E47
M02			V
M03			V
M23

Quantification of resveratrol: A standard curve was generated by injecting 1 μΙ_ of five known concentrations of standard resveratrol, ranging from 500 pg/ml to 10 pg/ml dissolved in 100% methanol. As shown in Table 3 resveratrol is a prédominant phytochemical and ubiquitously présent in ali extracts. The highest amount is found in E08 and M02 while it was î . i n.·. .

detected in relatively lower amounts in E46, E47 and M21.

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Table 3. Resveratrol content in selected plant extracts as determined by HPLC-DAD.

Plant Extracts	Yield (%)* ’ Ç-jk· ·	Resveratrol content in dried plant material (pfl'g) **
E01	20.2	258.6 ± 5.07
E08	16.5	2480 ±185.
E46	15.2	67.0 ±0.57
E47	13.8	1442. ±31.2
M02	14.2	455.4 ± 1.83
M03	8.7	133.8 ±0.55
M21	19.3	108.5 ±10.2
M23	15.5	187.4 ±1.17

The plant extracts were obtained after drying the supernatants to dryness by a rotary evaporator for 30 - 45 min. The water bath température was set at 40 degree Celsius. Yield was calculated as (weight of dried extract/weight of dried plant material) x 100 ** The values are average values ± stdev, n=3 and n=5 for E01 and E08 i

EXAMPLE 2: Identification and quantification of resveratrol by HPLC in G.africanum ’ --i ' jleaf and stem methanol extracts / .·*.··

EXPERIMENTAL

Stems and leaves fromi Gnetum africanum were received in dried unprocessed form and submitted to a methanol extraction. Gnetum africanum methanol extracts were separated using Lichrosphere 5RF-18™ column from Phenomax™ using mobile phase consisted of ! -1216367 water (A) and acetonitrile (B), both containing 0.1% acetic acid which were applied in gradient elution.

RESULTS

Under the experimental HPLC conditions vMich were selected, a standard sample 5 comprising a 1.0 pg of resveratrol had a rétention time of 39 min. A resveratrol peak having a rétention time 39 min was also identified within the 2 mg of leaf extract. Spiking the 2 mg of leaf extract with 1.0 pg of resveratrol'incréàsed the size of the resveratrol peak at 39 min.

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The spiking confirmed peak identity and provided an estimate for the quantity of resveratrol within this leaf extract.

| ΐ 10 It was calculated that about 1 pg resveratrol was présent in the 2 mg of leaf extract (i.e. 0.5 | pg/mg of dried leaves or 500 pg/g of dried leaves). Similarly, a resveratrol peak with a rétention time 39 minutes was identified within the 2 mg of stem extract and it was calculated that this 2 mg stem extract contained about 33 gg of resveratrol (i.e. 16.5 pg/mg of dried stem or 16 500 pg/g of dried stem).

EXAMPLE 3: Quantification of biochemicals in selected parts of Gnetum africanum Leaves and liana (stem) from Gnetum africanum growing either in Mayombe forest or the équatorial forest of Congo were, analyzed fbr their biochemical content. A summary of the results are shown in Table 4.

Table 4. Biochemical analysis of Gnetum africanum ; . h. <

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	Forest of Mayombe	Equatorial Forest
	Leaves	Liana	Leaves	Liana
Protein content	14,6%	11,3%	15,6%	9,37%
Oil content	3.2%	3.1%	6.4 %	1.6%
Oméga 6	9.4%	17.8%
Total trans fat	29.8%	39.4%
Cis fat	12.4%	; 21 ;1
Oméga 3	48.4	21.7
Minerais (mg/100g)
Bore (B)	1.07	0,485	N/A	N/A
Calcium (Ca)	0.75	0,19	1,23	0,79
Chrome (Cr)	0.06	0,025	N/A	N/A
Cobalt (Co)	0.01	0,015	N/A	N/A
Magnésium (Mg)	43	32,49	N/A	N/A
Manganèse (Mn)	12.15	4,58	N/A	N/A
Iron (Fe)	2.37	1,47	N/A	N/A
Copper (Cu)	0.63	1	N/A	N/A
Nickel (Ni)	0.055	0,05	N/A	N/A
Phosphorus (P)	44	49	N/A	N/A
Potassium (K)	1.45	1.44	0,62	1.02
Sélénium (Se)	50.49	49,15	N/A	N/A
Sodium (Na)	0.5	0.055	0.018	0,030
Zinc (Zn)	0.41	0,66	N/A	N/A
Lithium (Li)	1,8	3,7	N/A	N/A
Aluminium (Al)	10.61	17,2	N/A	N/A
Silicium (Si)	8	3,76	N/A	N/A
Titane (Ti)	0:21	0,145	N/A	N/A
Strontium (Sr)	0.88	1,31	N/A	N/A
Antimoine (Sb)	0.14	0,01	N/A	N/A
Rubidium (Rb)	1,4	2,56	N/A	N/A
Total fibers	66.5	. 81.9	48.6	N/A
Soluble fibers	0.8	0.1	10.7	N/A
Insoluble fibers	65.7	£111	37.9	N/A
Resveratrol (mg/100g)	20,8	18,2	15,8	250
Total sugar	5.8 %	2.0 %	6.5 %	6.7 %
Vitamin E mg/100 g	71.8	1.8	38.2	<1

Headings are included herein for reference and to aid in locating certain sections These headings are not intended to limit the scope of the concepts described therein under, and these concepts may hâve applicability in other, sections throughout the entire spécification Thus, the présent invention is nbt intended to be Iimited to the embodiments shown herein

-14- , but is to be accorded the widest scope consistent with the principles and novel features disclosed herein, O^-

As used herein and in the appended daims, the singular forms a, an, and the include plural référants unless the context clearly indicates otherwise. Thus, for example, reference 5 to a compound includes one or more of such compounds, and reference to the method includes reference to équivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

i

Unless otherwise indicated, ail numbers .expressing quantities of ingrédients, reaction conditions, concentrations, properties, and so forth used in the spécification and claims are 10 to be understood as being modified in ail instances by the term about. At the very least, each numerical parameter should at least be çonstrued in light of the number of reported significant digits and by applying ordinary ' rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the présent spécification and attached claims are approximations that maÿ’vary depending upon the properties sought to 15 be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the spécifie examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analyses and such.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the présent invention and scope of the appended claims.

Claims (17)

1. A processed plant material, wherein said processed plant material is dried and comprises at least about 30 pg resveratrol, or at least about 50 pg resveratrol, at least about 100 pg resveratrol, at least about 500 pg resveratrol, at least about 1 000 pg resveratrol, at least about 2 500 pg resveratrol, at feast about 5 000 pg resveratrol, at least about 10 000 pg, 5 at least about 15 000 pg resveratrol per gram of dried plant material.

2. The processed plant material of claim 1, wherein said dried plant material is derived from the plant Gnetum afncanum, such as the leaves or a stem of the plant Gnetum africanum.

3. The processed plant material according to claim 1 or 2, wherein said processed plant material further comprises materials selected from the group consîsting of resveratrol glycosides, lipids, minerais, fibers, sugars, vitamins, amino acids and proteins.

4. The processed plant material according to any one of ciaims 1 to 3, wherein said

J . . < , processed plant material is packaged in a sealed container, a pouch, a jar, a tube, a blister

..L ;. i pack, or a box. ';

5. A plant extract comprising at least about 0.002% w/w resveratrol, or at least about

15 0.005% w/w resveratrol, or at least about 0.01% w/w resveratrol, or at least about 0.05% w/w resveratrol, or at least about 0.1% w/w resveratrol, or at least about 0.5 % w/w resveratrol, or at least about 1% w/w resveratrol.

6. The plant extract of claim 5, wherein said plant extract dérivés from the plant Gnetum

20 afncanum, such as the leaves or a stem of the plant Gnetum africanum.

7. A method for obtaining a plant extract concentrated in resveratrol, comprising:

(i) providing plant material from Gnetum africanum, such as the leaves or a stem of the plant Gnetum africanum-, (ii) submitting said plant material to a solvent extraction; and (iii) obtaining a plant extract concentrated ïn resveratrol, said plant extract comprising at least about 0.005% w/w resveratrol.

8. The method of claim 7, wherein 1 g of dry^; plant material yield at least about 100 pg resveratrol.

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9. The method of claim 7 or 8, wherein said solvent is selected from the group consisting of methanol, éthanol, ethyl acetate and combinations thereof.

10. The method of any one of daims 7 to 9, wherein said plant extract further comprises a resveratrol glycoside selected from:. (i) a compound identifiable under détection conditions of

5 Table 1 by the presence of a peak having a rétention time of 9.1 min on a HPLC-APCI-MSD chromatogram; and (ii) a compound identifiable under détection conditions of Table 1 by the presence of a peak having a rétention time ôf 9.5 min on a HPLC-APCI-MSD chromatogram.

. ' 'Ίί' :t'

11. An edible product, comprising at least one of the following constituents:

(a) a processed plant material as defined in any one of daims 1 to 4;

10 (b) a plant extract as defined in daim 5 or 6; ' (c) a resveratrol glycoside selected from: (i) a compound identifiable under détection conditions of Table 1 by the presence of a peak having a rétention time of 9.1 min on a HPLC-APCI-MSD chromatogram; and (ii) a compound identifiable under détection conditions of Table 1 by the presence of a peak having a rétention time of 9.5 min on a HPLC-APCI-MSD chromatogram.

12. The edible product of claim 11, wherein said edible product is a food product selected from the group consisting of a dairy. product, a nutrition al bar, a herbal tea, and a drink.

13. The edible product of claim 11, wherein said edible product is a nutraceutical product . · · l selected from the group consisting of vitamins, minerais, nutritional suppléments, herbal 20 remédiés, homéopathie remedies, probiotics, amino acids, and essential fatty acids.

14. A cosmetic or pharmaceutical composition comprising at least one of the following constituents:

(a) a processed plant material as defined in any one of daims 1 to 4;

(b) a plant extract as defined in claim 5 or 6;

25 (c) a resveratrol glycoside selected from: (i) a compound identifiable under détection conditions of Table 1 by the presence of à peak having a rétention time of 9.1 min on a HPLC-APCI-MSD chromatogram; and (ii) a compound identifiable under détection conditions of Table 1 by the presence of a peak having a rétention time of 9.5 min on a HPLC-APCI-MSD chromatogram; .

30 in combination with a suitable cosmetic or pharmaceutically acceptable carrier, diluent or excipient. |

15. The composition of claim 14, wherein said composition is a cosmetic composition selected from the group consisting of facial creânls, lotions, and skin tonies.

-17t ·** X

16. The composition of claim 14, wherein said composition is a pharmaceutical composition for the prévention or treatment of a disease selected from the group consisting of skin diseases, cardiovascular diseases, inflammatory diseases, blood lipids related conditions, and pain.

17. Use of at least one of the following constituents:

(a) a processed plant material as defined in any one of claims 1 to 4;

(b) a plant extract as defined jn claim 5 or 6;» (c) a resveratrol glycoside selected from: /(i) a compound identifiable under détection conditions of Table 1 by the presence of a peak having a rétention time of 9.1 min on a HPLC-APCI-MSD chromatogram;'and (ii) a compound identifiable under détection conditions of Table 1,bythe presence pf a peak having a rétention time of 9.5 min on a HPLC-APCI-MSD chromatogram;

in the manufacture of medicine for the prévention or treatment of a disease selected from the group consisting of skin diseases, cardiovascular diseases, inflammatory diseases, blood lipids related conditions, and pain.