Dye composition comprising a combination of two plant extracts of <i>Lawsonia inermis</i>

Abstract

The disclosure relates to a dye composition comprising a combination of two extracts of Lawsonia inermis and a method for preparing thereof. The disclosure also relates to the cosmetic use of said composition for dyeing keratin fibers. Finally, the disclosure relates to a cosmetic method for dyeing keratin fibers comprising the application of such a composition.

Claims

1. A method for preparing a dye composition comprising the following steps: providing an extract A of the aerial parts of Lawsonia inermis containing from 7 to 60% by weight of lawsone relative to the total weight of the dry extract, wherein the lawsone results notably from enzymatic hydrolysis of glycosylated lawsone derivatives; and providing an extract B of the aerial parts of Lawsonia inermis containing less than 0.6% by weight of lawsone, at least 15% by weight of phenol compounds and at least 60% by weight of saccharide compounds, including polysaccharides, relative to the total weight of the dry extract; mixing extract A and extract B; obtaining a dye composition.

2. The method according to claim 1 wherein extract A of the aerial parts of Lawsonia inermis further comprises luteolin, apigenin and 2,3,4,6-tetrahydroxyacetophenone.

3. The method according to claim 1 wherein the extract A of the aerial parts of Lawsonia inermis further comprises coumaric acid.

4. The method according to claim 1 wherein the extract A of the aerial parts of Lawsonia inermis does not contain more than 2% by weight of proteins, peptides or amino acids relative to the total weight of the dry extract.

5. The method according to claim 1 wherein the dye composition comprises a standardized dry extract AN of Lawsonia inermis which comprises the extract A of the aerial parts of Lawsonia inermis and a carrier, the standardized dry extract AN comprising from 0.6 to 1.4% by weight of lawsone relative to the total weight of the standardized dry extract.

6. The method according to claim 5, wherein the standardized dry extract AN of Lawsonia inermis comprises, relative to the total weight of the dry extract: from 0.05 to 1.0% by weight of luteolin; from 0.01 to 0.5% by weight of apigenin; from 0.01 to 1.0% by weight of 2,3,4,6-tetrahydroxyacetophenone.

7. The method according to claim 1 wherein the extract A of the aerial parts of Lawsonia inermis is obtained by a process comprising the following steps: a) macerating the aerial parts of Lawsonia inermis in water, at a pH ranging from 4 to 8, in order for the glycosylated lawsone derivatives initially present in the aerial parts of Lawsonia inermis to be partially or totally hydrolyzed enzymatically, to provide an aqueous solution containing lawsone; b) adding an organic solvent to the solution obtained from step a), the organic solvent being chosen from C.sub.4-C.sub.12 linear, or branched, alcohols or solvents having a miscibility with water which is less than 10% by weight at 25° C., to provide an aqueous phase and an organic phase; c) recovering the organic phase obtained from step b); and d) concentrating the organic phase recovered from step c), to provide an extract A as recited in claim 1.

8. The method according to claim 7 wherein step a) is performed according to at least one of the following conditions: step a) is conducted at a temperature ranging from 20° C. to 60° C.; step a) is conducted at a pH ranging from 5 to 7.5; step (a) is performed under stirring for 15 min to 2 h; step (a) is conducted in a volume of water whose weight is 2 to 15 times greater than the weight of the aerial parts of Lawsonia inermis subjected to maceration.

9. The method according to claim 1 wherein the saccharide compounds of extract B further comprise monosaccharides, disaccharides and combinations thereof.

10. The method according to claim 1 wherein the extract B further comprises from 2 to 8% by weight of organic acids related to the total weight of the dry extract B.

11. The method according to claim 1 wherein the phenol compounds of extract B are selected from the group consisting of: gallic acid, coumaric acid, and/or heterosides of such compounds and combinations thereof; tannins, phloroglucinol derivatives; and flavonoid derivatives.

12. The method according to claim 1 wherein extract B is an aqueous, hydroalcoholic or alcoholic extract of Lawsonia inermis.

13. The method according to claim 1 wherein the extract A is mixed with extract B according to a B/A weight ratio greater than or equal to 1, the extract A being a standardized dry extract.

14. The method according to claim 1 wherein the extract A is mixed with extract B according to a B/A weight ratio less than 1, the extract A being a standardized dry extract.

15. A dye composition obtained by the method according to claim 1.

16. The dye composition according to claim 15 further comprising one or more cosmetically-acceptable excipients suitable for application on human keratin fibers.

17. A cosmetic method for dyeing human keratin fibers comprising the following steps: a) providing a composition according to claim 15 in powder form, b) preparing an aqueous composition, by adding to the powder of step a) an aqueous composition at a temperature between 20° C. and 98° C. and mixing, c) applying onto keratin fibers, optionally while heating the fibers thus treated, d) rinsing, e) optionally, repeating steps c) and d).

Description

FIGURES

(1) FIG. 1 represents the normalized UHPLC-UV chromatogram of an extract obtained by the process according to example 1.2 (sample E2).

(2) FIG. 2 represents the same chromatogram as in FIG. 1: the filled peaks correspond to the ones that can be seen in FIG. 1, while the plain line corresponds to a zoom of said chromatogram.

(3) FIGS. 3a, 3b, 3c, 3d and 3e represent the UV spectra of lawson, luteolin, apigenin, para-coumaric acid and 2,3,4,6-tetrahydroxyacetophenone, isolated from sample E2.

(4) FIG. 4 represents the normalized UHPLC-UV chromatogram of an extract obtained by the process according to example 1.4.

(5) FIG. 5 represents color difference dE* after 10 washes for product I1 (vertical lines) or for control T1 (horizontal lines)

CHARACTERIZATION

(6) A) Structural Analyses

(7) Material and Methods

(8) Chromatographic separations were performed on a Waters ACQUITY UHPLC system equipped with a quaternary pump, an auto-sample injector, an on-line degasser, an automatic thermostatic column oven and a DAD detector (200-500 nm). An ACQUITY UPLC BEH Shield RP18 column (100 mm×2.1, 1.7 μm) equipped with a Vanguard™ precolumn (5 mm×2.1) (Waters Corporation, Milford, USA) at 35° C. was used and the flow rate was set at 0.4 mL/min. The mobile phase consisted of a linear gradient system of (A) water with 0.1% formic acid and (B) acetonitrile and (C) methanol as wash solvent: 0-9 min, 2%-100% B; 9-9.55 min, maintain 100% B; 9.55-9.70 min, 0%-100% C; 9.7-10.2 min, maintain 100% C; 10.20-10.35 min, 0%-100% B; 10.35-10.85 min, maintain 100% B; 10.85-11 min, 0%-98% A; held at 98% A—2% B for 1 min for equilibration of the column. Compounds were identified by high-resolution mass spectrometry, 1D- and 2D-NMR experiments (.sup.1H NMR, .sup.11C NMR, DEPT, COSY, HMBC, HSQC).

(9) B) Quantitative Analysis: Experimental Conditions

(10) Luteolin, apigenin were titrated by analytical HPLC performed with a C18 column (XBridge 100 C18; 3.5 mm, 150 mm×4.6 mm) using gradient conditions (see below) with H.sub.2O/trifluoroacetic acid 0.1% (A) and Acetonitrile/trifluoroacetic acid 0.1% (B) as eluent: Gradient conditions: t0 A 18% B 82%; t1 min: A 18% B 82%; 10 min A 50% B 50%; 10.1 min: A 18% B 82%

(11) UV detection is at 340 nm for apigenin and 310 nm for luteolin. Flow rate was 1 mL/min and temperature 40° C. Pure luteolin, apigenin and p-coumarin were used for calibration.

Example 1: Synthesis of Extract A

Example 1.1: Isopropyl Acetate Extract According to the Invention

(12) 50 g of uncrushed leaves of Lawsonia inermis are extracted by 500 mL of water at 30-40° C. for 30 min. 600 mL of isopropyl acetate are added to this solution. This is mixed for 30 min. After decantation, the upper isopropyl acetate phase (480 mL) is recovered, the aqueous phase being separated because it is practically free of lawsone. The isopropyl acetate phase is filtered then dried with the Rotavapor. The residue is the dry henna extract.

(13) The plant contains 1.5 g of lawsone/100 g of dry plant.

(14) The isopropyl acetate upper phase contains 80.7% of the lawsone potential present in the plant.

(15) The dry henna extract contains 30.2% by weight of lawsone, i.e. 71% of the lawsone present in the plant.

(16) Stability study: sample stored at 25° C., 60% relative humidity and protected from light:

(17) At T0: lawsone content=30.2% by weight of lawsone relative to the weight of the dry extract.

(18) At T1 month: lawsone content=29.7% by weight of lawsone relative to the weight of the dry extract; so no significant loss within the meaning of the present invention.

Example 1.2: Isopropyl Acetate Extract No 2 According to the Invention

(19) 49.5 g of uncrushed leaves of Lawsonia inermis are extracted by 500 mL of water at 30-40° C. for 30 min. 600 mL of isopropyl acetate are added to this solution. This is mixed for 30 min. After decantation, the upper isopropyl acetate phase is recovered and filtered on Büchner (K900), and the residue is rinsed with 50 mL of isopropyl acetate. The resulting solution is then dried with the Rotavapor. The residue is the dry henna extract (sample E2). The dry henna extract (sample E2) contains 30.9 wt. % of lawsone.

(20) Results

(21) UHPLC-UV Chromatogram

(22) The obtained UHPLC-UV chromatogram is displayed in FIGS. 1 and 2. The peaks that can be observed on a zoom of said chromatogram (FIG. 2, plain line) have been associated with the following compounds:

(23) TABLE-US-00001 15 lawsone 1 2 gallic acid lalioside 3 4 myrciaphenone A catechin 5 6 2,3,4,6-tetrahydroxyacetophenone 1,2-dihydroxy-4-O- glycosyloxynaphtalene 7 10 luteolin-4′-O-glucoside para-coumaric acid 11 12 apigenin-7-O-β-glucoside luteolin-3′-O-glucoside 13 14 apigenin-4′-O-β-glucoside 3,4,5-trihydroxyacetophenone 19 20 3′,4′,5,7-tetrahydroxyflavanone luteolin 21 22 3′,5,7-trihydroxy-4′-methylflavone apigenin

(24) UV Spectra

(25) The UV spectra of the compounds corresponding to peaks No 15, 20, 22, 10 and 5 are displayed in FIGS. 3a, 3b, 3c, 3d and 3e respectively.

(26) B) Quantitative Analysis

(27) Material and Methods

(28) Batches Samples LP110: Henna extract AN, Ethyl acetate extract standardized with maltodextrine—industrial scale. ES310: Henna extract AN, Ethyl acetate extract standardized with maltodextrine—laboratory scale. JQ137A: Henna extract AN, Isopropyl acetate Henna extract with fructose—laboratory scale.

(29) The lawsone in each of the above standardized extract is equal to 1.1 wt. %.

(30) Results:

(31) TABLE-US-00002 Q.sub.inj(μg) Mass Vol V.sub.inj P-coumaric Sample (mg) (mL) (μg) luteolin apigenin acid LP110 1 215.5 20 5 0.0951 0.0132 0.0234 LP110 2 266.1 20 5 0.1191 0.0163 0.0281 LP110 3 233.1 20 5 0.1029 0.0157 0.0256 ES3310 4 265.8 20 5 0.2348 0.0484 0.0285 ES3310 5 217.25 20 5 0.195 0.0285 0.0238 ES3310 6 227 20 5 0.2376 0.0307 0.0262 JQ137A 7 235.9 20 5 0.1015 0.0178 0.0159 JQ137A 8 222.6 20 5 0.1039 0.0188 0.0161 JQ137A 9 227.7 20 5 0.1158 0.0195 0.0176 Mean content (wt. %) Extract luteolin apigenin p-coumaric acid LP110 0.18% 0.03% 0.04% ES3310 0.38% 0.06% 0.04% JQ137A 0.19% 0.03% 0.03%

Example 1.3: Ethyl Acetate Standardized Dry Extract According to the Invention

(32) 50 g of crushed leaves of Lawsonia inermis are extracted by 500 mL of water at 30-40° C. for 30 min. 600 mL of ethyl acetate are added to this solution. This is mixed for 30 min. After decantation, the upper ethyl acetate phase is recovered and the aqueous phase is removed because of its very low lawsone content.

(33) The lawsone content of the ethyl acetate phase is determined by H.P.L.C, and maltodextrin is added in sufficient quantity to obtain a mixture containing 1.3 wt % of lawsone, which is then lyophilized.

(34) The dry henna extract standardized with maltodextrin contains 1.1 wt. % of lawsone, i.e. 71% of the initial lawsone content in the plant.

Example 1.4: n-Butanol Extract According to the Invention

(35) 50 g of uncrushed leaves of Lawsonia inermis are extracted by 6 volumes of water at 30-40° C. for 30 min. Six volumes of n-butanol are added to this solution at room temperature. This mixture is stirred for 30 min. After decantation, the upper butanol phase is recovered, the aqueous phase being removed because it is practically free of lawsone. The organic phase is concentrated with passage over water.

(36) The lawsone content in the n-butanol phase is determined by HPLC. Maltodextrin is added in an amount sufficient to obtain a mixture comprising from 1.1 to 1.3 wt. % of lawsone.

(37) The concentrate is dried to obtain a powder.

(38) Results

(39) UHPLC-UV Chromatogram

(40) The obtained UHPLC-UV chromatogram is displayed in FIG. 5. The peaks that can be observed have been associated with the following compounds:

(41) TABLE-US-00003 Resolution time Compound 4.47 lawsone 3.38 2,3,4,6-tetrahydroxyacetophenone 5.34 luteolin 5.96 apigenin

(42) The presence of glycosylated luteolin, in particular of luteolin-6-C-neohesperidoside and coumaric acid, is noted.

Example 2: Synthesis of Extract B

Example 2.1

(43) Crushed Lawsonia inermis leaves are introduced into a 50-g reactor with 6 volumes of water. The paste obtained is stirred for 30 min at 50° C. Then add 16 volumes of ethanol to this mixture, i.e. a final alcohol content of 80% v/v, then shake vigorously for 30 min. The entire paste is filtered (on an AF15 filter) and the hydroalcoholic filtrate is recovered, sterilized and then concentrated by evaporation of ethanol. The aqueous concentrate is then dried. The dry product corresponds to the dry henna extract (yield by weight: 54%).

(44) The extract thus obtained comprises (% expressed in weight relative to the total weight of the dry extract): lawsone 0.6%; saccharide compounds: 69.9% (total sugars) (polysaccharides content 59.3%); apigenin 0.05%; luteolin 0.17%.

Example 2.2

(45) Crushed Lawsonia inermis leaves are introduced into a 50-g reactor with 6 volumes of water. The paste obtained is stirred for 30 min at 50° C. Stir vigorously for an additional 30 min. The entire paste is filtered (on an AF15 filter) and the aqueous filtrate is recovered, sterilized 20 min at 120° C. and then concentrated. The aqueous concentrate is then dried. The dry product corresponds to the dry henna extract (yield by weight: 60%).

(46) The extract thus obtained comprises 0.4% of lawsone,% expressed in weight relative to the total weight of the dry extract.

Example 3: Comparison of the Characteristic Compounds of Extracts A and B

(47) The characteristic compounds of the extracts obtained according to Example 1 or according to Example 2 are reported in the following table; the percents are expressed by weight relative to the total weight of the dry extract:

(48) TABLE-US-00004 TABLE 5 Standardized extract AN Extract B Compounds (Example 1) (Example 2) Lawsone   1-1.3 <0.6 Phenol compounds 0.2-3   >15 Tannins n.d. n.m. luteolin 0.05-1   0.1-0.3 Apigenin 0.01-0.5  0.05-0.5 Para-coumaric acid 0.01-0.1  n.m. 2,3,4,6- 0.05-1   0.02-0.1 tetrahydroxyacetophenone Gallic acid n.m. 0.1-0.3 3,4,5- n.m. n.d. trihydroxyacetophenone 1,3,6.7- n.d. n.m. trihydroxyacetophenone Saccharide compounds <0.5 70-75 Malic acid, succinic acid, n.d. 4-5 ascorbic acid

(49) n.d. means not detected, so the compound was not detected during analysis

(50) n.m. means not measured, so the compound was detected during analysis but was not measured

Example 4: Assessment of the Color Retention on White, Natural Human Hair Strands after Washing

(51) An extract A according to the invention (that of Example 1.2) is mixed with an extract B according to the invention (that of Example 2.2) in a B/A weight ratio of 1/1. This mixture is designated product I1. A conventional henna powder is also used (finely crushed Lawsonia inermis leaves (<250 μm)) as a control. This powder is called product T1.

(52) In each case, 10 g of powder are mixed with 40 g of water before application onto the strand. The two products I1 and T1 were assessed for their dye content following washing. All the strands underwent standardized washes up to 10 times with a neutral shampoo.

(53) For each product I1 and T1, the color of 5 natural white human hair test strands, color 10 (white) was measured using a reflectance colorimeter (CR400 Croma Meter, Minolta, France). The color parameters measured by this device, L*a*b*, describe the strand colors. The parameter assessed is a derivative of L*a*b, i.e., the color difference (dE*).
dE*=√((L.sub.1*−L.sub.2*).sup.2+(a.sub.2*−a.sub.1*).sup.2+(b.sub.2*−b.sub.1*).sup.2)  [Math 1]

(54) Where L.sub.1*, a.sub.1*, b.sub.1* are the coordinates in the CIELAB color space established in 1976 by the International Commission on Illumination of the first color to be compared and L.sub.2*, a.sub.2*, b.sub.2* those of the second.

(55) In our case, the “1” values are the Lab values of the dyed strands before washing, and the “2” values the values obtained after 10 washes.

(56) Statistical study concluded that the color difference with the combination according to the invention I1 is significantly less than the control T1 (*p<0,05).

(57) The results are reported in FIG. 5.

(58) Method 1: Lawsone Assay by HPLC

(59) This method can be applied for: A. the assay of lawsone in an extract B. the assay of the total lawsone present in the free form or form of glycosylated lawsone derivatives in the aerial parts of Lawsonia inermis, obtained by acid hydrolysis, and thus quantifying the lawsone potential in the plant, C. the assay of the lawsone formed by enzymes.

(60) Reagents

(61) Lawsone>97% (HPLC) SIGMA—ref: H46805

(62) Dichloromethane for analyses.

(63) Sulfuric acid for analyses.

(64) Methanol for analyses.

(65) HPLC-grade water.

(66) HPLC-grade acetonitrile.

(67) HPLC-grade trifluoroacetic acid.

(68) HPLC Conditions Column: XBridge C18, 3.5 μm, 4.6×150 mm Waters Furnace: 40° C. Solvents: S-A: 0.1% trifluoroacetic acid in water. S-B: 0.1% trifluoroacetic acid in acetonitrile. Gradient: T0 min 40% S-A; T 1 min 40% S-A; T 10 min 5% S-A; T 11 min 5% S-A; T 11.1 min 40% S-A. Wavelength: λ=278 nm. Flow rate: 1 mL/min Injection: 10 μL.

(69) Sample Preparation:

(70) For whole or roughly crushed leaves:

(71) 50 g of leaves are crushed then sieved through a 0.355 μm sieve.

(72) For leaf powders:

(73) Use 50 g of leaf powder as is.

(74) Preparation of the Solutions Control solutions:

(75) Lawsone solution at 0.3 mg/mL in 1/1 methanol/ethanol. Dilute to 1/10, 1/20, 1/100 in 1/1 methanol/water. Test solutions: Test solution A (assay of the lawsone present in an extract)

(76) Dissolve 50 mg of extract in 100 mL of 1/1 methanol/water. Dissolve with ultrasound. Filtration on Acrodisc GFGHP. Inject 10 μL. Test solution B (assay of total lawsone)

(77) Introduce 80 mg of leaf powder into a volumetric flask. Add 50 mL of 2N H.sub.2SO.sub.4. Heat to 97° C. for 30 min. Let cool. Add methanol qs 100 mL. Filter the solution on Acrodisc GF GHP 0.45 μm. Inject 10 μL of the filtrate. Test solution C (assay of the lawsone formed by enzymes)

(78) Introduce 80 mg of leaf powder into a volumetric flask. Add into 50 mL of demineralized water. Place in an ultrasound bath for 30 min between 30 and 40° C. Let cool. Add methanol qs 100 mL. Filter the solution on Acrodisc GF GHP 0.45 μm. Inject 10 μL of the filtrate.

(79) Results

(80) Use the regression line calculated with the control solutions to determine: A. the lawsone content of the extract, B. the total lawsone content, and/or C. the content in lawsone formed by the enzymes.

(81) Method 2: Assay of Nitrogen-Containing Compounds (Amino Acids, Proteins

(82) Free amino acids and proteins can be assayed before or after hydrolysis by ninhydrin spectrophotometry. The results are expressed in percentage of amino acids relative to asparagine.

(83) Assay of Total Proteins and Amino Acids

(84) Principle

(85) Colorimetric assay of amino acids by the ninhydrin reagent after acid hydrolysis. The results are expressed in percentage of total amino acids relative to asparagine.

(86) Reagents Citrate buffer (pH=5)

(87) Dissolve 2.1 g of citric acid in 20 mL of water, add 20 mL of 1 N sodium hydroxide and adjust to 50 mL with water. Ninhydrin reagent:

(88) Dissolve 0.08 g of tin (II) chloride (SnCl.sub.2, 2H.sub.2O) in 50 ml of citrate buffer (pH=5).

(89) Dissolve 2 g of ninhydrin in 50 mL ethylene glycol monomethyl ether (EGME).

(90) Mix the two solutions. 6N hydrochloric acid

(91) Dilute to 1/2 of concentrated hydrochloric acid (36%). Diluent

(92) Mix 100 mL of 1-propanol with 100 mL of water.

(93) Preparation of the Solutions Preparation of the calibration range

(94) Dissolve 17 mg of asparagine in 100 mL of water. Preparation of the test solutions

(95) Weigh approximately 30 to 200 mg of extract depending on the sample to analyze (pe.sub.1) in a screw thread tube, add 2 mL of 6N HCl.

(96) Hermetically seal then place for around 16 hours at 110° C.

(97) Neutralize with 3N sodium hydroxide (methyl red changes color) then adjust to 20 ml with water.

(98) Assay

(99) TABLE-US-00005 T 0.1 T 0.2 T 0.5 Test Blank Control solution (mL) 0.1 0.2 0.5 — — Test solution (mL) — — — 0.2 — Water (mL) 1 1 1 1 1 Ninhydrin reagent (mL) 1 1 1 1 1

(100) Stir and place in a water bath at 100° C. for 20 minutes. Cool in an ice bath. Adjust to 10 ml with diluent.

(101) Measure the absorbance at 570 nm of the different solutions against the blank.

(102) Calculations

(103) Construct the calibration curve. Deduce from it the total amino acid concentration (Q.sub.AAT), expressed in asparagine, in the test solutions. The total amino acid content (T.sub.AAT) of the extract is given by the following formula:

(104) $T_{\mathrm{AAT}}\left(\%\right)=\frac{Q_{\mathrm{AAT}}\times 100\times 20}{{\mathrm{pe}}_1}$

(105) with Q.sub.AAT in mg/ml et pe.sub.1 in mg

(106) Method 3: Weight Assay of Chlorophylls

(107) The chlorophyll content in the extract may be evaluated by the weight obtained after washing the extract with heptane. The extract is taken up by 10 volumes of methanol. After stirring for 15 min, the solution is filtered. The supernatant is dried and constitutes the fraction containing chlorophylls.

(108) Method 4: Spectrophotometric Assay of Phenol Compounds

(109) The content of phenol compounds in the extract can be evaluated by spectrophotometry according to the method of the European Pharmacopoeia, version 9.0, 2.8.14.

(110) The solutions to be tested are prepared by dissolving 25 mg of extract in 100 mL of water. The content of phenol compounds is expressed by reference to pyrogallol.

(111) Method 5: Colorimetric Assay of Saccharide Compounds Before and After Hydrolysis

(112) Principle: Colorimetric determination of saccharide compounds by dinitrosalicylic acid (DNS) compared to glucose before and after hydrolysis. The results are expressed as the percentage of saccharide compounds relative to glucose.

(113) Reagents:

(114) DNS reagents: dissolve 30 g of sodium and potassium ditartrate in 50 ml of water. Add 20 mL of 2N sodium hydroxide. Dissolve 1 g of dinitrosalicylic acid (DNS) while slightly heating. Make up to 100 mL with water.

(115) Preparation of Solutions:

(116) Preparation of the calibration range: dissolve 5 mg of glucose in 10 mL of water.

(117) Preparation of hydrolyzed test solutions (total saccharide compounds): Weigh about 1 g of extract (pe2). Add 1 mL of 4N H2SO4. Heat at reflux for 2 hours. Neutralize with 1N sodium hydroxide and transfer to a 20 mL volumetric flask. Make up to 20 mL with water.

(118) Preparation of non-hydrolyzed test solutions (free saccharide compounds=monosaccharides): weight about 10 g of extract (pe3) in a 20 mL volumetric flask. Make up to 20 mL with water.

(119) Dosage: the solutions are dosed according to the following table:

(120) TABLE-US-00006 T 0.5 T1 T1.5 T2 Tests Blank Control solution (ml) 0.5 1 1.5 2 — — Test solution (ml) — — — — 1 — Water (ml) 1.5 1 0.5 0 1 2 DNS 1 1 1 1 1 1

(121) Shake and then place for 5 minutes in a water bath at 100° C. Cool on an ice bath and make up to 10 mL with water. Measure the absorbance at 540 nm of the different solutions against the blank.

(122) Calculation:

(123) Construct the calibration curve.

(124) Deduce the concentration of total saccharide compounds (QSRT) and free saccharides (QSRL), expressed as glucose, in the test solutions. The titer in total saccharide compounds (TSRT) of the extract is provided by the following formula:

(125) $T_{\mathrm{SRT}}\left(\%\right)=\frac{Q_{\mathrm{SRT}}\times 100\times 20}{{\mathrm{pe}}_2}$

(126) With Q.sub.SRT in mg/ml and pe.sub.2 in mg

(127) The titer in free saccharide compounds (TSRL) of the extract is provided by the following formula:

(128) $T_{\mathrm{SRL}}\left(\%\right)=\frac{Q_{\mathrm{SRL}}\times 100\times 20}{{\mathrm{pe}}_3}$

(129) With Q.sub.SRL in mg/ml and pe.sub.3 in mg

(130) Method 6: HPLC Assay of Organic Acids

(131) Principle: HPLC assay of the organic acids from a plant extract. The organic acids assayed are: succinic, malic and ascorbic acids.

(132) Chromatographic System: Column: Atlantis dc18 5 μm 5 μm 250×4.6 mm Waters or equivalent; Temperature: 25° C.; Mobile phase: Solvent A: 50 mM phosphate buffer, pH 2.8; Solvent B: Acetonitrile; Gradient: T0% A: 100%; 7.5 min % A: 100%; 15 min % A: 90%; 16 min % A: 50%; 23 min % A: 50%; 24 min % A: 100%; 40 min % A: 100%; Flow rate: 1 mL/min; λ=200 at 400 nm (extraction at 214 nm); Injection volume: 5 μL.

(133) Preparation of the Solutions:

(134) Control solution: Weigh 10 mg of control and dissolve in 20 mL of purified water. Carry out the dilutions to 1/5, 1/10 and 1/20.

(135) Test solution: Weigh 250 mg of substance to be analyzed in a 20-mL volumetric flask. Fill with water—Run through ultrasound if necessary.

(136) Results: The chromatograms of the control and test solutions have several characteristic and identical peaks.

(137) The retention time of the controls are: Malic acid 5.5 min, Ascorbic acid: 7.7 min, Ascorbic acid: 11.7 min.

(138) Draw the calibration curve for each of controls and deduce the quantity of the sample.