Extract of the aerial parts of <i>Lawsonia inermis </i>and its preparation method

Abstract

The invention relates to an extract of the aerial parts of Lawsonia inermis, as well as its preparation method and the extract that can be obtained by said method. The invention also relates to a cosmetic dye composition comprising such an extract. The invention finally concerns a cosmetic method for dying keratin fibers comprising the application of such a composition.

Claims

1. An extract of aerial parts of Lawsonia inermis containing between 10 and 60% by weight of lawsone relative to the total weight of the dry extract, wherein a portion of the lawsone results from enzymatic hydrolysis of glycosylated lawsone derivatives, and wherein the extract further contains luteolin, apigenin, para-coumaric acid and 2,3,4,6-tetrahydroxyacetophenone.

2. The extract according to claim 1 containing not more than 2% by weight of proteins, peptides or amino acids relative to the total weight of the dry extract.

3. A composition comprising the extract of claim 1 and a carrier, wherein the composition contains between 0.6 and 1.4 wt. % of lawsone relative to the total weight of the composition wherein said standardized dry extract contains between 0.6 and 1.4 wt. % of lawsone, and wherein: the luteolin content is comprised between 0.05 and 1.0 wt. %, the apigenin content is comprised between 0.01 and 0.5 wt. %, and the para-coumaric acid content is comprised between 0.01 and 0.5 wt. % relative to the total weight of the composition.

4. A preparation method for a lawsone extract comprising the following steps: a) macerating the aerial parts of Lawsonia inermis in water, at a pH between 4 and 8 to partially or totally hydrolyze enzymatically the glycosylated lawsone derivatives, initially present in the aerial parts of Lawsonia inermis and to provide an aqueous solution containing lawsone; b) adding an organic solvent to the solution obtained from step a), the miscibility with water of said solvent being less than 10% by weight at 25° C., to form 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 obtain a lawsone—rich extract.

5. The preparation method according to claim 4, wherein the water temperature during step a) is comprised between 20° C. and 60° C.

6. The preparation method according to claim 4, wherein step a) is carried out at a pH between 5 and 7.5.

7. The preparation method for a lawsone—rich extract according to claim 4, wherein it does not include any step of changing the pH of the aqueous solution or the aqueous phase by addition of acid or base.

8. The preparation method for a lawsone—rich extract according to claim 4, wherein step a) is carried out under stirring, and the duration of the maceration is comprised between 15 minutes and 2 h.

9. The preparation method for a lawsone—rich extract according to claim 4, wherein in step a), the volume of water used is 5 to 15 times greater than the mass of the aerial parts of Lawsonia inermis subjected to maceration.

10. The preparation method according to claim 4, wherein the organic solvent added during step b) is characterized by a dipole moment less than 2.0 D, and is selected from the group consisting of alcohols, chlorinated solvents, ketones, ethers, esters and their mixtures.

11. The preparation method according to claim 4, wherein the organic solvent is a (C.sub.1-C.sub.6) alkyl acetate or a mixture of (C.sub.1-C.sub.6) alkyl acetates.

12. The preparation method according to claim 4, wherein the lawsone—rich extract obtained from step d) contains more than 50% of the lawsone initially present in the free form or in the form of glycosylated lawsone derivatives, in the aerial parts of Lawsonia inermis subjected to maceration in step a).

13. The preparation method according to claim 4 further comprising the step of: c′) adding a carrier between steps c) and d), and e) drying after step d) to obtain a standardized dry extract.

14. An extract that can be obtained by the method according to claim 4.

15. A cosmetic dye composition comprising an extract according to claim 1 and at least one cosmetically—acceptable excipient.

16. A standardized dry extract that can be obtained by the method according to claim 13.

17. A cosmetic dye composition comprising a standardized dry extract according to claim 3 and at least one cosmetically—acceptable excipient.

18. A cosmetic dye composition comprising a standardized dry extract that can be obtained by the method according to claim 13, and at least one cosmetically—acceptable excipient.

Description

FIGURES

(1) FIG. 1 represents the UHPLC-UV chromatogram of an extract according to the invention (sample E2) (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.; flow rate: 0.4 mL/min; mobile phase: linear gradient system of (A) water with 0.1% formic acid and (B) acetonitrile and (C) methanol (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.

(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) FIGS. 4a, 4b, 4c, 4d and 4e represent the .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of lawson isolated from sample E2.

(5) FIGS. 5a, 5b, 5c, 5d and 5e represent the .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of luteolin, isolated from sample E2.

(6) FIGS. 6a, 6b, 6c, 6d and 6e represent the .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of apigenin, isolated from sample E2.

(7) FIGS. 7a, 7b, 7c, 7d and 7e represent the .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of para-coumaric acid, isolated from sample E2.

EXAMPLES

(8) The examples that follow illustrate the invention.

(9) I. Extract, Standardized Dry Extract and Preparation Method of the Invention

Example 1: Isopropyl Acetate Extract No 1 According to the Invention

(10) 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 (sample E1).

(11) The plant contains 1.5 g of lawsone/100 g of dry plant. The isopropyl acetate phase contains 80.7% of the lawsone potential present in the plant.

(12) The dry henna extract (sample E1) contains 30.2% of lawsone, i.e. 71% of the lawsone present in the plant.

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

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

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

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

(16) 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 Buchner (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).

(17) The dry henna extract (sample E2) contains 30.9 wt. % of lawsone.

Example 3: Ethyl Acetate Standardized Dry Extract According to the Invention

(18) 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.

(19) 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.

(20) The dry henna extract standardized with maltodextrin (sample E3) contains 1.1 wt. % of lawsone, i.e. 71% of the initial lawsone content in the plant.

(21) For comparative purposes, counter-examples are described hereinafter.

Counter-Example 1: Acidic Isopropyl Acetate Extract No 1

(22) 20 g of uncrushed Lawsonia inermis leaves are extracted by 60 mL of isopropyl acetate saturated by phosphoric acid (1N H.sub.3PO.sub.4) at room temperature for 60 min. After solid/liquid separation on Buchner (K900) and rinsing of the residue by 20 mL of isopropyl acetate saturated in 1N H.sub.3PO.sub.4, the liquids obtained are collected and assayed (sample CE1)

(23) The plant contains 1.52 g of lawsone/100 g of dry plant. Sample CE1 contains 0.9% of the lawsone potential present in the plant.

Counter-Example 2: Acidic Methyl Ethyl Ketone Extract

(24) 20 g of uncrushed Lawsonia inermis leaves are extracted by 60 mL of methyl ethyl ketone saturated by phosphoric acid (1N H.sub.3PO.sub.4) at room temperature for 60 min. After solid/liquid separation in Buchner funnel (K900) and rinsing of the residue by 20 mL of methyl ethyl ketone saturated in 1N H.sub.3PO.sub.4, the liquids obtained are collected and assayed (sample CE2).

(25) The plant contains 1.52 g of lawsone/100 g of dry plant. Sample CE2 contains 0.9% of the lawsone potential present in the plant.

Counter-Example 3: Limewater Extract No 1

(26) 20 g of uncrushed Lawsonia inermis leaves are extracted by 80 mL of limewater at room temperature for 60 min. After solid/liquid separation in Buchner funnel (K900) and rinsing of the residue by 20 mL of limewater, the liquids obtained are collected and assayed (sample CE3).

(27) The plant contains 1.52 g of lawsone/100 g of dry plant. Sample CE3 contains 24% of the lawsone potential present in the plant.

Counter-Example 4: Limewater Extract No 2

(28) 19.75 g of crushed Lawsonia inermis leaves are extracted by a mixture of 80 mL of water and 20 mL of limewater (CaO(OH).sub.2, 50 g/L) at room temperature for 60 min. After solid/liquid separation in Buchner funnel (K900) and rinsing of the residue by 50 mL of water, the liquids obtained are collected and then dried with the Rotavapor. The residue is the dry henna extract (sample CE4).

(29) The dry henna extract (sample CE4) contains 1.8 wt. % of lawsone.

Counter-Example 5: Acidic Isopropyl Acetate Extract No 2

(30) 20.0 g of crushed Lawsonia inermis leaves are extracted by 60 mL of isopropyl acetate saturated by phosphoric acid (1N H.sub.3PO.sub.4) at room temperature for 60 min. After solid/liquid separation in Buchner funnel (K900) and rinsing of the residue by 20 mL of saturated isopropyl acetate, the liquids obtained are collected and then dried with the Rotavapor. The residue is the dry henna extract (sample CE5).

(31) The dry henna extract (sample CE5) contains 0.2 wt. % of lawsone.

Counter-Example 6: Aqueous Solution Extract

(32) 50 g of uncrushed Lawsonia inermis leaves are extracted by 150 mL of an acid aqueous solution (HCl, pH=2.5) at room temperature for 30 min. The mash obtained is separated into a pomace and an aqueous solution on Buchner without filter. The pomace is extracted with 1 L of an alkaline aqueous solution (NaOH 0.15%) at 50° C. for 3 h. After solid/liquid separation by filtration (on a gauze of size 100 mesh), the obtained solution is concentrated under vacuum to 1/15, cooled and acidified with HCl to pH=2.5. It is then centrifuged and oven-dried under vacuum at 40° C. The residue is the dry henna extract (sample CE6).

(33) The dry henna extract (sample CE6) contains 9.5 wt. % of lawsone.

(34) For ease of comparison, significant aspects of extracts E2, CE4, CE5 and CE6 are summarized below. Of note, the comparison of said extracts is of particular relevance, given that the same plant lot was used as starting material.

(35) TABLE-US-00002 E2 CE4 CE5 CE6 Starting plant material (g)   49.5   19.75   20.0   50.0 Lawsone content in the 896  357  362  905  starting material (mg) Extracted quantity of 580  41   0.48 213  lawsone (mg) Lawsone yield (Lawsone     64.7%     11.5%     0.1%     23.5% potential of the plant) Lawsone content in the     30.9%     1.8%     0.2%     9.5% dry extract

(36) As it appears clearly from the results displayed in the table above, the method of the invention allows to extract a much more important part of the lawsone potential of the plant than other methods. This is notably due to the specific maceration step a), during which the glycosylated lawsone derivatives originally present in the plant undergo an enzymatic hydrolysis.

II. Characterization Studies

(37) A) Structural Analyses

(38) Material and Methods

(39) The extract of the invention that was used for analyses is sample E2.

(40) 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.

(41) Compounds were identified by high-resolution mass spectrometry, 1D- and 2D-NMR experiments (.sup.1H NMR, .sup.13C NMR, DEPT, COSY, HMBC, HSQC).

(42) NMR experiments were performed on a Bruker Avance IIIHD 500 MHz spectrometer equipped with a BBO Prodigy 5 mm probe (Bruker Biospin, Karlsruhe, Baden-Wûttemberg, Germany). Deuterated. dimethylsulfoxide was used as solvent. Acquisition sequences were .sup.1H, .sup.13C, COSY, HSQC, HMBC. Chemical shifts were adjusted with the solvent as an internal standard (δ.sub.H 2,52 ppm and δ.sub.C 39,52 ppm).

(43) HRMS data were obtained on a Synapt G2Si with Masslynx v4.1 SCN957 software (Waters Q-TOF SYNAPT™, Waters Corp, Manchester, England). The MS source temperature was set at 125° C. and the desolvation temperature was set at 500° C. Nitrogen was used as the dry gas: the desolvation gas flow rate was set at 1000 L/h; the cone gas flow was maintained at 50 L/h. In both positive and negative modes, capillary and cone voltages were set at 0.5 kV and 50 V respectively. The mass spectra were recorded across the range of 100-1500 Da. To ensure stable and precise scanning, leucine enkephalin was used as reference compound (positive ion mode ([M+H]+=556.2771 and 278.1141) and negative ion mode ([M−H]−=554.2615 and 236.1035)).). All data were acquired in continuum mode with a resolving power of 25,000.

(44) Results

(45) UHPLC-UV Chromatogram

(46) 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:

(47) TABLE-US-00003 15 lawsone  1  2 gallic acid lalioside  3  4 myrciaphenone A catechin  5  6 2,3,4,6- 1,2-dihydroxy-4-O- tetrahydroxyacetophenone 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- luteolin tetrahydroxyflavanone 21 22 3′,5,7-trihydroxy-4′- apigenin methylflavone

(48) UV Spectra

(49) 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.

(50) NMR Spectra

(51) Peak 15

(52) The .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of the compound corresponding to peak 15 are displayed in FIGS. 4a, 4b, 4c, 4d and 4e.

(53) Said compound has been identified as lawsone.

(54) Indeed, the following attribution can be made with respect to the .sup.1H and .sup.13C NMR spectra:

(55) TABLE-US-00004 0 .sup.1H-NMR .sup.13C-NMR Position (500 MHz, CDCl.sub.3) (125 MHz, CDCl.sub.3)  1 — 181.74  2 11.7, se 160.02  3 5.8, s 111.49  4 — 181.74  5 8.01, d, 7.1 126.45  6 7.83, m 134.98  7 7.83, m 133.79  8 7.95, d, 7.2 125.95  9 — 131.10  10 — 132.38

(56) HRMS (ESI−) calcd for C10H5O3 [M−H]−: 173.0239, found: 173.0244 (0.5 mDa/2.9 ppm)

(57) HRMS (ESI+) calcd for C.sub.10H703 [M+H]+: 175.0395, found: 175.0405 (1.0 mDa/5.7 ppm)

(58) Peak 20

(59) The .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of the compound corresponding to peak 15 are displayed in FIGS. 5a, 5b, 5c, 5d and 5e.

(60) Said compound has been identified as luteolin.

(61) Indeed, the following attribution can be made with respect to the .sup.1H and .sup.13C NMR spectra:

(62) TABLE-US-00005 .sup.1H-NMR .sup.13C-NMR Position (500 MHz, CDCl.sub.3) (125 MHz, CDCl.sub.3)  1 — —  2 — 161.93  3 6.68, s 103.32  4 — 182.12  5 — 104.15  6 12.99 se 164.34  7 6.19, s  99.28  8 10.86, se 164.58  9 6.45, s  94.29 10 — 157.74  1′ — 121.95  2′ 7.40, d, 5 133.82  3′ 9.42, se 146.19  4′ 9.44, se 150.15  5′ 6.89, d, 9 116.47  6′ 7.43, d, 3 118.57

(63) HRMS (ESI−) calcd for C15H9O6 [M−H]−: 285.0399, found: 285.0398 (0.1 mDa/0.4 ppm)

(64) HRMS (ESI+) calcd for C15H11O6 [M+H]+: 287.0556, found: 287.0560 (0.4 mDa/1.4 ppm)

(65) Peak 22

(66) The .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of the compound corresponding to peak 15 are displayed in FIGS. 6a, 6b, 6c, 6d and 6e.

(67) Said compound has been identified as apigenin.

(68) Indeed, the following attribution can be made with respect to the .sup.1H and .sup.13C spectra:

(69) TABLE-US-00006 .sup.1H-NMR .sup.13C-NMR Position (500 MHz, CDCl.sub.3) (125 MHz, CDCl.sub.3)  1 — —  2 — 161.82  3 6.75, s 103.25  4 — 182     5 — 103.25  6 12.95 se 164.02  7 6.17, s  99.52  8 — 164.17  9 6.46, s  94.63 10 — —  1′ — 121.65  2′ 7.92, d, 9 128.91  3′ 6.93, d, 9 116.57  4′ — 157.90  5′ 6.93, d, 9 116.57  6′ 7.92, d, 9 128.91

(70) HRMS (ESI−) calcd for C15H9O5 [M−H]−: 269.045, found: 269.0462 (1.2 mDa/4.5 ppm)

(71) HRMS (ESI+) calcd for C15H11O5 [M+H]+: 271.0606, found: 271.0608 (0.2 mDa/0.7 ppm)

(72) Peak 10

(73) The .sup.1H, .sup.13C, COSY, HSQC and HMBC NMR spectra of the compound corresponding to peak 15 are displayed in FIGS. 7a, 7b, 7c, 7d and 7e.

(74) Said compound has been identified as para-coumaric acid.

(75) Indeed, the following attribution can be made with respect to the .sup.1H and .sup.13C spectra:

(76) TABLE-US-00007 .sup.1H-NMR .sup.13C-NMR Position (500 MHz, CDCl.sub.3) (125 MHz, CDCl.sub.3) 1 — 166.28 2 6.29, d, 16 113.64 3 7.48, s 142.51 4 — 123.58 5 7.52, d, 8.6 128.43 6 6.75, 8.6 114.06 7 9.96, se 157.92 8 6.75, d, 8.6 114.06 9 7.52, d, 8.6 128.43

(77) HRMS (ESI−) calcd for C9H7O3 [M−H]−: 163.0395, found: 163.0403 (0.8 mDa/4.9 ppm)

(78) B) Quantitative Analysis

(79) Material and Methods

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

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

Experimental Conditions

(82) 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:

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

(84) Results

(85) TABLE-US-00008 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

(86) TABLE-US-00009 Mean content (wt. %) p-coumaric Extract luteolin apigenin acid LP110 0.18% 0.03% 0.04% ES3310 0.38% 0.06% 0.04% JQ137A 0.19% 0.03% 0.03%

III. Assay Methods

(87) Method 1: Lawsone Assay by HPLC

(88) 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.

(89) Reagents

(90) Lawsone >97% (HPLC) SIGMA-ref: H46805

(91) Dichloromethane for analyses.

(92) Sulfuric acid for analyses.

(93) Methanol for analyses.

(94) HPLC-grade water.

(95) HPLC-grade acetonitrile.

(96) HPLC-grade trifluoroacetic acid.

(97) 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: X=278 nm. Flow rate: 1 mL/min Injection: 10 μL.

(98) Sample Preparation:

(99) For whole or roughly crushed leaves:

(100) 50 g of leaves are crushed then sieved through a 0.355 μm sieve.

(101) For leaf powders:

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

(103) Preparation of the Solutions

(104) Control Solutions:

(105) 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.

(106) Test Solutions: Test solution A (assay of the lawsone present in an extract)

(107) Dissolve 50 mg of extract in 100 mL of 1/1 methanol/water.

(108) Dissolve with ultrasound.

(109) Filtration on Acrodisc GF GHP.

(110) Inject 10 L. Test solution B (assay of total lawsone)

(111) Introduce 80 mg of leaf powder into a volumetric flask.

(112) Add 50 mL of 2N H.sub.2SO.sub.4.

(113) Heat to 97° C. for 30 min.

(114) Let cool.

(115) Add methanol qs 100 mL.

(116) Filter the solution on Acrodisc GF GHP 0.45 μm.

(117) Inject 10 μL of the filtrate. Test solution C (assay of the lawsone formed by enzymes)

(118) Introduce 80 mg of leaf powder into a volumetric flask.

(119) Add into 50 mL of demineralized water.

(120) Place in an ultrasound bath for 30 min between 30 and 40° C. Let cool.

(121) Add methanol qs 100 mL.

(122) Filter the solution on Acrodisc GF GHP 0.45 μm.

(123) Inject 10 μL of the filtrate.

(124) Results

(125) 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.

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

(127) 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.

(128) Assay of Total Proteins and Amino Acids

(129) Principle

(130) 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.

(131) Reagents

(132) Citrate Buffer (pH=5)

(133) 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.

(134) Ninhydrin Reagent:

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

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

(137) Mix the two solutions.

(138) 6N Hydrochloric Acid

(139) Dilute to ½ of concentrated hydrochloric acid (36%).

(140) Diluent

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

(142) Preparation of the Solutions

(143) Preparation of the Calibration Range

(144) Dissolve 17 mg of asparagine in 100 mL of water.

(145) Preparation of the Test Solutions

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

(147) Hermetically seal then place for around 16 hours at 110° C. Neutralize with 3N sodium hydroxide (methyl red changes color) then adjust to 20 ml with water.

(148) Assay

(149) TABLE-US-00010 T 0.1 T 0.2 T 0.5 Test Blank Control solution 0.1 0.2 0.5 — — (mL) Test solution (mL) — — — 0.2 — Water (mL) 1 1 1 1 1 Ninhydrin reagent 1 1 1 1 1 (mL)

(150) Stir and place in a water bath at 100° C. for 20 minutes.

(151) Cool in an ice bath.

(152) Adjust to 10 ml with diluent.

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

(154) Calculations

(155) Construct the calibration curve.

(156) Deduce from it the total amino acid concentration (Q.sub.AAT), expressed in asparagine, in the test solutions.

(157) The total amino acid content (T.sub.AAT) of the extract is given by the following formula:

(158) $T_{AAT}\left(\%\right)=\frac{Q_{\mathrm{AAT}}\times 100\times 20}{{\mathrm{pe}}_1}$
with: Q.sub.AAT in mg/ml pe.sub.1 in mg

(159) Method 3: Weight Assay of Chlorophylls

(160) 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 V of methanol. After stirring for 15 min, the solution is filtered. The supernatant is dried and constitutes the fraction containing chlorophylls.