Determination of Hair Staining Effect and Lawsone Content from Leaf Extracts of Lawsonia inermis Linn.

Thiri Hlaing, Khin Tar Yar Myint, San San Wai, Khine Sabel Aung, Aye Aye Phyu Mar Mar Myint, Aye Win Oo, Kyi Kyi Oo, Soe Myint Aye

Myanmar Health Sciences Research Journal, 2020; 32(2):159-165

Cosmetic beautification outcomes are associated with medicinal effects. Continuous usage of synthetic dye on natural hair causes many side effects such as skin irritation and erythema. The side effects of the synthetic dye limit its use by health conscious customers throughout the world. Concern for the environment has also created an increasing demand for natural dyes, which are friendlier to the environment than synthetic dyes. Thus, the aim of this study was to determine hair staining effect and Lawsone content from leaf extracts of Lawsonia inermis L. This study was done from January 2017 to December 2017.The dried leaf powder and different extracts were screened for phytochemical constituents. Hair staining effect of the three different extracts was evaluated by in-vitro test with human hair samples. Among them, hair staining effect of defatted 50% ethanolic extract was observed to be semi-permanent and good staining effect (4/5 to 4 experimental grade scales). In rabbit skin irritation study, defatted 50% ethanolic extract had negligible skin irritancy (PII<0.4). Isolated Lawsone compound was identified by UV-Vis spectrophotometry and FT-IR spectrometry.Quantitative determination of lawsone compound in three different extracts was done by TLC scanner 4 (CAMAGE). Lawsone contents in defatted 97% ethanolic extract, defatted methanolic extract and defatted 50% ethanolic extract were 2.2 μg/g, 1.4 μg/g and 0.8 μg/g, respectively. Therefore, the leaf extract of Lawsonia inermis L. can safely be used as an alternative to synthetic hair dye chemical of hair color cosmetics because it gives the beautiful and long-lasting hair color without any skin irritation.

When the plate was viewed, there was a significant spot in each of three different extracts which was the same as standard Lawsone spot in both of Rf value (0.44) and color (orange under visible light and dark green under short wave of UV)(Fig. 2& 3).

Based on the calculation of peak area from TLC scanner,Lawsone contents in defatted 97% ethanolic extract, defatted methanolic extract and defatted 50% ethanolic extractwere 2.2μg/g, 1.4 μg/g and0.8 μg/g,

Fig. 2.Thin layer chromatogram of (1) Lawsone standard (2) defatted 50% ethanolic extract (3) defatted methanolic extract and (4) defatted 97% ethanolic extract detected by visible and UV-254 nm

respectively,with a correlation coefficient (r) of 0.928 and the % recovery was found between 90-110%.The IR spectrum of isolated Lawsone was obtained by KBrpellet preparation.Similarity of relevantwave numbers was bserved with both the

Fig.3. 3D Chromatogram of Lawsone standards (1 μg/ml, 2 μg/mL, 3 μg/ml, 4 μg/ml, 5 μg/ml) and three different extracts by TLC scanner

standard and the reference values of OH stretching of phenolic -OH group (3410.15 cm1), C=O stretching of ketoenol(1680.00 cm-1), aromatic C=C vibrational bands of the naphthalene ring (1606.70 cm-1, 1573.91 cm-1) and aromatic C-O stretching (1211.30 cm-1). Its UV spectrum in methanol was agreed with literature value of 254 nm, 277 nm and 332 nm wavelength absorptions. In addition to spectral data, TLC result confirmed the isolated compound was Lawsone.

Table 1. Grades of color fastness of three different extracts to shampoo washing and sunlight

Experimental color grades are defined as (1) original grey, (2) pale yellow, (3) yellowish brown, (4) golden brown and (5) reddish brown, respectively Data pertaining to fastness properties of defatted 97% ethanolic extract and defatted methanolic extract had experimental coloring grades of 2/3 to 3 and 3/4 to 4, respectively where as they were poor when subjected to six and ten washing with shampoo. The defatted 50% ethanolic extract had the staining effect of 4/5 to 4 experimental grade scales up to 16 times washing with shampoo Table 1.

Lawsonia inermis L. has been used since the Bronze Age to dye skin (including body art), hair, fingernails, leather, silk and wool.3Hair staining power is mainly due to the presence of Lawsone. It belongs to the family Lythraceae and is a dicotyledonous, much branched glabrous herbaceous shrub or small tree (2 to 6 m in height). It is grown in dry tropical and subtropical zones. It is mainly cultivated for cosmetic purposes and as traditional medicine in all over the world. It has been reported to have anti-inflammatory, anti-oxidant, antimicrobial, anti-diabetic, wound healing and anticancer properties.4The standardized natural extracts are very much useful for textiles, food,pharmaceuticals and cosmetics.

Lawsone is the major active principle for coloring and pharmacological use.5Chemically, it is called as 2-hydroxyna-phthalene-1,4-dione (2-hydroxy-1, 4 napthoquinone) having molecular formula C10H6O3.6 Pure Lawsone is an orange powder, insoluble in water with optical absorption maximum of 452 nm.7It is soluble in ethanol, methanol, ethyl glycol and dimethyl formamide.8 A typical content of the leaves is 0.5-1% w/w of Lawsone. The concentration of this natural compound in leaves varies from place to place depending upon many of the environmental factors.9Due to lack of availability of precise technical knowledge on the extracting and dyeing technique, it has not commercially succeeded like the synthetic dyes.Therefore, this research work aimed to determine the hair staining effect and Lawsone content from leaf extracts of Lawsonia inermis L.

Plant collection and botanical identification

The leaf samples of Lawsonia inermis L. were collected from Amarapura Township, Mandalay Regionfrom October to January. The collected fresh specimens with their inflorescences were used for morphological identification. The anatomical and histological characters of the transverse section and epidermal preparation of the leaf and leaf powder were microscopically examined.

Preparation of extract

Dried leaf powder (100g) was defatted with 500 mL of petroleum ether (40-60°C).Then defatted samples were extracted with three different solvents. For 97% ethanol and methanol, extraction was performed in Soxhlet extractor at 60°C for 6 hours,respectively. In case of 50% ethanolic extraction,temperature controlled water bath shaker was used at a constant shaking speed of 50 rpm at 70°C for 8 hours. The acquired crude extracts were concentrated by using rotary evaporator and evaporated on water bath until constant weight was obtained.

Phytochemical analysis

Phytochemical screening of the leaf powder and extracts was performed by the Harbone method.10

Evaluation of hair staining effect (In-vitro studies)

Three different extracts were evaluated for dyeing efficiency in-vitro, on human white hair strands.11, 12

Dyeing of hair fibres

The collected grey human hair samples were thoroughly washed with shampoo and dried at room temperature prior to dyeing. In the present investigation, 0.5 gm of white human hair was kept in the extract solution for 2 hours. The dyed hair sample was washed with tap water; a second coat of dye was applied after 24 hours of first application, kept for 2 hours and then washed with tap water.

Color fastness test

The dyed samples were subjected to color fastness to light and washing. The change in color was perceived and grading was done according to the gray scale rating. Gray scales for alteration of color, consisting of grade 1 to 5 has been defined.

(a)       Test for color fastness to washing

In present investigation, the dyed hair fibres were washed with mild shampoo using tap water for about 32 dayson alternate days.

(b)      Test for color fastness to light

The dyed hair strands were placed on a white paper sheet covered with transparent sheet and then kept in sunlight at noon from 12 to 2 pm (for 2 hours) for 32 days at the interval of 2 days.

Skin irritation test

Draize modified scoring technique was used to evaluate the skin irritation on four adult albino rabbits from the Department of Medical Research (Head Quarter). The dorsal surface (approximately 4 cm2) of the rabbits was cleared, shaved and cleansed with rectified spirit. About 0.5 g of crude dye extract was directly applied to No.3
and No.4 rabbits. Positive control as 0.2% formalin and area of untreated skin as negative control were prepared in the same manner. Skin reaction at the site of application was subjectively assessed and scored according to post-test observation periods.13,14

The Score of Primary Irritation (SPI) was calculated for each rabbit.

Primary Irritation Index (PII) was calculated as the arithmetical means of the SPI values of the tested rabbits.15

Isolation, purification and identification

The defatted 97% ethanolic extract (about 5g) was dissolved in 50 mL toluene and poured into the separatory funnel. Then, 50 mL of 0.2 M sodium hydroxide (NaOH) was added to the solution and shaken for minutes. The aqueous phase was collected and acidified to pH 3 by 0.2 M hydrochloric acid (HCl). The filtrate was then extracted with diethyl ether (3×100 mL). The combined ethereal phases were washed with 30 mL water and dried over anhydrous magnesium sulphate (MgSO4). The ether was removed to leave a reddish brown residue (333mg). The crude Lawsone is purified  by using Preparative Thin Layer Chromatography (PTLC). The product was chromatographed over silica gel 60 F254 TLC plate by using mobile phase of toluene: ethyl acetate: formic acid (18:1:1 v/v/v). The compound from the extract which had the same Rf value of standard Lawsone was scrapped from the plate and dissolved in methanol. Then, it was centrifuged at 200 rpm for 10 minutes, filtered and dried in the air. It was dissolved in methanol and characterized by Ultraviolet-Visible (UV-Vis) spectrometry at wave-length 200-500nm and Fourier Transform Infra-Red (FT-IR) analysis between 4000 and 400 cm-1. Then, the spectrum was recorded and wave numbers of main peaks of isolated compound and reference compound were compared and identified.

Quantitative determination

Lawsone compound was detected at 254 nm and identified by comparison of Rf value and color with standard Lawsone. Quantitative estimation of Lawsone was carried out by the densitometric method using TLC scanner 4. Exactly 100 mg of the extract was dissolved in 1 ml methanol by vortex mixer. Thereafter, the mixture was centrifuged at 5000 rpm for 20 minutes and the clear supernatant was separated and read out at 254 nm. A calibration curve was prepared by plotting the concentration versus absorbance at the same wavelength in the concentration range 1-5 μg/ml of pure Lawsone (Sigma-Aldrich, Japan) applied as 1µL spot.All the observations were taken in triplicate. Amount of Lawsone in each extract was calculated by comparison of peak area measured for the sample to that of the standard. Calculations were carried out with the Microsoft Excel 2010 for Windows software package.

color shades were not obtained using the L. inermis extracts alone but off black to dark brown color (grades 1-3) initially with Indigoferat inctoria faded gradually to medium brown (grades 5 to 6) after 14 to 16 shampoo washes. The study of Vadivel,et al. stated that there was a continuous color decrease in the hair because of the exposure to the sunlight and washing with natural detergent also made a continuous decrease of color pigments in the hair.

In cases of the defatted 97% ethanolic and methanolic extracts’ staining tests, the slight green color may be due to the presence of the chlorophyll in the leaves and retention capacity was lower than the one of the defatted 50% ethanolic extract produced due to the water-soluble tannin and other compounds helping to retain the dyed color as natural mordant. The natural dyes having limited substantively for the fibre, require use of the mordant which enhances the fixation of the natural colorant on the fibre by the formation of the complex with the dye. The result concluded that the Lawsone alone is not responsible for dyeing properties of henna but presence of other compounds (eg. tannin, mucilage, gallic acid, etc.) along with it adds its dye quality.18

The Lawsone compound is a dye which can be seen with the naked eye and can also be viewed under UV light, because it also absorbs UV light. It was observed that the extracts contain Lawsone compound because it has the same retardation factor (Rf) value of 0.44 and color same to the standard Lawsone compound, which was visualized as orange color spot and as dark spot under UV-254 nm (short wavelength).

Isolation of Lawsone was done from defatted 97% ethanolic extract because it has the highest Lawsone content among three different alcoholic extracts. Crude Lawsone (reddish brown residue) showed about more than 4 spots on TLC plate and thus needed to be purified by PTLC to obtain purer compound. Although UV spectrum of Lawsone standard showed peaks at 332, 277 and 254 nm but the highest sensitivity (λmax)was at 254nm and then it was visualized at 254 nm wavelength in accordance with some literatures.19,20 The Rf value of 0.44±0.05 was obtained densitometrically. The difference in Lawsone content is due to the factors such as geographical conditions, environmental factors, time of collection, extraction methods and so on.

Conclusion

Diagnostic characters of both morphological and microscopical investigations may be useful to supplement existing information with regard to the identification and standardization of Lawsonia inermis L., even in the powdered form of the plant drug,to distinguish it from substitutes and adulterants.Results of phytochemical analysis are expected to be part of important herbal pharmacopoeias as a herbal drug. The present study investigated the dye ability and fastness qualities of the extracts on human hair. These studies are useful to provide as natural local available dye. The isolation studies are further going on to make Lawsone an efficient and potent drug molecule by complexing it with suitable legands. Quantitative determination of Lawsone content also suggested that this method is accurate and cost-effective for routine analysis of Lawsone.

The authors declare that they have no competing interests.