Dhiman A.

et al: Determination of Lawsone Content In Leaves Of Lawsonia inermis Linn

Journal of Pharmaceutical and Scientific Innovation

www.jpsionline.com
Research Article

DETERMINATION OF LAWSONE CONTENT IN FRESH AND DRIED LEAVES OF

LAWSONIA INERMIS LINN. AND ITS QUANTITATIVE ANALYSIS BY HPTLC
Dhiman Anju1*, Sharma Kavita2, Goyal Jugnu2, Garg Munish3, Sharma Asha4
1
Assistant Professor, Department of Pharmaceutical Sciences, M. D. University, Rohtak-124001. Haryana. India.
2
Research Scholar, Department of Pharmaceutical Sciences, M. D. University, Rohtak-124001. Haryana. India.
3
Associate Professor, Department of Pharmaceutical Sciences, M. D. University, Rohtak-124001. Haryana. India.
4
Assistant Professor, Department of Botany, M. D. University, Rohtak-124001. Haryana. India.
*Email: ad_mdu@rediffmail.com

Received on: 02/03/12 Revised on: 12/04/12 Accepted on: 17/04/12

ABSTRACT
The present study was carried out to determine the content of lawsone in fresh and dried leaves of L.inermis Linn. The measurement containing high lawsone
content was proceeded for further investigation by making hydro-alcoholic extract of the chosen plant part and its phytochemical analysis was also performed.
The test extract was further screened for quantitative estimation of lawsone content using high performance thin layer chromatography. The chromatograms
were developed using toluene: ethyl acetate: formic acid (5.5: 4.0: 0.5) as solvent system. Dried leaves of L.inermis was found to be a better source of lawsone
as compared to the fresh leaves. The fresh leaves of L. inermis had 5.3 mcg/ml lawsone content while in case of dried leaves, 6.9 mcg/ml lawsone content was
observed. The chromatogram of hydroalcoholic extract of leaves of L. inermis showed the presence of 4.56 mcg/ml lawsone.
KEYWORDS: Chromatography, extract, hydroalcoholic, lawsone, L. inermis Linn., quantitative analysis.

INTRODUCTION Therefore, it was considered that most of the active

Lawsonia inermis Linn (Lythraceae), most commonly known components including lawsone has been extracted out in the
as ‘henna’ invites attention of the investigators worldwide for hydroalcoholic extract. The test extract was further screened
its pharmacological profile ranging from anti-inflammatory for quantitative estimation of lawsone content using high
to anti-cancer activities.1 L. inermis is a much branched shrub performance thin layer chromatography.
that grows mainly in middle east of Africa.2 The leaves of MATERIALS AND METHODS
L.inermis having the highest yield of lawsone are confined to Synthetic lawsone was purchased from Yucca enterprises,
India, mainly in Punjab and Gujrat and to a small extent in Mumbai, India. All the chemicals used were of analytical
Rajasthan and Madhya Pradesh.3 The principal colouring grade.
substance of henna is a red-orange colored molecule Collection and authentication of plant material
(lawsone, 2-hydroxy-1, 4 napthaquinone) having molecular Fresh leaves of L.inermis were collected from botanical
formula, C10H6O3 and melting point of 190ºC, present in garden of Maharshi Dayanand University, Rohtak and
dried leaves in a concentration of 1-1.4% w/w.4,5,6 Lawsone is authenticated by Dr. H. B. Singh, Scientist F & Head, Raw
chiefly responsible for the colorant property of henna leaves.3 Materials Herbarium & Museum, National Institute of
Lawsone is available as yellow to mustard coloured powder Science Communication and Information Resources
which is practically insoluble in water at 0.2%, soluble in (NISCAIR) New Delhi, India (voucher specimen no:
95% ethanol at 0.5%, soluble in methanol at 1%. Lawsone is NISCAIR/RHMD/Consult/-2011-12/1990/290).
proposed to be used as a non-oxidising hair colouring agent
at a maximum concentration of 1.5% (typical concentration
1.26%) in the finished cosmetic product.7 The process which
is mainly used for the extraction of lawsone from leaves of
henna has been patented. Upadhyay et al in 2010, confirmed
that the quantitative estimation of leaves of L.inermis
collected in different seasons showed variations in the active
ingredient (lawsone). The leaves obtained in march season
contain less lawsone content in comparison to october –
november season leaves of L.inermis.8
Henna is not a sacred plant as such, but it is supposed to
symbolize prosperity, fertility and happiness. It is widely
used in a variety of religious and social ceremonies in India.9
The leaves of L.inermis have bitter bad taste and used in
vulnerary, diuretic, headache, hemicranias, lumbago,
bronchitis, boils, ophthalmia, syphilitis, sores, amenorrhoea,
scabies and spleen diseases and also favours hair growth.2 In
the present study, the content of lawsone in fresh and dried
leaves of L.inermis was detected and the measurement Figure 1. Fresh leaves of L. Inermis
containing high content of lawsone was proceeded for further
investigation by making hydro-alcoholic extract of the chosen
plant part since hydroalcoholic extract is known to haul out a
variety of chemical constituents (both polar and non-polar).

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 Dhiman A. et al: Determination of Lawsone Content In Leaves Of Lawsonia inermis Linn

concentration range of 10-200 mcg/ml. The sample solution

of hydro alcoholic extract of L.inermis was prepared at a
concentration of 10 mg/ml.
RESULTS AND DISCUSSION
As per the quantitative analysis of lawsone in fresh and dried
leaves of L.inermis, it was found that dried leaves contained
more lawsone as compared to fresh leaves. The absorbance of
fresh and dried leaves was found to be 0.549 and 0.688
respectively. From the standard plot of lawsone (Figure 3), it
was determined that dried leaves of L.inermis had more
lawsone as compared to the fresh leaves.
y = 0.0095x + 0.0635
R² = 0.9906
2.5

Figure 2. Dried leaves of L. inermis 2

Quantitative estimation of lawsone in fresh and dried
leaves of L.inermis 1.5
Quantitative estimation of lawsone in fresh and dried leaves
was carried out using spectrophotometric method in which
1
100 mg powdered leaves of L. inermis were soaked in 10 ml
methanol for 2 h. Thereafter, the mixture was centrifuged at
)
(m
ce
ran
so
b
A

5000 rpm for 20 min and the clear supernatant was separated 0.5
out into a test tube. The absorbance of supernatant was read
out at 452 nm. A calibration curve was prepared by plotting 0
the concentration versus absorbance at the same wavelength 0 50 100 150 200 250
in the concentration range 10-200 µg/ml of pure lawsone. Concentration(mcg/ml)
The lawsone content in fresh and dried leaves was then
Figure 4. Standard plot of lawsone by UV
calculated in µg/ml. All the observations were taken in
triplicate.
O
The naphthaquinone fraction of hydroalcoholic extract of the
leaves of L. inermis was characterized using HPTLC. The
OH
chromatograms were developed using toluene: ethyl acetate:
formic acid (5.5: 4.0: 0.5) as solvent system at 254 nm and
366 nm and were compared with the chromatograms
developed by pure lawsone. The chromatogram of
hydroalcoholic extract of leaves of L. inermis showed the
presence of 4.56 mcg/ml lawsone.
O
Figure 3. Structure of lawsone
Processing and preparation of hydro alcoholic extract of 6000 y = 24.053x + 466.29
dried L. inermis leaves R² = 0.9959
The leaves of L.inermis were shade dried as specified in 5000
WHO and dried leaves were grounded using pestle and
mortar and carefully packed in sterile polythene bags,
4000
weighed and stored under room temperature until used. The
powdered material was extracted with 50% ethanol using
soxhlet apparatus. After the extraction was complete, solvent 3000
was evaporated under reduced pressure in a rotary vacuum
evaporator to give a dark brown viscous mass. 2000
Quantification of lawsone in hydroalcholic extract of
cv
d
n
au
re
A

L.inermis using high performance thin layer 1000

chromatography (HPTLC)
The hydro alcoholic extract of L.inermis was subjected to 0
HPTLC analysis using HPTLC LINOMET 5 applicator and
0 50 100 150 200 250
CAMAG 3 scanner with the aid of wincat software for
characterization. The chromatograms were developed using Concentration(mcg/ml)
solvent system v.i.z. toluene: ethyl acetate: formic acid (5.5:
4.0: 0.5).10, 11
Preparation of standard and sample Figure 4. Standard plot of lawsone by HPTLC
For standard solutions, 10mg of pure lawsone was dissolved
in 10ml methanol. The solution was diluted in a

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 Dhiman A. et al: Determination of Lawsone Content In Leaves Of Lawsonia inermis Linn

Figure 5(a). Chromatogram of extract of leaves of Figure 5(b). Chromatogram of pure lawsone
L. Inermis (showing the presence of lawsone)

10mcg/ml Lawsone

20 mcg/ml Lawsone

50 mcg/ml Lawsone

100 mcg/ml
Lawsone

200 mcg/ml Lawsone

2 mcL of 1
mg/10ml extract

3 mcL of 1 mg/10
ml extract

Figure 6. Chromatogram of pure lawsone and hydroalcoholic extract of L. inermis at 254 nm.

Lawsone, the main component of L.inermis, is not only a
colouring agent but also possess diverse assortment of
activities. In the present study, quantitative analysis of
lawsone was carried out using fresh and dried leaves of
L.inermis. Before going for the extraction of leaves of
L.inermis, there was bewilderment whether to use fresh or
dried leaves. To some extent, this perplexity can be cleared
with the help of spectrophotometric analysis. The value for
the concentration of lawsone (in mcg/ml) in fresh and dried
leaves was calculated and found to be 5.3 mcg/ml and 6.9
mcg/ml for fresh and dried leaves respectively. On the basis
of these findings, the dried leaves had more lawsone content
as compared to fresh leaves. Further we also prepared the
hydro alcoholic extract of leaves of L.inermis and
phytochemical investigation of prepared extract showed the
presence of carbohydrates, gums and mucilage, tannins and
phenolic compounds, flavonoids, steroids and sterols. The
hydroalcoholic extract of dried leaves of L.inermis was
screened for quantitative analysis using HPTLC and its
content was found to be 4.56 mcg/ml.

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 Dhiman A. et al: Determination of Lawsone Content In Leaves Of Lawsonia inermis Linn
CONCLUSION
Henna finds applications in the treatment of a variety of
disorders like vulnerary, diuretic, headache, hemicranias,
lumbago, bronchitis, boils, ophthalmia, syphilitis, sores,
amenorrhoea, scabies and spleen diseases. The key role
behind its therapeutic efficacy is being played by its active
constituent, lawsone, which can be further explored by
incorporating this compound into novel dosage forms. The
studies are further going on to make lawsone an efficient and
potent drug molecule by complexing it with suitable legands.
Therefore, efforts must be put forward to identify novel,
natural and safe legands that may positively interact and
synergistically affect the therapeutic efficacy of lawsone.
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