wound healing properties of henna leaves - nopr:...
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Wound healing properties of Henna leavesD M Sakarkar1*, U M Sakarkar, V N Shrikhande1, J v Vyas1, S Mandavgade1, S BJaiswaP and R N Purohit
IVidyabharatiCollege of Pharmacy, Camp, Amravati - 444 602, Maharashtra
2ChaitanyaAyurvedicMedical College, Sakegaon, Post. Bhusawal, Dist.Jalgaon, Maharashtra3Gurunanak Technical Institute, Bezonbagh, Kadbi Chowk, Nagpur, Maharashtra
4AnuradhaCollege of Pharmacy, Chikhali, Dist. Buldhana, Maharashtra
*Correspondent author, Sakarkar Building, Namuna Lane - 2, Amravati-444 601, MaharashtraE-mail: [email protected]
Abstract
Wound healing potential of different extracts of henna leaves i.e.Lawsonia inermis Linn. was evaluated on the rat excision and incision wound
models. Lawsone isolated from the leaves was also screened for same
pharmacological activity.It was observed that the oral administration as well astopical application of ethanol extract of henna leaves and lawsone exhibited
significanthealing response in both the wound models. Further, it was found that
the topical application of ethanol extract as well as isolated lawsone was more
'effectivethan the same givenbythe oral route. Thus, topical application of ethanolextract can be successfully formulated for the wound healing activity.
Keywords: Lawsonia inermis, Mehendi, Henna leaves, Lawsone, Ethanolextract, Wound healing.
IPC Code; Int.cl,7 -A61P 17/02, C09B61/00
Experimental Work
Henna leaves were collected
from Chikhaldara forest area in Amravati
district, during September 2002 and were
authenticated at Chaitanya Ayurvedic
Medical College, Sakegaon, Bhusawal.
Henna leaves
The present study was aimed·.to
investigate the possible effect of differentextracts of the leaves of henna and its
formulations. The effectof the extracts on
wound repair was also investigated after
administering these extracts through oral
and topical routes. Thephytochemical and
pharm~cological action of the plant drug
through transdermal drug deliverysystem
on wound healing properties in rats was
also investigated.
Henna powder
Henna is reported to contain a
naphthaquinone, lawsone, which is a
natural dye and mainly responsible for
colouring the hair and skin of handsand feet. Lawsone has also been reportedto be an immunostimulant6• One of the
main constituents of henna leaves is
tlavonoids7,8.
Wound may be defined as a loss
or breaking of cellular and anatomic orfunctional continuity of living tissues. In
the traditional systems of medicine,various plants have been used to promote
wound healing1,2. The leaves ofLawsonia inermis Linn. syn. L. albaLam. (Family - Lythraceae),commonly called as henna (Hindi Mehendi) are used in the form of adecoction or ointment in the treatment of
burns, skin inflammations, wounds and
ulcers3, 4. The leaves also possess
antifungal and antibacterial activitiess.
Introduction
o ,406 Natural Product Radiance Vol 3(6) November-December 2004
Successive solvent
extraction - Air dried leaves (750g)
of henna were reduced to a fine powder,
which was subjected to hot continuous
extraction in a Soxhlet extractor,
successively with petroleum ether(40-60°C), chloroform and ethanol
(95%). Finallythe powdered material wasmacerated with chloroform water for 24
hours to obtain the aqueous extract. Each
time before extracting with next solvent,the powdered material was dried iIi' hotair oven below 50°C. Each extract was
then concentrated by distilling off the
solventfollowed byevaporation to dryness
on water bath. All extracts were kept in
desiccator and stored in refrigerator for
phytochemical and pharmacological
studies. Each extract was subjected to
qualitative chemical investigation for the
identification of phytoconstituents such as
sterols, glycosides, saponins,
carbohydrates, alkaloids, flavonoids,
tannins and proteins.
Isolation of lawsone9
About 75g of crushed fresh henna leaves
were extracted by agitation for 2 hourswith 200 ml of 20% sodium bicarbonate
solution. The extract was filtered, marc
Petroleum ether (40-60°) extract
Chloroform extract
Ethanol extract
Aqueous extract
Lawsone
was again extracted with 100 ml of samesolution for 1 hour, filtered and the
alkaline extracts were pooled together.The extract was acidified with dilute
sulphuric acid and crude productobtained on standing was reextracted with
sufficient quantity of ammonium
hydroxide and again acidified with dilute
hydrochloric acid. Theproduct was finally
extracted with two successive quantitiesof benzene (40 ml) and filtered. The
filtrate was distilled to yield yellowish
brown coloured crystals of lawsone
(mp192-193°C). Further it was
characterized by UV and IR spectralanalysis.
Preparation of ointments-The simpleointment base10 was chosen
as a vehicle for the topical application of,the extract and lawsone. Lawsone
(lOOmg) were mixed homogeneouslywith simple ointment base USP (1OOg)togetlawsoneointment (0.1% w/w). Ethanol
extract ointment (30% w/w) waspreparedby incorporating 30g of dried, powdered
ethanol extract in 100g of simple ointmentbase USPfor topical use.
,Pharmacologicalscreening- Mice of either sex of 90-days age,
Table 1: Acute toxicity studies
LD,.. (mWkg)
2200±101
1995±66
2200±101
2600±139
500
weighing 20-25g were used for acute
toxicity studies. Wistar rats of either sex
weighing 150-200g were selected for the
pharmacological study. Animal EthicsCommittee of the Institution (CPCSEA
Registration No.751) approved the studyprotocols.
Acute toxicity study - The
method suggested by Miller and Tainter(1944) 11 was used for determination of
lethal dose (LD50)' Gum acacia (1%) wasused as a vehicle to suspend the various
extracts and the suspension was
administered orally.One tenth of the LD50
was used as the maximum dose to test the
pharmacological effects possessed by theextractl2, 13 shown in Table 1.
Wound healing studies Wistar rats were divided into nine groups
consisting six in each and were labeled
alphabetically from A to I. Animals weredepilated at the desired site before
wounding. Theywere housed individually
with food and water given ad libitum,the basal food intake and body weights to
the nearest gram were noted. The animals
were starved for 12 hours prior to study.Excision wound - It was
inflicted on rats and the procedure was
220±10
199.5±6.6
220±10
26o±14
50
Natural Product Radiance Vol 3(6) November-December 2004 407-
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Results and Discussion
ethanol extract and lawsone, respectively,
applied topically once a day.
Statistical analysis -All the
results were analyzed by Student's t-test
and were expressed as mean ± standarderror of mean values. The level of
significance was set at p<O.OS.
The breaking strength in incision
wound model was evaluatedbycontinuous
constant water flow technique. All five
extracts showed increased mean breaking
strength, compared to control
(125.0± 12.1 g), when given orally once
a day. The maximum breaking strength
was seen in groups treated with lawsone
(216.0±12.5 g) and ethanol extract
(213.0±12.63 g), which were statistically
The average per cent yield of significant (p<O.OS) from control. Thepetroleum ether, chloroform, ethanol and results are shown in Table 3.
aqueous extracts were 3.93, 1.47, 31.0 The results of pharmacological
and S.31 w/w, respectively. Flavonoids, screening indicated that ethanol extract
lawsone, tannins were found to be present and lawsone have greatly contributed
in ethanol extract ind steroids, saponins towards wound healing activity(p<0.05),
in various other extracts as observed by when given orally. Therefore, in view ofthe qualitative chemical tests. The ease of treatment and comparing the
assessment of the wound healing activity efficacyof oral and topical administration,of henna, rat excision and incision wound both these extracts were studied in
mod~ls were selected and per cent wound excision and incision wound models by
closure, time to epithelization, size of scar applying_them topically in the form ofarea and breaking strength ofwound were ointment.
studied. 1\voroutes of drug administration Topical treatment - When
(oral and topical) were compared to compared to control (76.5±1.2%) on the
evaluate efficacyin wound repair. days 12, ethanol extract (98.3±0.SO%)Oral treatment-The results and lawsone (99.2±O.lS%) showed
were compared to control (86.4±1.8%) significant improvement (p<O.OS) in per
on the day 12. Lawsone (9S.40±0.6S%), cent wound closure when applied
petroleum ether extract and aqueous . topically (Table 3). Significant decrease
extract of henna showed significantly in time of epithelization (p<O.OS) was
better (p<O.OS)wound closure. Asimilar observed in groups treated withimprovement in wound closure was seen ethanol extract of henna and lawsone
with ethanol extract (96.3±O.S3%) ointment. The ethanol extract and
(p<O.OS) compared to control group. On lawsone showed complete epithelization
the other hand, the chloroform extract on 16.4±0.4S and 17.8±0.36 days,
(91.0± 1.39%) failed to show any respectively when compared to
significant difference from the control control (22.S±0.60 days) (Table 2).
(86. 4± 1.80%) (Table 2). Xhese' results Thissignifiesbetter wound healing activity.indicate that ethanol extract and lawsone The least scar areas were observed for
showed complete epithelization in ethanol extract (12.3±O.66 mm2)
18.8±0.49 and 18.6±0.82 days, followed by lawsone (14.5±1.42 mm2)
respectively (Table 2). which indicated a significant
carried out as per the method described
byMorton and Malone14• Acircular woundof about 2.5cm diameter was made on
depilated ethanol-sterilized dorsal
thoracic region of rats under light ether
anaesthesia and observed throughout the
study.The observations of per cent woundclosure were made on day 4, 8 and 12
post wounding and also for epithelizationperiod and size of scar area.
Resutured incision - The
method suggested by Ehrlich and Hunt15
was adopted. Under light ether
anaesthesia, two paravertebral incisions
of 6 cm were made through the entirethickness of the skin, on either side of thevertebral column of albino rats with the
help of a sharp blade. The incisions were
sutured using· 4-0 size silk threads with
the help of straight round-bodied needle.
On the eighth post-wounding day, sutureswere removed and the breaking strength
was determined on 10th post wounding
day by continuous constant water flow
technique of Lee16,
Drug treatment - Group A,
served as a control group and received
single daily dose of vehicle (gum acacia
1%) orally. Groups B, C, D, E and F weretreated groups and animals in these
groups received a single daily oral dose
of petroleum ether extract (220± lOmg/kg), chloroform extract (199.S±6.6mg/
kg), ethanol extract, (220±10mg/kg)
aqueous extract (260± 14 mg/kg) and
lawsone (SO mglkg), respectively. For
topical application group G, served as a
control group and 100 mg of vehicle
(simple ointment base USP), applied
topically once a day. Group H and Iwere
treated groups and received application
of 100 mg in quantity of the ointment of
Natural Product Radiance Vol 3(6) November-December 2004
L
improvement (p<O.05) when comparedto control (38.0±2.10 mm2). The results
are presented in Table 2.
The maximum means breaking
strength was seen in group treated with
ethanol extract ointment (445.0±7.5 g)
and followed by the group treated with
lawsone ointment (414.0±6.39 g) (Table3). These results substantiate the wound
healing activity (p<O.05) for the ethanolextract of henna, when compared to
control (126.o±6.3g).
The results obtained by oral
administration and topical application of
ethanol extract and lawsone were
compared with each other. The topicalroute of ethanol extract of henna and
lawsone was found to significantly
(p<O.05) promote the wound healing
activity when compared to the ethanol
extract and lawsone given by oral route
(Tables 2 and 3). Even in comparison
between topical application ethanolextract of henna and lawsone in the form
of ointment, the ethanol extract was found
effective(p<O.05) in promotion ofwound
healing than the lawsone ointment. Theresults are summarized in Table 4.
______ n n_ -----------
The phytochemical investigation
of ethanol extract revealed the presenceof both tlavonoids and the lawsone. The
tlavonoids are well-known for their role
in wound healing17• Moreover, there are
the reports that bioflavonoids have
pharmacological activities such asantimicrobiall8 and antioxidant activitiesl9•
Lawsoneis a polyphenolic compound and
is present in ethanol extract. The earlier
reports on such polyphenols revealed
significant healing effect in both wound
models used in this study20.
Table 2 :Effect of the extracts of Lawsonia inermis on the excision wound parameters--GroupCode% Wound contraction onPeriod ofMean size
givenepithelizationof scar
'Day 4DaySDay 12(days) area (mm2).----.-.--
--Oral Treatment
Control
A47.583.486.424.0 31.6±2.10
±1.66±1.86±0.45 ±1.57Petroleum ether
B59.591.695.519.5 . 22.7
(40-60°) extract
±1.20*±1.72*±1.82*±0.65*±1.79*CWorofonn extract
C56.383.391.020.5 25.3
±2.46±2.70±1.39±0.92 ±1.68
Ethanol extract
D61.893.597.818.8 10.5
±2.55*
±2.30*±0.53*±0.49* ±1.98*
Aqueous extract
E54.986.795.120.4 23.8±1.42*
±1.58±1.24*±0.72* ±1.85
Lawsone
F59.491.295.418.6 10.7
±2.57*
±1.48*±0.65*±0.82* ±1.l8*
Topical Treatment Control (simple
G42.775.376.5022.6 38.0
ointment base USP)
±1.24±1.25±1.2±0.50 ±2.81
f Ethanol extractH
41.694.898.3016.4 12.30ointment (30% w/w)±4.77
±0.67*±0.50*±0.45* ±0.66*
Lawsone ointment
I38.496.199.217.8 14.5
(0.1% w/w)
±1.24±0.62*±0.15*±0.36* ±1.42*
I Allvalues are given in Mean±S.E.; *p<0.05 = Significant Vs. Control.Natural Product Radiance Vol 3(6) November-December 2004
Table 3: Effect of the extracts of Lawsonia inermis on
the breaking strength of incision wounds
Code given
Oral Treatment
Control
IA 125.0±12.1
Petroleum ether (40-60°) extract
B196.7±10.7*
CWorofonn extract
C145.1±6.0
Ethanol extract
D213.0±12.6*
Aqueous extract
E178.0±7.2*
Lawsone
F216.0±12.5*
Topical Treatment Control (simple ointment base USP)
G126.0±6.3
Ethanol extract ointment (30% w/w)
H445.0±7.5*
Lawsone ointment (0.1 % w/w)
I414.0±6.2*
All values are given in Mean±S.E.; *p<0.05 = Significant Vs. Control.
Table 4: Comparison between topical application of ethanolextract ointment and Lawsone ointment
-;.;;;:. ..: -. ~ . 0.'·'.·' '~""o··m' ..'w' .."O"" "00'0
Excision Incision--
"A ,- Period ofSize of scar areaBJ;eaking strengthParameters
.~ .(mmZ)(g)on 4
1i
dayl2 (days)
Control (simple
ointment base USP)
77.6±1.1722.6±0.5038.0±2.1126.0±6.3
Ethanol extract ointment (30% w/w)
99.7±0.10*16.4±0.4S*12.3±0.66*444.0±7.5*
Lawsone ointment l 414.0±6:2*(0.1% w/w) 99.4±0.12*17.8±0.36*14.5±1.42*
~
-- - --- -----______ l.-_
All values are given in Mean±S.E.; *p<0.05= Significant Vs. Control.
Natural Product Radiance Vol 3(6) November-December 2004
Ar,;,I.
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Conclusion
The results of the present studyhave led to the conclusion that 4.
transdermal drug delivery systemcontaining ethanol extracts has exhibited
more prominent wound healing activitythan trans dermal drug delivery system
containing other extracts and also withisolated lawsone formulation, when given
by topical route. Similarlyethanol extract
and isolated lawsone were also 5significantly better than other extracts .
when given by oral route. This improvedwound promoting activity of ethanolextract could be attributed to the
additional presence oflawsone along withthe flavonoids that as alluded earlier have
significantwound healing activity.Further 6.in our studies, itwas found that the topicalapplication of ethanol extract as well asisolated lawsone was more effectivethan
the same given by the oral route.
References
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some plant products, J Nat Remed,2002,2, 11.
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Effect of Tridax procumbensextract on wound healing, PlantaMed, 1991, 57, 325.
3. Indian Materia Medica, by KM
Nadkarni (Popular Book Depot,Bombay & Dhootapapeshwar 9.
Prakashan Ltd, Panvel), Lawsoniaalba Lam.,L. spinosa; L. inermis,Linn., 3rd edn, revised and enlarged
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MR and Krishna DR, Bioflavonoids
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of wound healing activity of selected
traditional medicinal plants from
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Applications of Henna in Unani Systemsof Medicine
In Unani practices Henna (Lawsonia inermis Linn.) leaves, flowers and seeds are used in various diseases
except respiratory disorders (due to its adverse effect on lungs). Some of its uses are given below:
1. For the treatment ofleprosy in primary stage supernatant fluid (100 ml) ofleaves (420 g soaked in water over night)
mixed with 35 g sugar is taken daily for 20 days in the. morning.
2. Leaves are gr6und with Sutra nium (Orchis sp.) to make a paste and applied on hair. It gives very dark red
colour and protects hair falling.
3. Paste of leaves is applied on rough hands to make them soft.
4. Paste of leaves prepared in hot water, protects pustule formation of small pox and from skin marks.
5. Leaves alone or with Kishniz (Coriandrum sativum Linn.) are boiled in water and used as medicated footbath
or irrigation on burns and scald wounds and their marks. This preparation is also used for the treatment of gingivi
tis, stomatitis and excessive salivation.
6. Leaves are ground and mixed with rose oil to use locally to cure scabies and psoriasis.
7. Leaves ground with leaves of A rand (Ricinus communis Linn.) are used locally to fill kracked heel.
8. Leaves are used as substitute for Socotra Dragon' Blood Tree (Dracaena cinnabari Balf.f.) as antihaemorrhagic
agent. Leaves (12 g) and Gentian (Gentiana lutea Linn.) (24 g) are ground and mixed with water to make paste
and applied on hand and foot as styptic in excessive bleeding during lochia period.
9. Leaves ground with fat of goat milk are applied on nails as corrective to wounds and deformities of nails.
10. Leaves are ground widl Khall (Vinegar) to make paste and apply on skull as effective remedy for migraine and
epiphora.
12. Leaves (250 g) are boiled in water and supernatant fluid is used orally as effective medicine for jaundice, hepatitis,
nephrolithiasis, urolithiasis, dysurea and dysmenorrhoea. The fluid is also used in tub bath to cure all types of
uterine pain.
13. Leaves (4.5 g) paste mixed with honey is used orally to cure all type of headache and nasal secretion as rhinitis,
epitasis, etc.
14. Flower oil is used locally as complexion enhancer, rubefacient, resolvent for all types of abscess, hair grower and innerves weakness.
15. Seeds (4.5 g) ground and mixed with honey are used orally to give relief in high blood pressure and brain disorders
(Contributed by: Dr. Mohd. Danish Mal1fooz, TKDL,NISCAIR,New Delhi)
Natural Product Radiance Vol 3(6) November-December 2004