Wound healing potential of Tephrosia purpurea (Linn.) Pers. in rats

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Journal of Ethnopharmacology 108 (2006) 204–210

Wound healing potential of Tephrosia purpurea (Linn.) Pers. in rats

Santram Lodhi, Rajesh Singh Pawar, Alok Pal Jain, A.K. Singhai ∗Division of Pharmacognosy and Phytochemistry, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar 470003, MP, India

Received 15 February 2006; received in revised form 21 April 2006; accepted 8 May 2006Available online 23 May 2006

bstract

Tephrosia purpurea is a well-known herb for its hepatoprotective, anticancer, antiulcer, antibacterial and in healing bleeding piles, etc. The presenttudy was aimed for wound healing potential of ethanolic extract of Tephrosia purpurea (aerial part) in the form of simple ointment using three

ypes of wound models in rats as incision wound, excision wound and dead space wound. The results were comparable to standard drug Fluticasoneropionate ointment, in terms of wound contraction, tensile strength, histopathological and biochemical parameters such as hydroxyproline content,rotein level, etc. Histopathological study showed significant (P < 0.05) increase in fibroblast cells, collagen fibres and blood vessels formation.ll parameters were observed significant (P < 0.05) in comparison to control group.2006 Elsevier Ireland Ltd. All rights reserved.

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eywords: Tephrosia purpurea; Wound healing; Incision wound; Excision wou

. Introduction

Tephrosia purpurea (Linn.) Pers. (Leguminosae), commonlynown in Sanskrit as Sharapunkha is a highly branched, sub-rect, herbaceous perennial herb (Chopra et al., 1956). Accord-ng to Ayurveda literature this plant has also given the namef “Sarwa wranvishapaka” which means that it has the prop-rty of healing all types of wounds (Despande et al., 2003). It isn important component of some preparations such as Tephrolind Yakrifit used for liver disorders (Sankaran, 1980; Kumart al., 1997). In Ayurvedic system of medicine various parts ofhis plant are used as remedy for impotency, asthma, diarrhoea,onorrhoea, rheumatism, ulcer and urinary disorders. The plantas been claimed to cure diseases of kidney, liver spleen, heartnd blood (Kirtikar and Basu, 1956; Despande et al., 2003). Theried herb is effective as tonic laxative, diuretics and deobstru-nts. It is also used in the treatment of bronchitis, bilious febrilettack, boils, pimples and bleeding piles. The roots and seeds areeported to have insecticidal and piscicidal properties and alsosed as vermifuge. The roots are also reported to be effective in

eprous wound and their juice, to the eruption on skin. An extractf pods is effective for pain, inflammation and their decoctions used in vomiting (The Wealth of India, 1976). The ethanolic

∗ Corresponding author. Tel.: +91 7582264125.E-mail address: abhay [email protected] (A.K. Singhai).

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378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2006.05.011

ead space wound

xtract of this plant has been reported to have anticancer activ-ty against KB cells in culture (Zafar et al., 2004). The aqueousxtract of seeds has shown significant in vivo hypoglycaemicctivity in diabetic rabbits (Rahman et al., 1985). The ethanolicxtracts of Tephrosia purpurea possessed potential antibacte-ial activity. The flavanoids were found to have antimicrobialctivity (Gokhale and Saraf, 2000). The phytochemical inves-igations on Tephrosia purpurea have revealed the presence oflycosides, rotenoids, isoflavones, flavanones, chalcones, fla-anols, and sterols (Pelter et al., 1981).

In the present study, Tephrosia purpurea was found to beffective in healing external wounds. As this plant has beeneported to have gastric and duodenal ulcer healing activity, itas hypothesized that it should also be able to heal an externalound. We have selected different wound models using albino

ats. Successive solvent extraction of the aerial parts of plantas done and their respective formulations were made. Afterreliminary pharmacological screening appropriate extract waselected and studied for its wound healing property on differentodels.

. Materials and methods

.1. Plant material

Aerial parts of Tephrosia purpurea was collected from Uni-ersity campus Sagar (India) in the month of April–May 2005.

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macology 108 (2006) 204–210 205

he plant was an identified by Dr. A.S. Mishra, Department ofotany, Dr. H.S. Gour Vishwavidyalaya, Sagar (MP). A voucher

pecimen has been deposited in the Department of Botany, Uni-ersity of Sagar (MP). The plant material was dried in shade,owdered and sieved through 40-mesh size and material wastored in well-losed container.

.2. Extraction and preparation of formulation

The powdered aerial parts (100 g) were extracted in soxh-et apparatus for 24 h with ethanol then concentrated and driednder reduced pressure. The extract was weighed and the yieldas 7.22% (w/w). The semisolid mass (brown colour) wasbtained and used as ingredient for 5% ointment preparation.bout 5 g of semisolid extract was incorporated into the 100 gf simple ointment base B.P. (Anonymous, 1953). Simple oint-ent base was used as control group. Extract ointment was used

wice daily to treat different groups of animals.

.3. Animals

Healthy wistar rats of either sex (150–200 g) with no priorrug treatment were used for all the present in vivo studies. Thenimals were fed on a commercial pellet diet (Hindustan Lever,angalore, India), and water ad libitum. The animals were accli-atized to laboratory hygienic conditions for 10 days before

tarting the experiment. Animal study was performed in Divi-ion of pharmacology, Department of Pharmaceutical Sciences,r. H.S. Gour Vishwavidyalaya, Sagar (MP) with due permis-

ion from institutional animal ethical committee (registrationumber 379/01/ab/CPCSEA, India).

.4. Wound healing activity

.4.1. Grouping of animalsFor incision, excision and dead space wound model, 54

nimals of either sex weighed between 150 and 200 g wereivided into three groups in each model consisting of sixnimals as follows: group I – simple ointment base; group II –% ethanolic extract ointment of Tephrosia purpurea and groupII – 0.005% Fluticasone propionate (Glaxo SmithKline, India)intment was used.

.4.2. Excision wound modelAll animals in each group were anaesthetized by the open

ask method with anaesthetic ether before wound creation. An

able 2ffect of ethanolic extract and standard ointment on various wound parametersf incision wound model in rats

roups Hydroxyproline(mg/g tissue)

Protein content(mg/g tissue)

Tensile strength(kg/cm2)

37.45 ± 3.52 53.67 ± 2.58 0.487 ± 0.058I 66.0 ± 6.03a 82.70 ± 3.22b 0.667 ± 0.081c

II 69.92 ± 5.22a1 89.32 ± 4.51b1 0.695 ± 0.025c1

= 6 albino rats per group, tabular value represents mean ± S.D. a,a1P < 0.05;,b1P < 0.05; c,c1P < 0.05 (comparison of I with II and III).

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206 S. Lodhi et al. / Journal of Ethnopharmacology 108 (2006) 204–210

Table 3Effect of ethanolic extract and standard ointment on various wound parameters of dead space wound model in rats

Groups Hydroxyproline (mg/g tissue) Protein content (mg/g tissue) Tensile strength (kg/cm2) Granuloma dry weight (mg)

I 33.70 ± 4.32 26.53 ± 5.12 0.392 ± 0.048 20.71 ± 3.20II 55.21 ± 3.71a 49.70 ± 3.22b 0.665 ± 0.087c 51.66 ± 1.95d

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II 68.34 ± 3.12 52.32 ± 5.36

= 6 albino rats per group, tabular value represents mean ± S.D. a,a1P < 0.05; b,

xcision wound was inflicted by cutting away a 500 mm2 fullhickness of skin from a predetermined area, the wound waseft undressed to the open environment. The standard drug oint-

ent (0.005% Fluticasone propionate), simple ointment base.P. (Anonymous, 1953), Tephrosia purpurea ethanolic extractintment (5%, w/w) were applied topically to the standard group,ontrol group and treated group respectively, till the wound wasompletely healed. In this model wound contraction and woundlosure time was monitored. Wound contraction was measureds percent contraction in each 2 days after wound formation.

rom the healed wound, a specimen sample of tissue was iso-

ated from each rat for histopathological examination (Taranallit al., 2004).

ig. 1. Photomicrograph of rat skin on ninth day after post-wounding with sim-le ointment base treatment in daily showing less blood vessels formation andoor collagen fibres in incision method. Hematoxylin and eosin, ×100.

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0.742 ± 0.11 57.12 ± 4.17

0.05; c,c1P < 0.05; d,d1P < 0.05 (comparison of I with II and III).

.4.3. Incision wound modelAll animals were anaesthetized before wound creation and

wo paravertebral long incisions were made through the skin athe distance of about 1.5 cm from midline on each side of theepilated back of rat. No local or systemic antimicrobials weresed throughout the experiment. All groups were treated sames in excision model, the both edges kept together and stitchedith black silk surgical thread (no. 000) and a curved needle

no. 11) was used for stitching. The continuous threads on bothound edges were tightened for good closure of the wound.

fter stitching, wound was left undressed then simple ointmentase, extract ointment and standard ointment were applied dailyp to 9 days; when wounds were cured thoroughly the sutures

ig. 2. Photomicrograph of rat skin on ninth day after post-wounding with sim-le ointment base treatment in daily showing less blood vesseles (Bv) formationnd poor collagen fibres (C) in dead space method. Hematoxylin and eosin,100.

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S. Lodhi et al. / Journal of Ethn

ere removed on the day 9 and tensile strength of cured woundkin was measured using tensiometer (Hemalata et al., 2001).

.4.4. Dead space wound modelThis model was used for the study of granuloma tissue. Ani-

als were anaesthetized by light ether and wound was madey implantation of two polypropylene tubes (2.0 × 0.5), one onither side, in the lumber region on the dorsal surface in each ani-al. On the ninth post-wounding day, granuloma tissue formed

n an implanted tube was dissected out carefully. Granulomaissue from one tube was dried (60 ◦C) and stored in 10% forma-in for the biochemical parameters and histopathological study,hile the other part of granuloma tissue was used for determi-ation of tensile strength (Shirwaikar et al., 2003; Patil et al.,001).

.5. Wound healing evaluation parameters

.5.1. Wound contraction and epithelialization time

An excision wound margin was traced after wound creation

y using transparent paper and area measured by graph paper.ound contraction was measured in each 2 days interval, until

omplete wound healing and expressed in percentage of healed

ig. 3. Photomicrograph of rat skin on ninth day after post-wounding, whicheceived daily topical application of 5% ethanolic extract ointment. Figure showsncreased collagen fibres (C), fibroblasts cells (F) and new blood vesicles for-

ation (Bv) in incision method. Hematoxylin and eosin, ×100.

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macology 108 (2006) 204–210 207

ound area. The epithelialization time was measured from initialay (Rashed et al., 2003).

.5.2. Measurement of tensile strengthTensile strength is the resistance to breaking under tension. It

ndicates how much the repaired tissue resists to breaking underension and may indicate in part the quality of repaired tissue.utures were removed on the day 9 after wound creation and

he tensile strength was measured. For this purpose, the newlyormed tissue including scar was excised and tensile strengthas measured with the help of tensiometer, which is based onethod of Kuwano (Kuwano et al., 1994). In this method wound

reaking strength was measured as the weight of water at theime of wound breaking per area of the specimen.

.5.3. Histopathological studiesWound tissue specimens from control, test and standard

roups were taken after complete healing of incision and

ections were cut and stained with haematoxylin and eosinMcManus and Mowry, 1965). Sections were qualitativelyssessed under the light microscope and observed in respect of

ig. 4. Photomicrograph of rat skin on ninth day after post-wounding, whicheceived daily standard ointment. Figure shows increased collagen fibres (C),broblasts cells (F) and new blood vesicles formation (Bv) in incision method.ematoxylin and eosin, ×100.

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broblast proliferation, collagen formation, angiogenesis andpithelialization.

.5.4. Hydroxyproline estimationTissues were dried in a hot air oven at 60–70 ◦C to constant

eight and were hydrolysed in 6N HCl at 130 ◦C for 4 h in sealedubes. The hydrolysate was neutralised to pH 7.0 and was sub-ected to Chloramine-T oxidation for 20 min. The reaction waserminated by addition of 0.4 M perchloric acid and color waseveloped with the help of Ehrlich reagent at 60 ◦C (Woessner,961) and measured at 557 nm using a spectrophotometer.

.5.5. Protein estimation and granuloma weightFrom the collected tissue, protein estimation was done

Lowry et al., 1951). On day 9, skin sample of dead space woundas taken and granuloma mass was dried and weighed.

.6. Statistical analysis

Treated group was compared with the control group. Theesults were analyzed statistically using Student’s t-test to iden-ify the differences between the treated and control. The dataere considered significant at P < 0.05.

ig. 5. Photomicrograph of rat skin on ninth day after post-wounding, whicheceived daily topical application of 5% ethanolic extract ointment. Figure showsncreased collagen fibres (C), fibroblasts cells (F) and new blood vesseles for-

ation (Bv) in dead space method. Hematoxylin and eosin, ×100.

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macology 108 (2006) 204–210

. Results

.1. Wound contraction

The percentage wound contraction was determined using theollowing formula:

ercent wound contraction = Healed area

Total wound area× 100

Wound area was measured by tracing the wound marginsing a transparent paper in each 2 days interval and healedrea calculated by subtracting from the original wound area.n day 4, the wound contraction of standard and extract oint-ent treated groups was found to be significant (P < 0.05) in

omparison to simple ointment base treated group. On day 16,tandard ointment treated wound was completely healed whilextract ointment treated group was also almost at complete heal-ng stage. On day 18, extract ointment treated group healed 100%nd simple ointment base treated group showed 95.71% healing.

t was also observed that epithelialization period of treated andtandard group were less in comparison to simple ointment basereated group (Table 1).

ig. 6. Photomicrograph of rat skin on ninth day after post-wounding, whicheceived daily standard ointment. Figure shows increased collagen fibres (C),broblasts cells (F) and new blood vesicles formation (Bv) in dead space method.ematoxylin and eosin, ×100.

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.2. Tensile strength of excision and dead space wound

Tensile strength for the treated group on day 20 was foundo be significant (P < 0.05) than control group as shown inables 2 and 3.

.3. Histopathological study

.3.1. Healing of incision and dead space control groupsIn control wistar rats incision and dead space type of wounds

hown incomplete healing as in poor collagenation (C), fibrob-asts cells (F) and angiogenesis (Bv) in Figs. 1 and 2.

.3.2. Effect of ethanolic extract of Tephrosia purpurea and

.005% Fluticasone propionate ointmentIn wistar rats incision and dead space type of wounds shown

ignificant healing as in epithelialization, collagenation (C),broblasts cells (F) and angiogenesis (Bv) in Figs. 3–6.

.4. Hydroxyproline, protein estimation and granulomaeight

Treated group showed significant increased protein level andydroxyproline level when compared to control group (P < 0.05)n Tables 2 and 3. Granuloma weight of treated animal groupsas found to be increased when compared with control group.

. Discussions and conclusion

Wound healing process consists of different phases such asranulation, collagenation, collagen maturation and scar matu-ation which are concurrent but independent to each other. Hencen this study three different models were used to assess the effectf herbal ointment on various phases.

The result showed that ethanolic extract ointment possessesdefinite prohealing action. This was demonstrated by a sig-

ificant increase in the rate of wound contraction and bynhanced epithelialization. Significant increase (P < 0.05) in ten-ile strength, hydroxyproline content and collagen levels werebserved, which was further supported by histopathologicaltudies and gain in granuloma breaking strength. This indicatedmproved collagen maturation by increased cross-linking whilen increase in dry granuloma weight indicated higher proteinontent.

Phytochemical work concluded that ethanolic extract ofephrosia purpurea contains flavonoids (Gokhale and Saraf,000), which have been documented to have antibacterial activ-ty (Rao and Raju, 1978), potent antioxidant and free radicalcavenging effect (Devipriya and Shyamala Devi, 1999). Activexygens are effective as cytotoxic and to damage cells by inac-ivating and modifying cellular components. The low concen-ration of oxygen free radicals stimulate fibroblast proliferation,lso when free radical scavengers are added to cultured fibrob-

asts in absence of free radical generating system, they inhibitedhymidine incorporation, indicating the growth promoting actionf free radicals (Shukla and Patnaik, 1998). Ascorbic acid haseen shown to be involved in collagen gene expression. It was

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macology 108 (2006) 204–210 209

lso found in some burn injury cases ointment containing super-xide dismutase (SOD), which stimulates wound healing. It wasbserved that asiaticoside enhanced induction of antioxidantevels at an initial stage of healing, which may be importantontributory factor in the healing property (Shukla et al., 1999).

The antioxidant enzymes (superoxide dismutase and cata-ase) are known to quench the superoxide radical and thusrevent the damage of cells caused by free radicals (Shirwaikart al., 2003). So scavenging effect might be one of the mostmportant components of wound healing. Also plant reported toave antioxidant activity (Sleem et al., 1999) may be responsi-le to support wound healing. Thus the enhanced wound healingay be due to the free radical scavenging action of the plant asell as enhanced antioxidant enzyme level in granuloma tissues.The leaves extract of Chromolaena odorata contains mixture

f phenolic acids and lipophilic flavonoid aglycones (flavanones,avonols, flavones and chalcones) were found to be power-ul antioxidant to protect cultured skin cells against oxidativeamage (Phan et al., 2001). Tephrosia purpurea have also beeneported to contain same flavonoids, which may be one of theotential mechanisms contributing to enhanced wound healing.

The alcoholic stem extract of Tephrosia purpurea has beeneported for the antibacterial activity against Salmonella typhi,causal organism of typhoid fever (Gehlot and Bohra, 2000).he methanolic whole plant extract of serpentine environmentas also effective against Mycobacter phlei, a non-acid fast bac-

erium (Rajakaruna et al., 2002). The ethanolic and methanolicxtract of plant also has been reported for antibacterial activityMahajan et al., 1999).

In conclusion, the results of study showed that the ethanolicxtract ointment of Tephrosia purpurea effectively stimulatesound contraction; increase tensile strength of incision and dead

pace wounds as compared to control group. These finding couldustify the inclusion of this plant in the management of woundealing.

cknowledgement

The authors would like to acknowledge University Grantommission, New Delhi for giving Junior Research Fellowship.

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