Journal of Ethnopharmacology 142 (2012) 714–722

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

0378-87

http://d

n Corr

E-m

journal homepage: www.elsevier.com/locate/jep

Evaluation of the wound healing activity of methanol extract ofPedilanthus tithymaloides (L.) Poit leaf and its isolated activeconstituents in topical formulation

Soma Ghosh a, Amalesh Samanta a, Nirup Bikash Mandal b, Sukdeb Bannerjee b,Debprasad Chattopadhyay c,n

a Division of Microbiology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, Indiab Division of Natural Product Chemistry, Indian Institute of Chemical Biology, Jadavpur, Kolkata, Indiac ICMR Virus Unit, I.D. and B.G. Hospital, General Block 4, First Floor, 57 Dr Suresh C Banerjee Road, Beliaghata, Kolkata 700 010, India

a r t i c l e i n f o

Article history:

Received 11 January 2012

Received in revised form

10 May 2012

Accepted 25 May 2012Available online 7 June 2012

Keywords:

Pedilanthus tithymaloides

Wound healing

Epithelialization

Tensile strength

Hydroxyproline

41/$ - see front matter & 2012 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.05.048

esponding author. Tel.: þ91 23537425; fax:

ail address: debprasadc@yahoo.co.in (D. Chat

a b s t r a c t

Ethnopharmacological relevance: Pedilanthus tithymaloides leaves are widely used in Indian medicine to

heal wounds, burn, mouth ulcers. However, systematic evaluation of these activities is lacking. Thus,

the present study aimed to assesses the wound healing activity of Pedilanthus leaves and its isolated

constituents in topical ointment formulation.

Materials and Methods: Bioassay-guided chromatographic fractionation of the methanol extract of

leaves resulted in the isolation of 2-(3,4-dihydroxy-phenyl)-5,7-dihydroxy-chromen-4-one and 1, 2-

tetradecanediol, 1-(hydrogen sulfate), sodium salt. The ointment formulation of methanol extract (2.5%,

5% w/w) and isolated compounds (0.25% w/w) was prepared and evaluated on excision, incision and

dead space wound models in rats. The effects of formulations on wound healing were assessed by the

rate of wound closure, period of epithelialization, tensile strength, granulation tissue weight, hydro-

xyproline content and histopathology.

Results: Significant wound healing activity was observed with methanol extract and isolated consti-

tuents. Topical application of isolated compound ointments caused faster epithelialization, significant

wound contraction (95.41%), and better tensile strength (565.33 g) on 16 post-wounding day, while 5%

extract showed wound epithelialization with 95.55% contraction on 18th post-wounding day, better

than the control group (76.39% on 22 day). The tensile strength of incision wound was significantly

increased in extract and compound treated animals. Moreover, in dead space model the extract

significantly increased granuloma tissue weight, tensile strength and hydroxyproline content. The

tissue histology of ointment treated groups showed complete epithelialization with increased

collagenation, compared to the povidone–iodine group.

Conclusions: The results validated the traditional use of Pedilanthus tithymaloides for cutaneous wound

management.

& 2012 Elsevier Ireland Ltd All rights reserved.

1. Introduction

Pedilanthus tithymaloides L. Poit. (Euphorbiaceae) is a lowtropical shrub, known as Rang-chita in Bengali and devil’s-back-bone in English, grown in different parts of India. The plant isknown as Brihatgokshura, Trikantaka, Gokantaka and Bhakshan-taka in Ayurveda (http://www.mpbd.info/ plants/pedilanthus-tithymaloides.php). In Indian folklore Pedilanthus tithymaloides isused for antiviral, antibacterial, antihemorrhagic, antitumor,abortive, anticancer and anti-inflammatory (Bunyapraphatsara

d Ltd All rights reserved.

þ91 23537424.

topadhyay).

and Chokchaichareonporn, 2000) activity. Traditionally teabrewed from Pedilanthus tithymaloides leaves has been used inasthma, mouth ulcers, and venereal disease; while tea brewedfrom root has abortifacient activity; and the sap is topically usedto treat ringworm, skin cancer, and warts (Nellis, 1997). TheNyshi community of Arunachal Pradesh used its latex to curepiles (Doley et al., 2010), while islanders of the Indian ocean(Madagascar, Mauritius, Comoros, Mascarenes) used the stem asabortifacient and latex to cure venereal diseases (Jain andSrivastava, 2005). In Vidarbha District of Maharashtra the aerialpart is used for skin disorder (Kumar and Chaturvedi, 2010). Arecent report showed that the tincture of American species hasanti-inflammatory and antioxidant activity (Abreu et al., 2006).Earlier studies revealed that Pedilanthus tithymaloides contain

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722 715

triterpenes (Misra and Khastgir, 1969), long-chain alcohol(Mukherjee et al., 1989, 1992), carotene derivatives azafrin(Upadhyay and Hecker, 1974), anticancer-diterpene pedilstatin(Pettit et al., 2002), antioxidants like kaempferol 3-O-b-D-gluco-pyranoside-6-(3-hydroxy-3-methylglutarate), quercitrin, isoquer-citrin and scopoletin (Abreu et al., 2008). Interestingly, the latexof the plant yielded a protease, pedilanthain, having oral anti-inflammatory activity (Dhar et al., 1988) and a mitogenic galac-tose-specific lectin (Seshagirirao, 1995) having anti-diabetes(Nagda and Deshmukh, 1998) and anti-tubercular (Ankushet al., 2003) activity. A recent study showed that the ethanolextract of leaves and some of its phytoconstituents have anti-bacterial activity against Staphylococcus aureus, Bacillus subtilis,Pseudomonas aeruginosa and Escherichia coli (Vidottia et al., 2006).

Traditionally Valaiyan community used a handful of leaveswarmed on fire and tied around the affected wound and fire burnsfor relief and healing (Sandhya et al., 2006); while its latex(Vilayti-sher) is used by people of Sangli District, Maharashtra,for healing wounds (Patil et al., 2009). Recently the ethanolextract of Pedilanthus tithymaloides was evaluated on excisionwound model (Sriwiroch et al., 2010). However, there is nosystematic scientific or clinical evaluation of the wound healingproperty of the crude extract of Pedilanthus tithymaloides leaves orits phytoconstituents. Thus, the present study for the first timeaims to assess the wound healing activity of Pedilanthus tithyma-

loides leaves and its isolated constituents in ointment form,compared to the standard formulation with povidone–iodine.

2. Materials and methods

2.1. Plant material

The Pedilanthus tithymaloides (PT) leaves was collected fromthe suburbs of Kolkata, India, and identified by a taxonomist atIndian Botanic Garden, Botanical Survey of India, Howrah, India. Avoucher specimen (No. CNH/-1-1(56)/2006/Tech-11/1450) hasbeen deposited at the Division of Microbiology, Department ofPharmaceutical Technology, Jadavpur University, Kolkata.

2.2. Extraction and isolation

The collected leaves were thoroughly washed in running tapwater, air dried under shade and powdered in a mechanical grinder.The air dried powdered leaves (2 kg) were defatted at roomtemperature with petroleum ether (60–80 1C, 3�4L), and thensuccessively extracted with chloroform (3�4L) and methanol(3�4L) by cold maceration for 72 h to afford petroleum etherextract (150 g), chloroform extract (105 g) and methanol (85 g)extract. These three fractions when subjected to wound healingactivity only the methanol fraction showed the potent activity. Thus,methanol extract was subjected to activity guided fractionationand isolation by column chromatography (CC) using silica gel(60–120 mesh) and partitioned between n-butanol and water satu-rated with n-butanol (n-butanol is more polar than methanol, somost of the organic molecules get dissolved in butanol). However,before discarding, the aqueous part was further extracted with n-butanol to make sure that no organic molecules remain in aqueousextract. The n-butanol-soluble fraction (46 g) was then subjected tosilica gel CC with gradient of petroleum ether (100:0) to petroleumether (60–80 1C)–CHCl3 (25:75), CHCl3 (100:0) to CHCl3–MeOH(70:30) to obtain five major fractions. Among these five majorfractions, studied in thin layer chromatography (TLC), fractions4 and 5 were further fractionated, as fractions 1, 2 and 3 did notshow any activity. All the fractions were identified by TLC in everystep using b-sitosterol or b-sitosterol glucoside as the marker

compounds. Fraction 4 eluted with CHCl3–MeOH (100:0–90:10)was assembled and rechromatographed on silica gel CC (petroleumether–CHCl3 50:50 to CHCl3–MeOH 85:15) to yield seven sub-fractions (using silica gel of 100–200 mesh). The sub-fractions 4.1–4.4 showed the presence of b-sitosterol, while 4.5–4.7 were otherthan b-sitosterol. Thus, subfractions 4.5–4.7 was added together andfurther purified on SiO2 CC (CHCl3–MeOH 100:0–90:10) to affordcompound-1 (30 mg; yellow solid. m.p. 330–332 1C; m/z 309).Similarly, fraction 5 eluted with CHCl3–MeOH (80:20–65:35) wasgrouped and submitted to silica gel CC (petroleum ether–CHCl350:50 to CHCl3–MeOH 75:25) to get 10 sub-fractions, of which sub-fractions 5.1–5.4 showed the presence of b-sitosterol glucoside andthe rest was other than b-sitosterol glucoside. Thus, subfraction 5.5–5.8 was combined together and further purified on SiO2 CC (CHCl3–MeOH 95:5–85:15) to afford compound-2 as white crystals (35 mg;colorless solid, m/z 355.1535). The structures of the isolated com-pounds were determined by the spectral analysis of IR, NMR, andHR-ESIMS. The other fractions (petroleum ether and chloroformfraction) mainly contain mixture of straight chain compounds, b-sitosterol and b-sitosterol glucosides (methanol fraction) showed nosignificant activity, and hence not used in further study.

2.3. General procedures

Melting points measured on a Yanagimoto Micro melting pointapparatus are uncorrected. IR spectra were recorded onJASCO7300 FTIR spectrometer. 1H and 13C NMR spectra wererecorded at 600 MHz and 150 MHz, respectively, using BrukerAVANCE 600 spectrometer with TMS as internal standard inC5D5N and or MeOD. ESI-MS and HR-ESI-MS were performed ona Q-TOF-micromass spectrometer. Silica gel (60 mesh, Merck,Germany) was used for CC, while TLC was carried out on silicagel 60 F254 (Merck, Germany) and spots were visualized byspraying Liebermann–Burchard reagent followed by heating. Pre-parative TLC was carried out on precoated silica gel 60 plates(thickness: 0.5 mm; E. Merck, Germany). All other chemicals andsolvents were purchased from SRL, Mumbai, India.

2.4. Preparation of formulations

Formulations of extract and its constituents were prepared toevaluate its efficacy, in comparison with povidone–iodine oint-ment, USP. Ointment base was prepared by mixing the ingredi-ents (wool fat 5 g, hard paraffin 5 g, cetostearyl alcohol 5 g, softwhite paraffin 85 g) as per British Pharmacopoeia (1980) in abeaker at 65 1C water bath. After cooling, the mixture washomogenized by a homogenizer at 1500 rpm for 10–15 min. Themost stable ointment base was selected for the preparation of fiveformulations (four test groups and one control). The stability wasfurther evaluated at accelerated conditions to obtain the moststable formulations. Two types of topical formulations wereprepared, one containing ointment base (100 g) plus methanolextract (2.5 g and 5.0 g) of PT leaf and another with ointment base(100 g) plus isolated compound (0.25 g). The ointment base wasmixed with extract or isolated compound and stirred to get thehomogeneous ointment preparation. Thus, the formulation con-taining 2.5 g and 5.0 g of extract with 100 g of ointment baseyielded 2.5% and 5% w/w of active extract. Similarly, 25 mg ofisolated constituents mixed with 10 g of ointment base yielded0.25% w/w isolated active compound(s) in ointment formulation.The drug formulations were freshly prepared on every fifth day.

2.4.1. Stability of formulation

The stability of the ointment formulations was evaluatedaccording to the guidelines of the International Conference on

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722716

Harmonization (ICH, 1993). The physical stability of ointmentformulations was evaluated by testing the physical changes likephase separation, color, odor, and consistency. Samples of theointment formulations were kept at different temperatures(40 1C, 37 1C, room temperature) for 45 days and observedperiodically for changes like phase separation, development ofobjectionable color, odor, etc. Centrifugation at 10,000 rpm for10 min is used to evaluate the accelerated deterioration ofointments with an Eppendorf centrifuge as described byCockton and Wynn (1952). The formulations, which were resis-tant towards physical stability and centrifugation, were selectedfor spreadability. The spreadability was determined by modifiedwooden block apparatus consisted of a wooden block with fixedglass slide and a pulley at one end, and a pan attached to anotherglass slide (movable) with a string at other end. A measuredamount of ointment(s) was placed in the fixed glass slides, andthe movable slide with the pan was placed over the fixed slide, sothat the ointments were sandwiched between the two slides for5 min. A 50 g weight was placed on the top of two plates and thetime required by the top plate to cover a distance of 10 cm wasrecorded (Prasad and Dorle, 2006). The spreadability was deter-mined by the formula: S¼M/T, where S is the spreadability in g/s,M is the mass in grams and T is the time in seconds. A shorterinterval indicates better spreadability.

2.5. Experimental animals

Male Wistar albino rats of either sex, weighing about150–180 g were selected for the study. Animals were maintainedin polypropylene cages with free access to food and water adlibitum. The study was carried out with the approval of Institu-tional Animal Ethics Committee (Reg. no. 0367/01/C/CPCSEA) andwas in accordance with the guidelines of CPCSEA. Healthy Swissalbino mice of either sex weighing 20–25 g were used to deter-mine the toxicity and safety profile of the extract. The animalswere weighted before and after experiments.

2.6. Acute oral toxicity study

Acute toxicity study was performed by stair case method(Jalalpure et al., 2003). The Swiss albino mice were randomlydivided into 11 experimental and one control group of six animalseach. Control animals was administered with 0.3% w/v carbox-ymethyl cellulose in water (1 ml/kg) and the experimentalgroups were orally fed with the graded doses of methanol extract(0.1–5 g/kg) and the isolated compound(s) (0.05 and 0.1 g/kg,body weight). The animals were observed continuously in the first2 h for toxic symptoms and up to 72 h for mortality (Litchfieldand Wilcoxon, 1949).

2.7. Sub acute oral toxicity study

As none of the orally fed animals were died in acute toxicitystudy, the administration of extracts was continued till 28 days.The animals were randomly divided into four groups (n¼6), andanimals of group I received the vehicle (0.3% w/v carboxymethylcellulose in distilled water) as control; while groups II, III, and IVwere treated daily with methanol extract of PT leaves at 0.5, 1.0,1.5 g/kg body weight respectively, for 28 days. Feed and waterconsumption per group were recorded daily. Clinical signs andsymptoms such as weakness or aggressiveness, movements, foodintake, loss of body weight, discharge from eyes and ears, noisybreathing, and number of death were monitored carefully. For theestimation of hematological and serum biochemical parametersfresh blood was collected in heparinized and non-heparinizedtubes, from each group on the 29th day by cardiac puncture.

Subsequently, the animals were sacrificed by cervical dislocationto collect liver spleen and kidney for measuring organ weight andmicroscopic analysis, and then preserved in 10% formalin forhistopathological examination.

2.8. Acute skin irritation test

This was carried out by the method of Gfeller et al. (1985) onrats. About 500 mm2 area on the dorsal fur of each animal wasshaved. The shaved area was cleaned, and the ointment formula-tions were applied to different groups of animals. After 4 h ofointment applications, the skin of each animal was observed forsign of inflammation.

2.9. Evaluation of wound-healing activity

The excision, incision and dead space wound models were usedto evaluate the wound-healing activity of methanol extract of PTleaves and its isolated constituents. The rats were divided into sixgroups, each containing six animals, for excision and incisionwound models. Fifty milligrams of formulated ointments wereapplied topically to each animal once a day. The animals of group Ireceived ointment base (control), while group II were treated witha 5% w/w povidone–iodine ointment. The animals of groups III andIV were treated with 2.5% and 5% w/w of methanol extractointments, while groups V and VI received 0.25% w/w ointmentof the isolated compounds 1 and 2, respectively. For the deadspace wound model, four groups, each containing six animals,were used. The animals of group I (control) were treated tropicallywith simple ointment base. Group II was treated with 5% w/wpovidone–iodine ointment USP; while groups III and IV weretreated with methanol extract ointments 2.5% and 5% w/w,respectively. The animals were anaesthetized with ketaminehydrochloride (100 mg/kg, i.m.) prior to and during infliction ofthe wound. All the animals were closely observed for any infec-tion, so that the infected animals can be excluded from the study.

2.9.1. Excision wound

The animals were anaesthetized prior to and during thecreation of experimental wounds (Nayak et al., 2009), withketamine hydrochloride (100 mg/kg body wt.). Rats are theninflicted with excision wound as described by Morton andMalone (1972). The dorsal fur of the animals was shaved withelectric clipper and full thickness of excision wound of 500 mm2

was created along the marking using toothed forceps, a surgicalblade and pointed scissor. The entire wound was left open. Allgroups of animals were treated in the similar manner as men-tioned above. The healing of wound was assessed by tracing thewound on first, third, sixth, ninth, 12th, 15th, 18th, 21st post-wounding days using transparency paper and a marker, and therecorded wound areas were measured graphically.

2.9.1.1. Rate of wound contraction. The rate of wound contractionwas measured as percentage reduction of wound size at every2 day interval. Progressive decrease in the wound size wasmonitored periodically using transparency paper and a marker,and the wound area was assessed graphically to monitor thepercentage of wound closure, which indicates the formation ofnew epithelial tissue to cover the wound. Wound contraction wasexpressed as reduction in percentage of the original wound size.The Percentage (%) wound contraction ¼ (wound area on day 0�wound area on day n)/wound area on day 0�100.

2.9.1.2. Epithelialization time. Falling of eschar without any rawwound area was considered as complete healing of wound and

Table 1Spreadibility of ointment formulations on different days at various temperatures.

Day Temperature(1C)

Spreadability (g/s)

2.5% Extractointment

5% Extractointment

Compound 1ointment

Compound 2ointment

0th 34 5.314 5.523 5.451 5.73237 5.526 5.267 5.230 5.66740 5.190 5.449 5.120 5.412

7th 34 5.220 5.310 5.321 4.56837 5.189 4.921 5.234 5.10240 5.265 5.120 5.116 5.262

15th 34 6.212 5.224 5.334 5.34237 5.721 5.127 5.189 6.10440 5.449 5.342 4.983 5.349

30th 34 5.810 6.027 5.231 5.34737 5.891 5.537 5.543 5.21940 5.127 5.278 5.157 5.176

45th 34 5.627 5.452 5.309 4.82737 6.293 5.104 5.219 5.29340 6.130 5.235 5.234 5.348

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722 717

the number of days required for falling of eschar without anyresidual raw wound was calculated as a period of epithelialization(Morton and Malone, 1972; Nayak et al., 2009).

2.9.2. Incision wound

In this test rats were anesthetized with ketamine hydrochlor-ide (100 mg/kg) prior to and during the creation of experimentalwounds (Udupa et al., 1994). The dorsal fur of the animals wasshaved with electric clipper and two paravertebral long incisionof 6 cm length were made through the skin at a distance of about1.5 cm from the midline on each side of the depilated back of theanimals as described earlier (Ehrlich and Hunt, 1968). Afterincision, the parted skin was stitched together at intervals ofone centimeter using surgical thread (No. 000) and curved needle(No. 11). The wounds were then left undressed. All groups ofanimals were treated as described above. Sutures were removedon eighth post-wounding day and the treatment was continued.The skin breaking strength of healed wound was measured on the10th day by the method of Lee (1968). The breaking strength isthe strength of a healing wound which is measured by theamount of force required to disrupt it.

2.9.2.1. Determination of wound breaking strength. Breakingstrength is the most crucial phase in dermal wound healing, andthe restoration of tissue strength is a critical outcome of repairprocess. As the mechanical properties of the skin depend on thecollagen and elastic fiber networks of dermis, thus breakingstrength of the healed wound is measured as the minimumforce required to break the incision apart, which indicate theefficacy of the healing process, strength of wound tissues and thedegree of healing (Shetty et al., 2008). After removal of skinsutures on post-operative day 7, gradually increasing weight wasapplied to one side of the wound while the other side was fixed.The weight that completely separated the wound from theincision line is considered to be the breaking strength. Thesutures were removed on the eighth post-wounding day andthe breaking strength was measured on 10th day. The meanbreaking strength on the two para-vertebral incisions on bothsides of the animals was taken as the measure of the breakingstrength of individual animal.

2.9.3. Dead space wound model

Dead space wounds were created by subcutaneous implanta-tion of sterile cylindrical grass pith (2.5 cm�0.3 cm), on eitherside of the lumber region on ventral surface of each rat (Turner,1965). On the 10th post-wounding day, animals were sacrificedunder ketamine (100 mg/kg body weight, i.m.) anesthesia, andthe granulation (wound) tissues formed on the grass pithswere excised. After recording the wet weight, the granulationtissues were dried at 60 1C for 12 h in an oven to obtain constantdry weight (Dispasquale and Meli, 1965). Simultaneously, thedried tissue was hydrolyzed with 6N HCl (5.0 ml) for 24 h at110 1C, and then neutralized (pH 7). The neutralized hydrolysatewas used for hydroxyproline estimation as described by Neumanand Logan (1950). About 200 ml of hydrolysate was mixedwith 0.01 M copper sulfate solution (1 ml) followed by 2.5NNaOH (1 ml) and 6% H2O2 (1 ml). Samples were then kept at80 1C (for 5 min) on a shaking incubator, cooled and mixedwith 4 ml of 3N H2SO4 with agitation. Finally, 2 ml of 5% p-dimethylaminobenzaldehyde was added to the mixture, incu-bated (70 1C for 16 min), cooled (20 1C) and the absorbance wasmeasured at 540 nm in a colorimeter. The amount of hydroxypro-line present in the sample(s) was calculated from a standardcurve prepared with pure L-hydroxyproline (Gurung and Skalko-Basnet, 2009).

On 10th post-wounding day, granulation tissue was excised fromthe sacrificed animals. A part of wet tissue was preserved (10%formalin), embedded (5–6 mm paraffin blocks), sectioned, andstained with Van Geison’s stain (Gal et al., 2006), and finally evaluatedby histopathological examination of inflammatory cells, blood ves-sels-marker, collagen formation and quantification of collagen.

2.10. Antimicrobial screening by agar and tube dilution method

The antimicrobial activity of PT leaf extract was evaluatedagainst few pathogenic bacteria, such as Staphylococcus aureus,Bacillus subtilis, Escherichia coli, Shigella dysenteriae, and Pseudomonas

aeroginosa; and the fugal isolates like Candida albicans, Candida

tropicalis and Cryptococcus neoformans. Bacteria were cultured over-night at 37 1C in Mueller Hinton (MH) broth and fungi in PotatoDextrose broth (PDB) at 28 1C for 72 h. The overnight culture wastested to determine the visible growth on agar plates or microplatewells containing different concentrations of the extract (0–1000 mg/ml) or its isolated constituents and standard antibiotics by dilutionmethod (Chattopadhyay et al., 2001). For disk diffusion test sterilefilter paper disks (6 mm diameter) containing 10–1000 mg/disk of PTleaf extract or its constituents were placed on the agar surface andincubated overnight at 37 1C for evaluation of growth inhibition.MIC was defined as the lowest concentration of extract or isolatedconstituents that inhibit visible growth on agar surface or turbidityin microwell broth.

2.11. Statistical analysis

The results were expressed as mean7SE. The statisticalsignificance was evaluated by one-way ANOVA followed byDennett’s ‘t’ test (compared differences between experimentalgroups with control) using the ‘‘INSTAT 3’’ software. Po0.001 andPo0.05 were considered statistically significant.

3. Results

3.1. Stability of the formulation

The evaluation of stability parameters showed that therewas no phase separation, objectionable odor or any physicalinstability. The effect on storage at varying temperature onspreadability of ointments presented in Table 1 indicates thatall the formulated ointments are nearly the same in terms of

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722718

applicability or spreading capability. The storage of the formula-tions at accelerated stability conditions does not influence itsstability. Thus, the formulations are satisfactory with respect toits physical parameters.

3.2. Isolation and identification

The shade dried powdered leaves of Pedilanthus tithymaloides

were first defatted with petroleum ether (60–80 1C) and thensuccessively extracted with chloroform and methanol at ambienttemperature. Biologically active methanol extract was partitionedbetween n-butanol and water saturated with n-butanol. Then-butanol fraction of methanol extract was separately chromato-graphed over a silica gel column resulted in the isolation of one2-(3,4-dihydroxy-phenyl)-5,7-dihydroxy-chromen-4-one (com-pound-1) and a new tetradecanediol, sodium salt (compound-2).The identity of isolated compound-1 was confirmed by comparingthe data in the literature (Hirobe et al., 1997; Chiruvella et al.,2007), which indicated that this flavone 2-(3,4-dihydroxy-phe-nyl)-5,7-dihydroxy-chromen-4-one is reported for the first timefrom Pedilanthus tithymaloides.

Compound-2 was isolated as colorless solid with the molecularformula C14H29O5SNa, based on the observed pseudo-molecularion peak appeared at m/z 355.1535 attributable to [MþNa]þ

(calculated for C14H29 Na2O5S 355.1531). The 1HNMR spectrumshowed a triplet at d0.90 (J¼7.0 Hz) indicating the presence of amethyl group in a long chain, supported by the presence of abroad methylene peak centered at d1.29. The spectrum showedone 1H multiplet at d3.78 for a CHOH group flanked by methylenegroups, along with two double doublets integrating for oneproton each at d3.93 (J¼4.2, 10.2 Hz) and 3.88 (J¼7.0, 10.2 Hz),suggesting the presence of an esterified primary alcoholic groupnext to a methine group. This account for the mass spectralmolecular composition, where the presence of a long chain 1,2diol sulfated at the primary alcoholic group could be inferred. Inthe 13C NMR spectrum one methyl signal appeared at d14.6; onemethylene carbon signal at d73.1 and one methane peak at 71.0supported the presence of the sulfated 1, 2-diol structure asdeduced from 1H NMR and mass spectral analysis. Combining allthe above evidences, possible identity of the compound is 1,2-tetradecanediol, 1-(hydrogen sulfate), sodium salt and the mole-cular structure was deduced to be C14H29O5SNa. It is noteworthythat synthetically derived tetradecanediol, 1-(hydrogen sulfate),sodium salt was reported in the literature (Piepmeyer, 1966; Cahnet al., 1966). However, this is the first report of isolation oftetradecanediol, 1-(hydrogen sulfate), sodium salt from naturalsources. The chemical structures of the isolated compounds areillustrated in Fig. 1.

3.3. Toxicity study

The toxicity study aimed to investigate the safety profile ofmethanol extract of PT leaves. The results of the acute toxicity

Fig. 1. Structure of com

study of methanol extract (oral) indicated that, the extract up to5 gm/kg body weight did not produce any sign of toxicity ormortality in any of the groups during or after treatment. Inter-estingly, isolated compounds 1 and 2 up to 100 mg/kg bodyweight did not produce any toxicity or mortality. In sub-acutetoxicity study the food and water intakes were unaltered during28 day of treatment like control group, and even there was nomortality during the study period. The oral administration ofmethanol extract over 28 day caused no significant changes inweight of liver, kidney, heart, lung and spleen, compared to thecontrol group. Moreover, the methanol extract failed to induceany significant changes on blood cell count, hemoglobin contentand biochemical parameters like SGPT, SGOT and cholesterolcontent, as compared to control mice. Histopathology of the livershowed moderate number of normal hepatocytes and bloodsinusoids; while in kidney tubular and glomerular structureswith the signs of edema are seen in the cortex without anydamage (histopathology of liver and kidney is presented inSupplementary file, Figs. 2 and 3). Thus, the present studyestablishes the reliable safety profile of the methanol extract ofPT leaves, administered orally in Swiss mice.

3.4. Acute skin irritation test

The formulated ointments of methanol extract of PT leaves andits isolated constituents did not show any type of irritation andnoticeable inflammation, swelling or any other change onthe skin.

3.5. Wound healing activity study

To evaluate the wound healing ability of the prepared for-mulations, we measured parameters like: (1) rate of woundcontraction and epithelialization time (excision wound), (2) ten-sile strength of newly formed tissue (incision wound), and(3) hydroxyproline content in newly formed tissue along withthe histopathological studies of healed tissues (dead space woundmodel).

3.5.1. Wound contraction and epithelialization time (excision

wound)

Wound contraction indicates the rate of reduction of unhealedarea during the healing process. Thus, the fast rate of wound closerindicates the better efficacy of medication. The progressive reduc-tion in wound area of different groups of animals over 18 day, bymethanol extract of PT leaves and its isolated constituents ispresented in Fig. 2 (wound healing figures are presented inSupplementary file, Fig. 4). The fastest healing of wound wasobserved in animals which received ointment of compounds 1 and2, along with complete healing (100% wound contraction) within 18day, as compared with the animals treated with standard 5% w/wpovidone–iodine ointment. On 18th post-wounding day 5% extract

pounds 1 and 2.

Table 2Effect of topical application of methanol extract of Pedilanthus tithymaloides and its compounds on period of epithelialization, breaking strength, and healing of

dead wound.

Parameters Treatment with ointment

Control (ointment base) Extract Standard(5% povidone–iodine)

Compounds

2.5% w/w 5.0% w/w 1 2

Epithelialization time (excision wound) 22.0070.1 20.0070.39nn 19.570.52nn 15.5070.28nn 17.1670.4nn 17.2570.25nn

Breaking strength (incision wound) 372.1373.23 525.3374.32nn 512.0075.77nn 572.5073.4nn 565.1073.1nn 561.1273.9nn

Wet weight (in g) 8572.30 107.3372.90n 117.6671.07n 121.1571.85n

Dry weight (in g) 1372.30 8572.30n 15.8670.59n 19.170.55n

Hydroxyproline content (mg/g) 163.672.90 156.673.10n 192.3371.45n 207.6671.45n

Values are expressed as mean7SE. nnPo 0.05; n Po0.001 versus control (one-way ANOVA, followed by Dennett’s ‘t’ test).

Fig. 2. Effect of topical application of methanol extract of Pedilanthus tithymaloides and its constituents expressed as percentage wound contraction. Values are expressed

as mean7SE of six animals in each group. nPo0.001 and nnPo0.05 versus control (one-way ANOVA, followed by Dennett’s ‘t’ test).

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722 719

treated animals showed significant reduction in the wound area(95.88%) with faster epithelialization (19.570.39), while animaltreated with 2.5% extract showed moderate degree of healing(93.85% wound area, 20.070.52 epithelialization time). Within18th post-wounding day the complete epithelialization was noticedin the animals treated with standard drug (5% w/w povidone–iodine) having 100% wound contraction. The lowest rate of woundhealing with highest epithelialization time (22 day) was observed invehicle control group. While in methanol extract treated groupwound remained unhealed even after 15th day of treatment,although the unhealed area was smaller than the control group.Thus, compared to the methanol extract the formulation of isolatedcompounds heals the wound faster (epithelialization time 17.25–17.16 day), and nearly similar to the 5% povidone–iodine ointmenttreated group (Table 2).

3.5.2. Measurement of tensile strength or wound breaking strength

(incision wound)

The wound healing measured by the tensile strength of thehealing skin, treated with different formulations on 10th day,

revealed that the wound treated with ointment base had theminimum strength (372.13). While the tensile strength of thetissue treated with other formulations was substantially higherthan control group (512–565.10). Interestingly 5% and 2.5%extract ointments exert nearly similar result on the tensilestrength of the healing tissue (Table 2). The tensile strength ofwound treated with povidone–iodine ointment is comparable(572.50) to that of ointment formulations containing compounds1 (565.10) and 2 (561.12). This observation confirms that metha-nol extract as well as its isolated phytoconstituents possessesexcellent wound healing property.

3.5.3. Estimation of hydroxyproline content and histopathology of

healed tissues (dead space wound)

Collagen is the predominant extracellular protein in thegranulation tissue and hydroxyproline is an integral part ofcollagen fiber. Increased hydroxyproline content indicatesincreased collagen synthesis, which in turn leads to enhancedwound healing. Table 2 represents the wet weight (g), dry weight(g) and hydroxyproline content (mg/g) of the granulation tissue of

Table 3Minimum inhibitory concentration of methanolic extract and its constituents of

Pedilanthus tithymaloides leaf on selected bacteria and fungi by agar dilution

method.

Organisms Concentrations in mg/ml

Methanolic extract Compound-1 Amoxicillin

Staphylococcus aureus 100 32 1

Bacillus subtilis 185 64 1

Escherichia coli 175 128 10

Shigella dysenteriae 100 64 128

Pseudomonas aeruginosa 200 64 64

Fungi Methanolic extract Compound-1 Fluconazole

Candida albicans 312 256 2

Candida tropicalis 128 128 4

Cryptococcus neoformans 512 256 8

Values are expressed as mean7SE. Values for the triplicate assays.

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722720

the animals received different formulations up to 10 day. The dryand wet weights of control animals (13 and 85 g) was increased in5% extract ointment treated group (19.1 and 177.66 g), whereasthe standard drug treated group showed maximum weight (19.1and 121.15 g). Significant increase in hydroxyproline content wasnoted in animals that received povidone–iodine (207.66 mg/g);while nearly comparable values were observed in animals thatreceived 5% extract ointment (192.33 mg/g). The animals whichreceived only ointment base had lowest content of hydroxypro-line content (163.6 mg/g). Thus, these results corroborated thedata obtained from the wound contraction test.

Histopathology of granulation tissue, collected on 10th post-wounding day, was examined for cellular epithelial regenerationand matrix organization. The results provide a good evidence ofsuitability of the formulations in promoting wound healing (Fig. 5 ofSupplementary file). The granulation tissue section of control animalsshowed decreased epithelialization, fibrosis and more aggregation ofmacrophages with less collagen fibers, indicating incomplete healingof wounds (Fig. 5a of Supplementary file). The tissue section fromanimals treated with povidone–iodine ointment showed completehealing with prominent epithelialization and increased collagenationand fibroblast (Supplementary file, Fig. 5b). While the tissue sectionsfrom animals treated with 5% and 2.5% extract ointment showedproliferation of epithelial tissue covering the wound area (Fig. 5c andd in Supplementary file) with multiplication of fibrous connectivetissue, without proper union, in dermis region. Interestingly theanimals treated with ointment formulations of compounds 1 and 2showed fibrous connective tissue with well-collagenation and com-plete healing (Fig. 5e and f of Supplementary file).

3.6. Antimicrobial activity

The MIC of methanol extract of PT leaves and the standardantibiotics presented in Table 3 indicated that the extract havemoderate antibacterial and antifungal activity compared to thestandard antibiotics. Further assay with the isolated compounds 1and 2 showed that compound-1 had a better antibacterial spectrumwith MIC of 75–140 mg/ml for bacteria, and 110–450 mg/ml forfungal isolates tested. Interestingly the antibacterial or antifungalefficacy of compound-2 and methanol extract is nearly similar.

4. Discussion

Wound healing is a complex and dynamic process of restoringtissue structure in damaged tissue as closely as possible to its

normal state. Healing depends upon the repairing ability of thetissue, type and extent of damage, and general state of the host’shealth. It is characterized by hemostasis, re-epithelialization,granulation, remodeling of the extracellular matrix and scarformation (Mary et al., 2002). The aim of the present work is toverify, for the first time, the traditional use Pedilanthus tithyma-

loides leaves for wound healing (Sriwiroch et al., 2010) and theability of healing wounds by the formulation of PT leaf extract andits isolated constituents. No single method is adequate to repre-sent the various components of wound healing process(Shirwaikar et al., 2003), thus, the present work aimed to evaluatewhether the ointment formulations containing methanol extractor its isolated constituents can help in wound healing process inthree different wound models.

Herbal ointment containing different medicinal plants areused in the folk medicine and have been reported to be beneficialin wound care, promoting wound healing, minimizing pain,discomfort and scarring of the patient (MacKay and Miller,2003; Odimegwu et al., 2008). Ointment is the obvious choice ofdosage form due to its convenience of topical application. Thus,before preparing formulation we tested the stability of theointment during storage, by studying the physical parameters,spreadability and consistency and found that the prepared for-mulation, as per British Pharmacopoeia (1980), is stable andsuitable for topical application.

The process of shrinkage of wound area depends on therepairing abilities of the tissue type, extents of the damage andstates of the tissue health (Priya et al., 2004; Anuar et al., 2008). Invivo studies revealed the enhanced rate of wound contraction inanimals treated with ointment containing isolated compoundsfrom Pedilanthus tithymaloides leaves, as compared to controlgroup. This might be due to enhanced epithelialization in shortertime, because Pedilanthus tithymaloides promoted epithelializa-tion either by facilitating the proliferation or by increasing theviability of epithelial cells.

The breaking strength is the strength of healing woundmeasured by the amount of force required to disrupt it. In thebeginning of healing process a wound have little breakingstrength, but as it heals the breaking strength increases rapidlydue to synthesize of collagen and formation of stable intra- andintermolecular cross linking (Madden and Peacock, 1968). Anincrease in skin breaking strength of the animals treated with thePT leaf extract and its constituent, explained that probably theextract or its phytoconstituents help in the increased synthesis ofaldehyde groups of collagen fibers responsible for cross linkagesresulting in greater tensile strength (Chithra et al., 1998).

The granulation tissue formed in the proliferative phaseconsists of fibroblast, collagen, inflammatory cells and smallblood vessels. Wound contraction is a fibroblast-dependentmethod involving the deposition and maturation of collagen.The tensile strength of the granulation tissue increases propor-tionally with the collagen deposition. The increase in granulationtissue weight in PT leaf extract or its constituents treated animalssuggests higher protein content and enhanced collagen matura-tion due to increased cross-linking of collagen fibers (Azad, 2002).However, healing phases like coagulation, inflammation, macro-phagia, fibroplasias, collagenation, contraction and epithelializa-tion are intimately interlinked. Therefore, a treatment couldinfluence the healing process by intervening one or more phasesof healing. Here, the animals treated with the methanol extract ofPT leaves and its phytoconstituents showed a significantlyincreased amount of hydroxyproline of granulation tissue, indi-cating increase collagen turnover. Collagen is the predominantextracellular protein in the granulation tissue of wounds (Chithraet al., 1998) and hydroxyproline is an integral part of collagenfiber, act as a biochemical marker. The present study, between

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722 721

two test groups, showed that the wound healing activity of thecompounds 1 and 2 from the methanol extract of PT leaves ishigher than the methanol extract alone. This is because the crudemethanol extract contain less amount of compounds 1 and 2 and/or some other chemical constituents of PT leaves may hinderedeach other’s activity.

The chemical analysis of bioactive extract of PT leaves yieldeda flavone 2-(3,4-dihydroxy-phenyl)-5,7-dihydroxy-chromen-4-one, isolated for the first time from this plant, while the anothercompound is a tetradecanediol, sodium salt, isolated for the firsttime from a plant source. The isolated flavone is a yellowcrystalline powder, sparingly soluble in water, also known asluteolin having anti-inflammatory (Jang et al., 2008), antioxidant,antimicrobial and immunomodulating activities. Luteolin exertsits anti-inflammatory activity by inhibiting thromboxane andleukotriene enzyme of arachidonic acid pathway, along withscavenging of hydrogen peroxide, due to ortho-dihydroxy groupsat its B ring and OH substitution at C-5 position of A ring(Odontuya et al., 2005). The antibacterial activity observed bythe PT extract and its isolated compound also agreed well withearlier reports that flavone luteolin had bacteriostatic activityagainst Staphylococcus aureus (Liu and Matsuzaki, 1995; Tsuchiyaet al., 1996), Helicobacter pylori, and Neisseria gonorrhoeae due tothe inhibition of arylamine N-acetyltransferase (Tsou et al., 2001).Moreover, the ability of luteolin to inhibit IL-6 (Jang et al., 2008),phosphodiesterase (Yu et al., 2010), and multiple sclerosis(Theoharides, 2009), as well as neuroprotection through rebalan-cing of pro-oxidant-antioxidant level (Zhao et al., 2012) and theactivation of monoamine transporter (Zhao et al., 2010) maycontribute to the faster wound healing potential of this plant.Modulation of ROS, inhibition of topoisomerases I and II, reduc-tion of NFkB, stabilization of p53, and inhibition of PI3K, STAT3,IGF1R, and HER2 are possible mechanisms for the putativebioactivities of luteolin (Lopez-Lazaro, 2009). While the secondcompound tetradecanediol, sodium salt is not reported from plantyet, but the synthetic tetradecanediol have detergent activity(Piepmeyer, 1966). Thus, the observed wound healing activity ofthe isolated flavone from methanol extract of PT leaves isprobably due to the activation of multiple factors related to thewound healing process and inflammatory pathways.

5. Conclusion

The present study for the first time demonstrated that topicalapplication of methanol extract of Pedilanthus tithymaloides L. Poitleaves and its isolated compounds 2-(3,4-dihydroxy-phenyl)-5,7-dihydroxy-chromen-4-one and tetradecanediol, 1-(hydrogen sul-fate), sodium salt may promote wound healing activity in rats,probably due to their ability to scavenge free radicals, inhibitionof some mediators of inflammatory pathway, reduction of NFkB(Lopez-Lazaro, 2009), and inhibition of some bacteria harboringthe wound (Tsuchiya et al., 1996; Vidottia et al., 2006). Thus, theresult of the present study offers pharmacological evidence of thefolk use of Pedilanthus tithymaloides for wound healing.

Acknowledgments

The authors are grateful to the Indian Council of MedicalResearch, New Delhi, for Senior Research Fellowship to one ofthe author (S.G.). The Officer-In-Charge of the ICMR Virus Unit,Kolkata, and the Registrar of Jadavpur University are acknowl-edged for providing all the necessary facilities.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.jep.2012.05.048.

References

Abreu, M.P., Matthew, S., Gonzalez, T., Vanickova, L., David, C., Gomes, A., Marcela,A.S., Fernande, E., 2008. Isolation and identification of antioxidants fromPedilanthus tithymaloides. Journal of Natural Medicine 62, 67–70.

Abreu, P., Matthew, S., Gonzalez, T., Costa, D., Segundo, M.A., Fernandes, E., 2006.Anti-inflammatory and antioxidant activity of a medicinal tincture fromPedilanthus tithymaloides. Life Science 78, 1578–1585.

Ankush, R.D., Nagda, K.K., Suryakar, A.N., Ankush, N.R., 2003. Tuberculosis alteringerythrocyte membrane topology—a lectin haemagglutination study. IndianJournal of Tuberculosis 50, 95–98.

Anuar, N.S., Zahari, S.S., Taib, I.A., Rahman, M.T., 2008. Effect of green and ripeCarica papaya epicarp extracts on wound healing and during pregnancy. Foodand Chemical Toxicology 46, 2384–2389.

Azad, S., 2002. Essentials of Surgery. Paras Medical Publications, Hyderabad, pp. 1.British Pharmacopoeia, 1980, vol. II, pp. 1096.Bunyapraphatsara, N., Chokchaichareonporn, A., 2000. Medicinal Plants Indigen-

ous to Thailand. Prachachon, Bangkok, pp. 691.Cahn, A., Konort, M.D., Goldberg, M.A., 1966. 2- Hydroxyalkyl Sulfate as Dentifrice

Detergents, US 3256155 19660614. 5 pp.Chattopadhyay, D., Maiti, K., Kundu, A.P., Chakrabarty, M.S., Bhadra, R., Mandal,

S.C., Mandal, A.B., 2001. Antimicrobial activity of Alstonia macrophylla: folkloreof Bay Islands. Journal of Ethnopharmacology 77, 49–55.

Chiruvella, K.K., Mohammed, A., Dampuri, G., Ghanta, G.R., Raghavan, C.S., 2007.Phytochemical and antimicrobial studies of methyl angolensate and luteolin-7-O-glucoside isolated from callus cultures of Soymida febrifuga. InternationalJournal of Biomedical Science 3, 269–278.

Chithra, P., Sajithlal, G.B., Chandrakasan, G., 1998. Influence of Aloe vera on theglycosaminoglycans in the matrix of healing dermal wounds in rats. Journal ofEthnopharmacology 59, 179–186.

Cockton, J.R., Wynn, J.B., 1952. The use of surface active agents in pharmaceuticalpreparations: the evaluation of emulsifying power. Journal of Pharmacy andPharmacology 4, 959–971.

Dhar, S.N., Ray, S.M., Roy, A., Dutta, S.K., 1988. Oral anti-inflammatory activity ofpedilanthain—a new proteolytic enzyme from Pedilanthus tithymaloides Poit.Indian Journal of Pharmaceutical Sciences 50, 281–283.

Dispasquale, G., Meli, A., 1965. Effect of body weight changes on the formationof cotton pellet induced granuloma. Journal of Pharmacy and Pharmacology17, 379.

Doley, B., Gajurel, P.R., Rethy, P., Singh, B., Buragohain, R., Potsangbam, S., 2010.Lesser known ethno medicinal plants used by the Nyshi community ofPapumpare District, Arunachal Pradesh. Journal of Biosciences Research 1,34–36.

Ehrlich, H.P., Hunt, T.K., 1968. Effect of cortisone and vitamin A on wound healing.Annals of Surgery 167, 324–328.

Gal, P., Toporcer, T., Vidinsky, B., Mokry, M., Novotny, M., Kilik, R., Smetana, K.J.R.,Gal, T., Sabo, J., 2006. Early changes in the tensile strength and morphology ofprimary sutured skin wounds in rats. Folia Biologica 52, 109–115.

Gfeller, W., Kobel, W., Seifert, G., 1985. Overview of animal test methods for skinirritation. Food and Chemical Toxicology 23, 165–168.

Gurung, S., Skalko-Basnet, N., 2009. Wound healing properties of Carica papayalatex: in vivo evaluation in mice burn model. Journal of Ethnopharmacology121, 338–341.

Hirobe, C., Qiao, Z.S., Takeya, K., Itokawa, H., 1997. Cytotoxic flavonoids from Vitexagnus-castus. Phytochemistry 46, 521–524.

ICH, Guidelines, Stability Testing of Now Drug Substances and Products, 27October 1993.

Jain, S.K., Srivastava, S., 2005. Traditional uses of some Indian plants amongislanders of the Indian ocean. Indian Journal of Traditional Knowledge 4,345–357.

Jalalpure, S.S., Patil, M.B., Prakash, N.S., Hemalatha, K., Manvi, F.V., 2003. Hepato-protective activity of fruits of Piper longum L. Indian Journal of PharmaceuticalSciences 65, 363–366.

Jang, S., Kelley, K.W., Johnson, R.W., 2008, Luteolin reduces IL-6 production inmicroglia by inhibiting JNK phosphorylation and activation of AP-1. Proceed-ings of the National Academy of Sciences, USA 105, 7534–7539.

Kumar, G.P., Chaturvedi, A., 2010. Ethnobotanical observations of euphorbiaceaespecies from Vidarbha region, Maharashtra, India. Ethnobotanical Leaflets 14,674–680.

Lee, K.H., 1968. Studies on the mechanism of action of salicylates, effect of vitaminA on the wound healing retardation action of aspirin. Journal of Pharmaceu-tical Sciences 57, 1238–1240.

Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose–effectexperiments. Journal of Pharmacology and Experimental Therapeutics 96,99–135.

S. Ghosh et al. / Journal of Ethnopharmacology 142 (2012) 714–722722

Liu, M., Matsuzaki, S., 1995. Antibacterial activity of flavonoids against methicillinresistant Staphylococcus aureus (MSRA). Dokkyo Journal of Medical Sciences22, 253–261.

Lopez-Lazaro, M., 2009. Distribution and biological activities of the flavonoidluteolin. Mini-Reviews in Medicinal Chemistry 9, 31–59.

MacKay, D.J., Miller, A.L., 2003. Nutritional support for wound healing. AlternativeMedicine Review 8, 359–377.

Madden, J.W., Peacock, E.E., 1968. Studies on the biology of collagen during woundhealing. Surgery 64, 288–294.

Mary, B., Priya, K.S., Gnanamani, A., Radhakrishnan, N., 2002. Healing potential ofDatura alba on burn wound in albino rats. Journal of Ethnopharmacology 83,193–199.

Misra, D.R., Khastgir, H.N., 1969. Terpenoids and related compounds. Chemicalinvestigation of Pedilanthus tithymaloides. Journal of the Indian ChemicalSociety 66, 843–844.

Morton, J.J.P., Malone, M.H., 1972. Evaluation of vulnerary activity by an openwound procedure in rats. Archives Internationales de Pharmacodynamie 196,117–126.

Mukherjee, K.S., Datta, S.K., Mehrotra, A., Bhattacharjee, P., 1989. Phytochemicalinvestigation of Pedilanthus tithymaloides (L) Poit. Journal of the IndianChemical Society 66, 213–214.

Mukherjee, K.S., Laha, S., Manna, T.K., Chakraborty, C.K., 1992. Chemical investiga-tion on Limnophila rogusa and Pedilanthus tithymaloides. Journal of the IndianChemical Society 69, 411–412.

Nagda, K.K., Deshmukh, B., 1998. Hemagglutination pattern of galactose specificlectin from Pedilanthus tithymaloides in diabetes mellitus. Indian Journal ofExperimental Biology 36, 426–428.

Nayak, B.S., Sandiford, S., Maxwel, A., 2009. Evaluation of the wound-healingactivity of ethanolic extract of Morinda citrifolia L. Leaf. Evidence-BasedComplementary and Alternative Medicine 6, 351–356.

Nellis, D.W., 1997. Poisonous Plants and Animals of Florida and the Caribbean.. :Pineapple Press, Sarasota, FL, pp. 182.

Neuman, R.E., Logan, M.A., 1950. The determination of hydroxyproline. Journal ofBiological Chemistry 184, 299–306.

Odimegwu, D.C., Ibezim, E.C., Esimone, C.O., Nworu, C.S., Okoye, F.B.C., 2008.Wound healing and antibacterial activities of the extract of Dissotis theifolia(Melastomataceae) stem formulated in a simple ointment base. Journal ofMedicinal Plants Research 2, 11–16.

Odontuya, G., Hoult, J.R.S., Houghton, P.J., 2005. Structure–activity relationship forantiinflammatory effect of luteolin and its derived glycosides. PhytotherapyResearch 19, 782–786.

Prasad, V., Dorle, K.A., 2006. Evaluation of ghee based formulation for woundhealing activity. Journal of Ethnopharmacology 107, 38–47.

Patil, B.S., Naikwade, N.S., Kondawar, M.S., Magdum, C.S., Awale, V.B., 2009.Traditional uses of plants for wound healing in the Sangli district, Maharash-tra. International Journal of PharmTech Research 1, 876–878.

Pettit, G.R., Ducki, S., Tan, R., Gardella, S.R., McMahon, B.J., Boyd, R.M., Pettit, R.G.,Blumberg, M.P., Lewin, E.N., Doubek, L.D., Williams, D.M., 2002. Isolation andstructure of pedilstain from a Republic of Maldives Pedilanthus Sp. Journal ofNatural Products 65, 1262–1265.

Piepmeyer, J.A., 1966. Dry-Cleaning Detergent Compositions, 4 pp. US 325402919660531.

Priya, K.S., Arumugam, G., Rathinam, B., Wells, A., Babu, M., 2004. Celosia argenteaLinn. leaf extract improves wound healing in a rat burn wound model. WoundRepair and Regeneration 12, 618–625.

Sandhya, B., Thomas, S., Isabel, W., Shenbagarathai, R., 2006. Ethnomedicinalplants used by the Valaiyan community of Piranmalai hills (reserved forest),Tamil Nadu, India: a pilot study. The African Journal of Traditional, Comple-mentary and Alternative Medicines 3, 101–114.

Seshagirirao, K., 1995. Purification and partial characterization of a lectin fromPedilanthus tithymaloides latex. Biochemical Archives 11, 197–201.

Shetty, S., Udupa, S., Udupa, L., 2008. Evaluation of antioxidant and wound healingeffects of alcoholic and aqueous extract of Ocimum sanctum Linn in rats.Evidence-Based Complementary and Alternative Medicine 5, 95–101.

Shirwaikar, A., Shenoy, R., Udupa, A.L., Udupa, S.L., Shetty, S., 2003. Wound healingproperty of ethanolic extract of leaves of Hyptis suaveolens with supportiverole of antioxidant enzymes. Indian Journal of Experimental Biology 41,238–241.

Sriwiroch, W., Chungsamarnyart, N., Chantakru, S., Pongket, A., Saengprapaitip, K.,Pongchairerk, U., 2010. The effect of Pedilanthus tithymaloides (L.) Poit crudeextract on wound healing stimulation in mice. Kasetsart Journal (NaturalScience) 44, 1121–1127.

Theoharides, T.C., 2009. Luteolin as a therapeutic option for multiple sclerosis.Journal of Neuroinflammation 6, 1–3.

Tsou, M.F., Chen, G.W., Hung, C.F., Yeh, F.T., Chang, H.L., Lu, H.F., Chung, J.G., 2001.Luteolin inhibits the growth and arylamine N-acetyl-transferase activity inNeisseria gonorrhoeae. Microbios 104, 87–97.

Tsuchiya, H., Sato, M., Miyazaki, T., Fujiwara, S., Tanigaki, S., Ohyama, M., 1996.Comparative study on the antibacterial activity of phytochemical flavanonesagainst methicillin-resistant Staphylococcus aureus. Journal of Ethnopharma-cology 50, 27–34.

Turner, R.A., 1965. Inflammatory agent in screening methods of pharmacology,second ed. Academic Press, New York.

Udupa, S.L., Udupa, A.L., Kulkarni, D.R., 1994. Anti-inflammatory and woundhealing properties of Aloe vera. Fitoterapia 65, 141–145.

Upadhyay, R.R., Hecker, E., 1974. Azafrin from roots of Pedilanthus tithymaloides.Phytochemistry 13, 752–753.

Vidottia, J.G., Zimmermann, A., Sarragiotto, M.H., Nakamura, C.V., Benedito, P.,Filho., D., 2006. Antimicrobial and phytochemical studies on Pedilanthustithymaloides. Fitoterapia 77, 43–46.

Yu, M.C., Chen, J.H., Lai, C.Y., Han, C.Y., Ko, W.C., 2010. Luteolin, a non-selectivecompetitive inhibitor of phosphodiesterases 1–5, displaced 3H-rolipram fromhigh-affinity rolipram-binding sites and reversed xylazine/ketamine-inducedanesthesia. European Journal of Pharmacology 627, 269–275.

Zhao, G., Qin, G.W., Wang, J., Chu, W.J., Guo, L.H., 2010. Functional activation ofmonoamine transporters by luteolin and apigenin isolated from the fruit ofPerilla frutescens (L.) Britt. Neurochemistry International 56, 168–176.

Zhao, G., Yao-Yue, C., Qin, G.-W., Guo, L.-H., 2012. Luteolin from purple Perilla(Perilla frutescens L., Britton) mitigates ROS insult particularly in primaryneurons. Neurobiology of Aging 33, 176–186.