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10.

FORMULATION OF OI NTMENT

10.1. Introduction

Herbal drugs are formulated in the form of ointment and are

used topicall y for several purposes, e.g. as protectants, antiseptics,

emollients, antipruritic, keratol ytics and astringents. Ointment bases

are always anhydrous and g enerall y contain one or more

medicaments in suspension or solution or dispersion. Ointment

bases may be hydrocarbon (oleaginous), absorption, water

removable and water soluble t ype. On the basis of their level of

action, they are classified as: epidermatic , endodermatic and

diadermatic (Carter, 1987). A wound healing ointment is aimed to

heal the incised and excised wounds. In an earlier study, medicinal

plants have been reported to be very beneficial in wound care,

promoting the rate of wound healing with minimal pain, discomfort,

and scarring to the patient (Odimegwu et al., 2008)

The objective of the study was to formulate and evaluate the

original herbal extract ointment and Nanotised herbal extract from

the local medicinal plant for its wound healing propert y.

Ointments are semisolid preparations that are thicker than

creams but thinner than pastes. The ointment formulation consists of

active and inactive ingredients added to a base. For herbal

formulations, an ointment is made by emulsifying oil pha se in water

while the mixture is warm, then letting the formulation congeal at

room temperature. During the preparation, the oil is heated to

approximatel y 70 ƕ C and the water to 75 ƕ C. Usuall y the water phase

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contains an extract of the herb to be incorporated into the mixture.

When added together and mixed in the presence of an emulsifying

agent, the two phases quickl y form a stable emulsion. The mixing

continues at room temperature until the formulation begins to

congeal. Upon mixing the two phases together, the mixture initiall y

looks milky in appearance, and unless colouring agents are added to

the formulation, the final ointment is normall y white to off -white in

colour. Since the ointment is kept over a period of up to several

weeks, preservatives such as benzoic acid or its salt, sodium

benzoate, must be used in ointment preparations. Dispensation may

be in a plastic, but preferabl y, glass jar. Shelf life can be improved

by storing the ointment in a refrigerator or in a cool place. If heat is

deleterious to the herbal components, then the ointment can be

prepared without heat by incorporation method. A possible method

for incorporating an aqueous herbal extrac t into an ointment base is

to mix the extract with a small quantit y of lanolin (wool fat) using a

porcelain mortar and then incorporating the resulting mixt ure into

white petrolatum (petroleum jell y) using the same mortar for

mixing.

The herbal extract an d nano herbal extract were incorporated

to the base separately and named as extract ointment (EO) and Nano

ointment (NO).

The ointment of the Urena lobata was prepared by using the simple

ointment base I.P.

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Table: 10.1 Composition for the formulation of U rena lobata

Extract Ointment Quantity Nano extract Ointment Quantity

(EO) (g) (NO) (g)

White bees wax 2 White bees wax 2

Hard paraffin 3 Hard paraffin 3

Cetosteryl alcohol 5 Cetosteryl alcohol 5

White soft
90 White soft paraffin 90
paraffin

Urena lobata Urena lobata Nanotised


5 5
alcoholic extract alcoholic extract

Procedure

The extract and the nano extract separatel y were incorporated

into the molten simple ointment base and allowed to congeal b y

stirring. After the ointment was formulated, they we re packed in

collapsible tube separatel y.

10.1 Ointment formulation of original and nano extracts

A A - Original Extract
B C B - Extract ointment
C - Nano ointment

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10.2. Quality control of the formulated Ointment

Physical evaluations: Preliminary evaluation of formulations at

different concentrations was carried ou t as follows:

Organoleptic parameters : Organoleptic parameters like colour,

odour of the formulations were carried out by visual examination.

Loss on drying

This is employed in IP and USP. Although the loss in weight,

in the sample so tested, principall y is due to water and small amount

of other volatile material will be contribute the weight loss 1gm of

ointment is placed digital moisture balance instrument set the

temperature 105 o C and run the instrument up to constant weight.

Finall y read out the percen tage loss on drying automaticall y.

pH: The pH of various formulations was determined by using

Digital pH meter (Digital pH meter 335, S ystronics, Noroda,

Ahmedabad). The 0.5 g of the weighed formulation was dispersed in

50 mL of distilled water and the pH was (Panigrahi et al., 1997)

noted.

Homogeneity: All the developed ointments were tested for

homogeneit y by visual inspection. They were tested for their

appearance with no lumps (Panigrahi et al., 1997).

Viscosity: The measurement of viscosit y of prepared ointments was

carried out with Brookfield Viscometer (model LV -DV-II, Helipat h

spindle t ype S -96). The values of each formulation were done in

triplicate and average values were depicted in Table 10.2. The

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viscosit y values are expressed as Mean ± Stan dard deviation

(Kim et al., 2003).

Spreadability: Spreadabilit y of the formulation was determined by

an apparatus suggested by Mutimer et al. (1956) which was suitabl y

modified in the laboratory and used for the study. The experiment

was performed as described by Wood et al. (1963).

Spreadabilit y was determined by using the formula

S = MxL/T.

Where S = spreadabilit y, M = Weight tied to upper slide, L =

Length of glass slides and T = Time taken to separate the slides

completel y from each other. In this present experiment, M = 80 g,

L = 10 cm and T was recorded (Ehrlich and Hunt, 1968 ).

Acute skin irritation study: This test was performed on albino rats

weighing between 150 -200g. The animals were given standard

animal feed and had free access to water ad libitum. The total mass

was separated into four groups, each batch containing five animals.

Dorsal hair at the back of the rats were removed one day prior to the

commencement of the study and kept individuall y in cages to avoid

contact with the other rats. Two groups of each were used for

control and standard irritant. Other two groups were used as test.

The 50mg of the each formulation were applied over one s quare

centimetre area of whole and abraded skin to different animals.

Aqueous solution of 0.8 % formalin was used as standard irritant.

The animals were observed for seven days for any signs of oedema

and erythema ( Marzulli and Maibach, 1997 ).

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Stability studies: The stabilit y studies were carried out in all

formulations at different temperature conditions (4°, 25° and 37°C)

for 3 months. All the evaluation parameters i.e. , pH, viscosit y,

spreadabilit y, consistency and phase separation were studied at

different time intervals i.e., 15, 30, 60 and 90th days ( Shinde et al.,

2005; Mohanta et al., 2007).

Table: 10.2 Stability studies of the ointment of the Urena lobata extracts

Physicochemical
Formulation
parameters

Colour Light green

Odour Characteristic

Loss on Drying 9.5 % w/w

pH 6.5 (5.95 - 6.56)

Spreadabilit y(Seconds) 15 (between 18 sec)

Diffusion study 0.9 cm (between 1 cm)

No skin irritation was


Skin irritation study
observed

Storage( 4 ° ,24 ° ,37 ° C) Stable

In-vitro drug release study:

In In-vitro diffusion study of the ointment was carried out on

Franz diffusion cell having 57 m l capacit y. Whatman filter paper

no.41 was used as diffusion membrane. Pieces of Whatman filter

paper no.41 were soaked in phosphate buffer (PB) pH 6.0 for 24

hours, prior to experiment. Diffusion cell was filled with phosphate

buffer pH 6.0, Whatman filter paper no.41 was mounted on cell. The

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temperature was maintained at 37 ± 0.5°C. The formulation was

spread on the filter paper as a thin layer. The time point fo r

ointment was different. A sample of 1ml was withdrawn at

predetermined time intervals, the solution was filter ed with 0.45

micron filter paper and make up the volume with 5 ml of PB pH 6.0

and equivalent amount of fresh dissolution fluid equilibrated at

same temperature was replaced. The sample was diluted to 5 ml with

PB at pH 6.0. The standard also was prepared as the same

concentration of that of sample. The amount of drug permeated was

determined using a UV - spectrophotometer at 340 nm. (Linearit y

range = 10-20µg/ml, R2 = 0.9988)

Weight of the ointment-10 mg

Volume of solution (7.4 pH): 100 ml

Amount of sample withdrawn: 5 ml

Absorbance: 304 nm

Table: 10.3 In-vitro drug release study

NANO OINTMENT

Time Absorbance

15 mts 0.114

30 mts 0.183

45 mts 0.214

1 hour 0.302

2 hours 0.41

3 hours 0.522

4 hours 0.602

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5 hours 0.64

6 hours 0.712

EXTRACT OINTMENT

Time Absorbance

15 mts 0.232

30 mts 0.262

45 mts 0.284

1 hr 0.362

2 hours 0.488

3 hours 0.52

4 hours 0.612

5 hours 0.632

6 hours 0.702

Fig 10.2 Percentage drug release of the extract ointment and

nano ointment

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10.3. RESULTS AND DISCUSSION

The various physicochemical parameters utilized to evaluate

the prepared ointment formulations are shown in Table 10.2.

¾ The pH of the formulations lies in the normal pH range of the

human skin (6.5 ± 1).

¾ All the formulations did not produce any skin irritation, i.e.,

erythema and edema for about a week when applied over the

skin.

¾ The rheological behaviours of the different formulations of

ointments in Rotational Brookfield Viscometer indicated that

the speed of spindle increases viscosit y decre ases. A

comparative study of viscosit y and spreadabilit y showed that

the viscosit y of the formulations increases, spreadabilit y

decreases and vice versa.

¾ These formulations did not produce any skin irritation for

about a week when applied over the skin.

¾ From the stabilit y studies, Ointments showed no changes in

pH, viscosit y, spreadabilit y, consistency and phase separation

after keeping at different temperatures for 90 days.

Discussion

The mechanical evaluation parameters like pH, viscosit y,

spreadabilit y, homogeneit y are important tests to evaluate

pharmaceutical ointment formulations. The result of all the

formulations near to pH 6.5 ± 1 indicates better chemical

compatibilit y of ointments with skin. The results of viscosity gives

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an idea about measureme nt of strength and the result of

spreadabilit y denote the extent of area to which the prepared

formulations readil y spreads on application to skin or affected part

and homogeneit y confirms no lumps. The results of stabilit y study

indicate that there was no change in results of evaluation parameters

of prepared ointments during the treatment period. The absence of

erythema and edema for about a week when the ointments are

applied over the skin for skin irritation test indicates patient

compliance and fewer s ide effects. The results of the physical

evaluation of ointment preparation with ethanolic extract and the

nano extract of Urena lobata indicated the suitabilit y of method for

the production of ointments ( Ansel et al., 2005).

188
References:

¾ &DUWHU 6-  &RRSHU DQG *XQQ¶V 'LVSHQVLQJ IRU

Pharmaceutical Students: Ointments, Pastes and Jellies. 12 t h

Edition, C BS Publishers and Distributors, India,

¾ Chakole CM, Shende MA, Khadatkar SN. ³Formulation and

evaluation of novel combined halobetasol propionate and

fusidic acid ointment ´. International Journal of Chem -Tech

Research. 2009 Jan-March ;1(1):103 -106

¾ Ehrlich, H.P. and T.K. Hunt, 1968. ³Effect of cortisone and

Vitamin A on wound healing ´. Ann. Surg., 167: 324 -328.

¾ Fites et. al. J. Pharm. Sci. 1970; 59(2): 610-11.

¾ Gupta GD and Gaud RS. ³Release rate of Nimesulide from

different gellants ´. Ind. J. Pharm. Sci . 1999; 61(4): 227 -30.

¾ Indian pharmacopoeia, microbiological test and assay, 1996,

1, Appendix 4: A. 88 -A.11.

¾ Kim, J.Y., J.Y. Song, E.J. Lee and S.K. Park, 2003.

³Rheological properties and microstructure of carbopol gel

network s ystem ´. Colloid Polymer Sci ., 281: 614-623.

¾ Marzulli, F.N. and H.I. Maibach, 1997. ³Advance in Modern

Toxicology´. Hemisphere Publishing Corporation, London,

pp: 193-210.

¾ Mohanta, G.P., M. Jamal and S. Umadevi, 2007. ³Formulation

and evaluation of a pol y herbal wound healing cream ´. Indian

Drugs, 44: 281-281.

189
¾ Mutimer, M.N., C. Riffikin, J.A. Hill, E. Marry and C.N.G.

Glickman, 1956. ³Synthesis of methylsil yl derivat ives of

procaine and their diffusion ´. J. Am. Pharm. Assoc. Sci ., 45:

212-212.

¾ Odimegwu, D.C., Ibezim, E.C., Esimone, C.O ., Nworu, C.S.,

Okoye, F.B.C., ³Wound Healing and Antibacterial Activities

of the Extract of Dissotis theifolia (Melastomataceae) Stem

Formulated in a Simple Ointment Base ´. J Medicinal Plant

Res 2008,2(1): 011-016.)

¾ Panigrahi, L., T. Jhon, A. Shariff and R.S. Shobanirani,

³Formulation and evaluat ion of lincom ycin HCL gels ´. Ind. J.

Pharm. Sci., 1997, 59: 330-332.

¾ Peruru D. et. al. ³Isolation of eumelanin from Sepia

officinalis and investigation of its antimicrobial activit y by

ointment formulation ´. Int.J.Pharm.2012;2(2):67-72.192-210.

¾ Purushothamrao K, Khaliq K, Sagare P, Patil SK, Kharat SS,

Alpana.K. ³Formulation and evaluation of vanishing cream

for scalp psoriasis ´. International Journal of Pharma Sci

Tech. 2010; 4(1):33-41.

¾ Shinde, A.J., S.B. Bhise, R .J. Jarag and N.R. Jadhav, 2005.

³Preparation of cream containing Tridax procumbens,

Curcuma longa and Azadirachta indica and its evaluation for

wound healing propert y´. Indian Pharm., 4:107-110.

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¾ United States Pharmacop oeial Convention. Pharmacop oeial

Forum, Rockville (MD). Topical and transdermal drug

products-product quality tests 3, 2009; 35(3): 1-26.

¾ United States Pharmacopoeial Convention. Pharmacopoeial

Forum, Rockville (MD). Topical and transdermal drug

products -product performance tests 725, 2009; 35(3): 1-28.

¾ Wei Liu, Meiling Hu,Wenshuang Liu, Chengbin Xue, Huibi

Xu, XiangLiang Yang, International Journal of

Pharmaceutics ., 2008, 364, 135±141.

¾ Wood, J.H., G. Catacalos and S.V. Liberman, 1963.

³Adaptation of commercial viscometers for special

applications in pharmaceutical rheology II. Severs extrusion

rheometer´. J. Pharm. Sci ., 52: 375-378

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11. WOUND HEALING STUDY OF THE FORMULATIONS OF

Urena lobata

11.1. Introduction

Wound healing processes are well organized biochemical and

cellular events leading to the growth and regeneration of wounded

tissue in a special manner. Healing of wounds is an important

biological process involving tissue repair and regeneration. It

involves the activity of an intricate network of blood cells,

cytokines, and growth factors which ultimatel y leads to the

restoration to normal condition of the injured skin or tissue (Clark,

1991). The aim of wound care is to promote wound healing in the

shortest time possible, with minimal pain, discomfort, and scarring

to the patient and must occur in a physiologic environment

conducive to tissue repair and regeneration (Bowler et al., 2001).

Wound healing are convenientl y classified into any of three

types, healing by first intention, healing by second intention and

healing by third intention, depending on the nature of the edges of

the healed wounds. Whereas the edges of wounds healed by first

intention are smoothly closed that no scar is left, wounds healed by

second intention involve formation of granulation tissues which fill

up the gaps between the wound edges and are associated with

significant loss of tissue, leaving little scars. Wounds healed by

third intention are usuall y those wounds left for three to five days

until granulation bed falls before they are sutured re sulting in

extensive scars formation (Thomas, 1997). Four distinct stages of

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wound healing have also been identified -inflammatory,

debridement, proliferation, and remodelling maturation stages.

Wound healing processes are known to be influenced by

among other factors by infections, nutritional status, drugs and

hormones, t ype and sites of wound, and wasting diseases like

diabetes (Karl et al, 1995). In folklore medicine, medicinal plants

have been used widel y in facilitating wound healing with high

degree of successes. This has inspired many researches which are

aimed at validating the claims and discovering mechanisms which

possibl y explains the potentials of these herbs on wound repair

processes.

In our investigation the two different formulations in the form

of Ointments (original extract ointment (EO) and nanotised extract

ointment (NO) from the plant Urena lobata were evaluated using the

incision and excision models with Swiss albino rats.

Chemicals

All chemicals and reagents used were of anal ytical grade.

Experimental animals

Swiss Albino rats of 200 to 250 g body weight were used in

the study. Animals were procured from BiogenLaboratory Animal

Facilit y (CPCSEA -Reg no. 971 /bc/ 06), Bangalore.

All animals were kept in pol yacrylic cages and main tained

under standard housing conditions (room temperature 24 Ǧ27°C and

humidit y 60Ǧ65% with 12:12 light: dark cycles. Food was provided

in the form of dry pellets and water ad libitum.

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All experiments involving animals complies with the ethical

standards of animal handling and approved by Institutional Animal

Ethics Committee (IAEC/XXXIV/SRU/284/2013).

Model design

The design of wound healing activit y was performed b y two modelsǦ

1) Excision wound model

2) Incision wound model

11.2. Materials and Methods

Male Wister rats were used for excision and incision wound

models and the ointments were applied topicall y and animal were

divided into the following groups.

GroupǦ I : No wound was created and served as control.(6 animals)

GroupǦ II: wound was created and served as positive control.

(6 animals)

GroupǦ III: 5%w/w Extract ointment (EO) was applied once dail y.

i) Excision-4 animals

ii) Incision -4 animals

GroupǦ IV: 5%w/w Nanotised extract ointment (NO) was

applied once dail y.

i) Excision- 4 animals

ii) Incision- 4 animals

GroupǦ V: Betadine ointment (5 gm) was applied once dail y

i) Excision-4animals

ii) Incision-4 animals

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1) Excision wound model

The back of each rat was shaven under ether anaesthesia

and prepared for operation. Thereafter open circular wound of

500 mm 2 area was produced in each rat b y excising the skin.

For this purpose a marker was used to mark the area to be

excised.

The wounded animals were kept separately.

Rats wound were left undresse d to the open environment; this

model was used to monitor wound contraction and epithelisation

time. The standard drug (Betadine ointment (0.5 g m), simple

ointment; ethanolic extract ointment (EO) 5 %w/w and 3 %w/w

Nanotised extract ointment (NO) were applied everyday till the

wound was completel y healed.

Incision Wound Model

Animals were grouped which was divided into four groups

same as followed in excision wound model. The incision wound

model was studied under light ether anesthesia the anim al was

secured to operation table in its natural position. A longitudinal

paravertebral incision of 6 cm long was made through the skin and

cutaneous tissue on the back. After complete haemostasis, the

wound was closed by means of interrupted sutures place d at

equidistance points about 1 cm apart. Animals were treated dail y

with drugs, as mentioned above under excision wound model from

0 t h day to 9 t h post wounding day. Wounds were cleaned with 70 %

alcohol soaked with cotton swabs. They were kept in separat e cages.

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All the sutures were removed on the 9 t h post wounding day. On 10 t h

day the tensile strength was measured by continuous constant water

suppl y technique.

All the above mentioned treatments were started from the day

of operation and continued till 20 t h day of healing. On 2 n d , 4 t h , 8 t h ,

10 t h , 12 t h , 14 t h , 16 t h , 18 t h and 20 t h days the wound area of each

rat was traced on a graph paper. Animals were sacrificed by

cervical dislocation on day 30. Liver, spleen, stomach and skin were

collected and processed for histopathological studies.

The following physical and biochemical parameters were studied

11.2.1. Physical Evaluation

¾ Body weight

¾ Wound size and area

¾ Tensile strength

11.2.2 Antioxidant Studies

¾ Superoxide Dismutase (SOD) activity (Mark lund and

Marklund, 1974)

¾ Glutathione Peroxidase activit y (GPX) (Moren et al., 1973)

¾ Estimation of Reduced glutathione (GSH) (Moren et al.,

1979)

¾ Activit y of Catalase (Asru K Sinha, 1987)

11.2.3 Biochemical studies

¾ Myeloperoxidase activit y (Bradley et al., 1982)

¾ Total protein by Biuret method (Biuret et al., 1948)

¾ Hydrox y proline content (R.E.Newman et al, 1950

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11.2.1 Physical evaluation

1. Measurement of wound area

The progressive changes in wound area were measured planim

etricall y by tracing the wound margin on a graph paper every altern

ate dayThe changes in healing of wound i.e the measurement of wou

nd on graph paper was expressed as unit (mm 2 ).

Wound contraction was expressed as percentage reduction of

original wound size.

Wound area - Unhealed Area


% Wound Contraction = ǦǦǦǦǦǦǦǦǦǦǦǦǦǦǦǦǦǦǦ ----------- X 100
Wound area
2. Measurement of Wound Breaking Strength of Incised Wounds

Measurement of wound breaking strength was pe rformed by

using the following method with certain modifications. A board was

placed on the table, on which the anaesthetized animal was made to

lie on its abdomen. Two clamps were clamped on either sides of

healed wound at a distance 0.5 cm. the left clam p was fastened

tightl y to stand by means of thread. The right clamp was connected

to a leak proof pol yt hene container through a pulley, by means of a

thread. A reservoir containing water was placed at a suitable height

and connected to a pol ythene bag by m eans of a rubber tube. The

position of the board was adjusted so that, the pol ythene bag was

hanging freel y. Water was added to pol ythene bag rapidl y at

constant rate from the reservoir until the wound opened. Amount of

water in pol ythene bag was measured (in ml) and was considered as

tensile strength of the wound.

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11.2.2 Antioxidant Studies

1. Superoxide Dismutase (SOD)

0.05 ml of sample is added to 0.3 ml of sodium pyrophosphate

buffer (0.025 M, pH 8.3), 0.025 ml of phenazonium methosulphate

(186 µM) and 0.075 ml of Nitroblue tetrazolium chloride (300 µM

in buffer of pH 8.3).The reaction was started by addition of 0.075

ml of reduced nicotinamide adenine dinucleotide (780 µM in buffer

of pH 8.3).After incubation at 30º C for 90 seconds, the reaction

mixture was stirred vigorousl y and shaken with 2.0 ml of n -butanol.

The mixture was allowed to stand for 10 minutes and centrifuged.

1.5 ml of n-butanol alone was served as blank. The colour intensit y

of the chromogen was read at 560 nm using thermo scientific

multiscan spectrometer, USA (Kakkar et al., 1984).

Enz yme activit y (1 unit) =50% inhibition/minute

2. Glutathione Peroxidase (GPX)

Glutathione peroxidise (GPX) was assayed by taking 200 µl of

tris HCl buffer (0.4 M), 200µl K.EDTA (0.4 mM) along with 100 µl

of sodium azide and 200 µl of sample and mixed well. Thereafter,

200µl of reduced glutathione solution (2 mM) followed by 0.1ml of

H 2 O 2 were added and allowed to incubate for 10 min at 37º C. The

overall reaction was then arrested by adding 0.5 ml of 10 %

trichloroacetic acid (TCA).The precipitate was removed by

centrifugation at 1500 rpm for 10 minutes. To 0.2 ml of the

supernatant, 0.5 ml of saline and 1.0 ml of di -thio nitro benzoic acid

(DTNB) were added and the colour intensit y formed was absorb ed at

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412 nm using Thermo Scientific Multiskan Spectrophotometer,

USA, (Rotruck,et al.,1973)

3. Reduced Glutathione (GSH)

Reduced glutathione was assayed by taking 0.25 ml of sample

to equal volume of ice cold 5 % TCA. The precipitate was removed

by centrifugation at 3500 rpm for 10 minutes.1 ml of the

supernatant was mixed with 0.25 ml of 0.2 M phosphate buffer, pH

8.0 and 0.5 ml of DTNB (0.6 mM in 0.2 M phosphate buffer, pH

8.0) were added and mixed well.The absorbance was read at 412 nm

using Thermo Sci entific Multiskan Spectrophotometer,USA (Moren

et al.,1979).

4. Activity of Catalase:

The assay mixture contained 100 µl of sample, 0.4 ml of H 2 O 2

(2 mM) and 0.5 ml of phosphate buffer (10mM, pH 7.4).The above

mixture was stirred well and incubated at 37° C for 5 min and then

dichromate acetic acid reagent (5 % potassium dichromate in water,

glacial acetic acid mixed in 1:3 ratio) was added and absorbance

was taken at 570 nm using Thermo Scientific Multiskan

spectrophotometer, USA. 2 ml of dichromate acetic acid reagent

acts as blank (Asru K Sinha , 1987).

5. Myeloperoxidase activity

1ml of sample was mixed with equal volume of hexadecyl

trimethyl ammonium bromide (HTAB) buffer and centrifuge at 4ºC

for 15 minutes at 3000 rpm. Then 0.5 ml of supernatant was taken

and added to 3.5 ml of phosphate buffer (pH 6.0) followed by 1.5 ml

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of dinizidine. The absorbance was read at 450nm at 0 sec and 60 sec

against blank using Thermo Scientific Multiskan spectrophotometer,

USA (Bradley et al., 1982).The results were exp ressed in terms of

units (Nanomoles H 2 O 2 lib/min/mg protein).

6. Total Protein by Biuret method

0.6 ml of saline was mixed with 50 µl of sample followed by

addition of 1.25 ml of working biuret reagent. The tubes were

incubated at room temperature for 15 min.The color

intensit y was read at 540nm using Thermo Scientific

Multiskanspectrophotometer, USA, (Lubran et al., 1948).

7. Hydroxyproline (HPR) (R.E.Newman et.al., 1950).

To each tube, 0.3 mL of hydrol ysate, 2.5 N NaOH, 0.01 M

CuSO 4 , and 6 % H 2 O 2 were added. Tubes were shaken vigorousl y

and placed immediatel y in water bath at 80 ° C. After 15 minutes,

tubes were removed and cooled for 5 minutes in cold water. 0.6 m L

of freshl y prepared 5 % solution of paradimethyl amino -

benzaldehyde in n -propanol and 1.2 m l of 3 N H 2 SO 4 was added.

The test tubes were once again placed in a hot water bath at 75 ° C

for 15 minutes and then cooled for 5 minutes under running stream

of water. Color intensit y was measured at 540 nm against the blank.

Hydrox yproline content in the tissue was estimated as per standard

curve prepared with standard 4 -Hydrox y-L-proline (HiMedia

Laboratories Pvt. Ltd., Mumbai, India), from 75 to 900 g/0.3 mL

using 3 mg/m L working solution

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Results

Table: 11.1 Tensile Strength

NO= Nano extract ointment

EO= original extract ointment

All values are mean SEM ± n =6, **P < 0.001 indicates

extremel y significant compared to the control

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Fig: 11.1 Tensile strength of the tissue of wound treated rats

600

500

400

300

200

100

0
Control STD NO EO

Table: 11.2 Comparison of wound area Using

³.UXVNDO-:DOOLV7HVW´

Days Groups Mean SD Chi P value

Square

Day 1 Group 2 136.166 95.1155 5.261* 0.154*

Group 3 173.148 55.4694

Group 4 252.703 112.347

Group 5 188.750 63.6295

Day 4 Group 2 104.777 64.0765 2.842* 0.417*

Group 3 143.036 63.6552

Group 4 161.333 77.7134

Group 5 113.041 37.1494

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Day 7 Group 2 60.7777 46.6074 1.580* 0.664*

Group 3 87.5554 55.7997

Group 4 83.0740 42.9695

Group 5 68.3376 24.4996

Day 10 Group 2 29.6111 13.0833 5.070* 0.167*

Group 3 54.7036 30.8330

Group 4 53.1481 28.2970

Group 5 41.5416 12.6866

Day 13 Group 2 23.1666 8.79582 4.972** 0.174**

Group 3 37.8148 23.0579

Group 4 36.9999 15.6720

Group 5 27.0833 10.9047

Day 16 Group 2 11.6666 9.33333 10.48** 0.015**

Group 3 22.4443 9.14694

Group 4 17.18512 3.909089

Group 5 12.0208 2.07869

Day 19 Group 2 3.77778 3.02336 5.648** 0.130**

Group 3 6.11111 2.47206

Group 4 5.03704 3.27636

Group 5 3.95833 3.93774

*p value < 0.05 -Significant ** p value< 0.01 -Highly significant

203
1. Effect of Extract ointment and Nano ointment on Protein and

Hydrox y Proline content on Dry Connective Tissue

Table: 11.3 Estimation of Protein & Hydroxy proline

Groups Protein Hydrox yproline

mg / g dry tissue g / mg protein

Control 198.1 ± 12.4 132.2 ± 10.6

Std 272.3 ± 11.3* 197.2 ± 9.02**

NO 297.5 ± 15.0* 193.1 ± 10.2**

EO 305.6±11.3* 203.6±9.9**

Values are me an ± SEM of 6 rats in each group. * <0.05, **<0.01

compared to respective control group (statistical anal ysis was done

by using Mann Whitney test).

1. Effect of Extract ointment and Nano ointment on Protein and

Myeloperoxidase content on Wet granulation Tissu e

Table: 11.4 Estimation of Protein & Myeloperoxidase

Groups Protein mg / g wet Myeloperoxidase

tissue mU/ mg protein

Control 38.6 ± 2.25 22.1 ± 0.57

Std 53.7 ± 2.12* 17.9 ± 0.29**

NO 62.1 ± 2.98* 13.8 ± 0.37**

EO 66.20±2.66* 12.9±0.29**

Values are m ean ± SEM of 6 rats in each group. *<0.05, and

** <0.001 compared to respective control group (statistical anal ysis

was done by using Mann Whitney test).

204
Fig 11.2 Wound healing progression in treated rats

Results and Discuss ion

Collagen is the predominant extracellular protein in the

granulation tissue of a healing wound and there is a rapid increase

in the s ynthesis of this protein in the wound area soon after an

injury. Breakdown of collagen liberates free hydrox yproline a nd its

peptides. The observed increase in hydroxyproline, specific marker

of collegan and an important component of extra cellular

granulation tissue matrix in the wounds treated with Urenalobata

clearl y indicated the rapid collegan turnover and accumulati on,

explains the increased rate of wound contraction (Geethalakshmi et

al 2013).

Studies on the antioxidant, free radicals and MPO status

revealed that Urenalobata had significant antioxidant activit y,

reduced MPO and free radical stress facilitated in pr evention of

inflummation and oxidative damage thereby promoting healing

process (Murthy et al 2013).

205
The wounds treated with ointments showed significant

increase in tensile strength which confirms collagen formation

thereby promotes wound healing (Pickett BP et al 1996).

The estimation of a ntioxidants such as GSH, SOD, CAT is

relevant because the antioxidant s have been reported to hasten

wound healing (Murthy et al 2013).

Scavenging of free radical and reactive ox ygen species

promote contraction of the wound and increasing the formation of

capillary vessels and fibroblasts (Murthy et al 2013).

Increasing total protein confirms the tissue synthesis and

fighting infection and body tissue (James TJ et al 2000).

Table 11.5 Antioxidant Studies

Treatment LPO (mg/dl) SOD (IU/mg CAT(µM/mg GSH(µg/mg

protein tissue) tissue)

NC 12 ± 1.58 5.59 ±1.17 26.94 ± 5.28 3.24 ± 0.29

PC 14.05 ± 0.81 1.70 ±0.48 12.90 ± 2.01 1.32 ±0.32

STD 10.22 ± 1.06*** 3.49± 1.80** 25.08 ±3.45*** 2.51 ± 0.45*

NO 8.04 ± 1.16*** 2.80±1.73* ** 19.85 ±5.14*** 1.88 ±0.33***

EO 9.25 ±1.54*** 3.21±0.60*** 20.81 ±2.57*** 2.52 ± 0.22***

Values are mean SEM of 6 rats in each group. * -P value is <0. 05,

**-P value is <0. 01 and *** -P value is <0. 001 compared to control by

.UXVKNDOZDOOL¶VWHVW

206
Fig: 11.3 LPO on wound treated rats

Values are expressed in mean± SEM of 6 rats in each group

Fig: 11.4 SOD on wound treated rats

Values are expressed in mean± SEM of 6 rats in each group

207
Fig: 11.5 Catalase on wound trea ted rats

Values are expressed in mean± SEM of 6 rats in each group

Fig: 11.6 GSH on wound treated rats

Values are expressed in mean± SEM of 6 rats in each group

208
11.3. Histopathology

Procedure:

Wound was created in the rat skin by incision and excision

method. On day 30, all rats from each group were euthanized using

anesthetic ether. The skin from the incised and excised region were

collected and fixed in 10% neutral buffered formalin for 48 hours.

The skin tissues were subjected to dehydration in series of graded

alcohol and embedded in paraffin wax. Tissue sections of 4 to 5

micron thickness were obtained and stained with Hematox ylin and

Eosin (Bancroft and Gamble, 2008 ) for light microscopic

exaPLQDWLRQ 7KH VDPSOHV ZHUH DOVR VWDLQHG ZLWK 0DVVRQ¶V WULFKRPH

staining for specific evaluation of fibrous tissue proliferation.

Histopathology studies of treated rats for both extract and nano

ointment

Fig: 11.7

Hypodermis
Muscle
Hair follicles

Dermis

Control group MST 4x Control group Skin H & E 10x

209
Fig: 11.7.1

Hair follicles

Skin erosion

Induced group ± Epithelial erosion and mono nuclear

infiltrates in the dermis region

H& E 40 X

Fig: 11.7.2

Induced group ±Epithelial erosion dermal Infiltration and

increased fibroblast Proliferation H& E 10 x

Fig: 11.7.3

Induced group region of the skin - MST 10 x

210
Fig: 11.7.4

Moderate degree of erosion with dermis infiltration - H& E 40 x

Fig: 11.7.5

Nano extract ointment ±neovascularisation and Infiltration in

dermis H & E 40 x

Fig: 11.7.6

Nano extract ointment MST 47 X

211
Fig: 11.7.7

Hair follicles

Gland

Nano extract ointment ±H & E 10 X

Fig: 11.7.8

Extract ointment ±Dermal fibrosis with infiltration H& E 40X

Fig: 11.7.9

Extract ointment-Inflammatory cells in the dermis - H& E 40X

212
Fig: 11.7.10

Extract ointment ±MST 4x

Fig: 11.7.11

Epidermis

Dermis

Secretory glands

Extract ointment ±Fibrosis in healed region -H&E 10x

Histopathology report on the evaluation of wound healing

activityof herbal ointment in rats

Wound was created in the rat skin by incision and excision

method. On day 30, all rats from each group were euthanized using

anesthetic ether. The skin from the incised and excised region were

collected and fixed in 10 % neutral buffered formalin for 48 hours.

The skin tissues were subjected to dehydration in series of graded

alcohol and embedded in paraffin wax. Tissue sections of 4 to 5

micron thickness were obtained and stained with Hematox ylin and

Eosin (Bancroft and Gamble, 2008 ) for light microscopic

213
H[DPLQDWLRQ 7KH VDPSOHV ZHUH DOVR VWDLQHG ZLWK PDVVRQ¶V WULFKRPH

(MST) staining for specific evaluation of fibrous tissue

proliferation.

The skin from the control group revealed normal histology of

epidermis, dermis and hypodermis.

The H&E stained skin section from the induced group

revealed moderate degree of epithelial erosions, ballooning

degeneration of epithelium, incoherent and flaky layers of keratin,

moderate degree of pol ymorphonuclear leucocytes, mononuclear

cells infiltration in the dermis and increased fibroblast proliferation

along with neovascularization.

The skin tissues of standard group revealed absence of

epithelial erosions, re -epithelisation but increased mononuclear

cells infiltration along with fibroblast proliferation in the heale d

wound region of skin.

The skin treated with Nano herbal and extracts ointment

revealed re-epithelisation, thin epidermis, mild to moderate degree

of dermal fibrosis with cutaneous adnexal atrophy (loss of hair

follicles and sebaceous glands) in wound he aled region along with

neovascularization and mild degree of inflammatory infiltrates in

the dermis region composed of mononuclear cells.

The skin wound treated with nanoherbal and extract ointment

revealed re-epithelisation, fibrosis in the healed regio n and the

inflammatory infiltration persists but the proportion of

214
inflammatory cells was reduced than those observed in the standard

and induced group.

These histopathology findings suggest that nanoherbal and

extract ointment treated group revealed bet ter wound healing

propert y as evident by fibrosis and the reduced inflammatory

infiltrates when compared to that of the standard. The fibrosis was

IXUWKHU FRQILUPHG E\ 0DVVRQ¶V WULFKRPH VWDLQLQJ LQ VNLQ WLVVXHV RI

induced, standard, nanoherbal and extract ointment treated groups.

215
216
SCORING OF WOUND FROM 0TH DAY TO 21ST DAY

Each area represents values in sq.cm


Each row represents groups

217
Each area represents values in sq.cm
Each row represents groups

218
Each area represents values in sq.cm
Each row represents groups

219
Each area represents values in sq.cm
Each row represents groups

220
Each area represents values in sq.cm
Each row represents groups

221
Each area represents values in sq.cm
Each row represents groups

222
Each area represents values in sq.cm
Each row represents groups

223
Each area represents values in sq.cm
Each row represents groups

224
Each area represents values in sq.cm
Each row represents groups

225
Each area represents values in sq.cm
Each row represents groups

226
Each area represents values in sq.cm
Each row represents groups

227
Each area represents values in sq.cm
Each row represents groups

228
Each area represents values in sq.cm
Each row represents groups

229
Each area represents values in sq.cm
Each row represents groups

230
Each area represents values in sq.cm
Each row represents groups

231
Each area represents values in sq.cm
Each row represents groups

232
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