WOUND HEALING AND ANTIOXIDANT POTENTIAL OF CHLOROFORM EXTRACT OF SARACA ASOCA (ROXB.)

WOUND HEALING AND ANTIOXIDANT POTENTIAL OF CHLOROFORM EXTRACT OF SARACA ASOCA (ROXB.) 

Deepti Bandarupalli ^*1, Santhrani Thaakur ², Srinivasa Babu Puttugunta ¹ and RLC Sasidhar ³

Vignan Pharmacy College, Vadlamudi, Andhra Pradesh, India

Institute of Pharmaceutical Technology, Sri Padmavathi Mahila University ², Tirupathi, India

Chebrolu Hanumaiah Institute of Pharmaceutical Sciences ³, Guntur, Andhra Pradesh, India

ABSTRACT: The chloroform extracts of S. asoca bark were studied for the wound healing potential. The wound healing activity of S. asoca bark by excision, incision and dead space wound models in albino Wistar rats and antioxidant profile of this bark extract were also studied as there is an increase in the levels of free radicals during the damage of tissue. The parameters like wound  contraction,  period  of  epithelialisation,  tensile  strength  and hydroxyproline  content were evaluated in three wound models, namely; excision, incision and dead space wound models. Besides, their influences on oxidative stress in three wound models were also estimated. The  results obtained were  compared with standard  drug nitrofurazone and control  in  terms  of  wound  contraction,  period  of  epithelialisation,  tensile strength  and hydroxyproline  content. The chloroform extract of S. asoca bark showed significant results in all models when compared to standard and control. A subservient study made on the levels of superoxide-dismutase, catalase, glutathione, vitamin C and lipid peroxidation are recorded and a significant increase in the levels of these antioxidant enzymes and decrease in the levels of lipid peroxidation was observed.  The histopathological studies have shown the well-formed dermis with minimal inflammatory cells. Increased wound contraction, collagen migration, granulation tissue formation is attributed to the phytochemical constituents present in this plant extract. Increased levels of antioxidants favour the regeneration of wounds by quenching free radicals. Phytochemical  constituents  present  in  S. asoca  may be  responsible  for  the  wound  healing  activity.

Saraca asoca, Standard drug-Nitrofurazone, Tensile  strength,  Wound contraction, Antioxidants

INTRODUCTION:    Wounds  are  the  physical injuries  that  result  in  an  opening  or  breaking  of  the  skin  and  appropriate  method  for  healing  of  wounds  is  essential  for  the  restoration  of  disrupted  anatomical  continuity  and  disturbed the functional  status  of  the  skin ¹.

Healing  of  wounds  starts  from  the  moment  of  injury  and  can  continue  for  varying  periods  of  time  depending  on  the  extent  of  wounding  and  the  process  can  be  broadly  categorized  into  three  stages;  inflammatory  phase,  proliferative  phase,  and  finally  the  remodelling  phase  which  ultimately  determines  the  strength  and  appearance  of  the  healed  tissue  ².

The  basic  principle  of  optimal  wound  healing  is  to  minimize tissue  damage  and  provide  adequate  tissue  perfusion  and  oxygenation ³.  Antioxidant  activity  was  determined  since  the  release  of  oxygen  radicals  kills invading  foreign  organisms  and clears the wound of  fibrin  matrix,  thus  enhancing the healing process, whereas  fibroblasts  play  a  crucial  role  in  wound  healing  by  initiating  the  proliferative  phase  of  repair . More  than  80%  of  the  world’s  population  still  depends  upon  traditional  medicines  for various  skin  diseases ⁴.

Various  plant  species  have  served  as  a  source  of  medicine  for people  all  over  the  world. S. asoca (family: Caesalpinaceae) is a small evergreen tree 7-10 cm high. It occurs up to the altitude 750 meters. Bark is blackish brown in colour and rough due to warty protuberances and transversely arranged lenticles.  It is of astringent taste and odourless character. S. asoca is one of the universal plant having medicinal activities.

Ashoka is ancient and reliable source of medicine. It is used in the treatment of uterine bleeding, dysmenorrhoea, depression in women, uterine fibroids, uterine sedative, leucorrhoea, piles, ulcers, astringent. Different extracts of S. asoca bark were screened against the enteric pathogen isolates, namely, Escherichia coli, Shigella sonnei and Salmonella enteritis.

All the extracts other than aqueous extract showedantimicrobial activity with the methanol extract having the highest percentage of activity ⁵. The methanolic extract of S. asoca bark was also evaluated for analgesic and anti-inflammatory activity and it is reported to show significant activity ⁶.

As  global  scenario  is  now  changing  towards the  use  of  nontoxic  plant  products  having  traditional  medicinal  uses,  S. asoca  is  evaluated  in the hastening of wound  healing by promoting maturation of fibroblasts and in the management of hypertropic scars. The removal of reactive oxygen species that rises during the inflammatory phase of wound healing is essential to hasten the wound healing process.

Therefore, the estimation of antioxidants like vitamin c, glutathione, superoxide dismutase, catalase, lipid peroxidation is also relevant because these antioxidants hasten the process of wound healing by destroying the free radicals.

MATERIALS AND METHODS: Inbred  house  Albino  and  Wistar  rats  of  either  sex  were  used  in  the  study.  The range of the weight of the animals is between 200-250g. They were housed individually in standardized environmental conditions.  All the animals were provided with food ad libitum.

Chemicals: Chloroform, O-dianisidine, hydrogen peroxide, riboflavin, 5% & 6% Trichloro acetic acid, Dinitrophenylhydrazine, Thiobarbituric acid were purchased from Sigma chemicals. Other chemicals and reagents used are of analytical grade.

Plant material and extraction procedure: Bark  of  S. asoca  was  collected  from  the  village  of  Vadlamudi,  Guntur district,  AP  India. It was identified by Dr. M.Raghuram (Department of Botany & Microbiology) Acharya Nagarjuna University, Guntur, Andhra Pradesh. They were shade dried and coarsely powdered. The  powdered material (100g)  was  placed  in  soxhlet  extractor  and  it  is  extracted  with  chloroform.  The extract is evaporated and the dried residue (15g) i.e; drug is collected.    

Phytochemical  screening: Various  chemical  tests  were  performed  to  identify  the  phytochemical constituents  present  in  the  chloroform  extract  of  S. asoca ^{7, 8}.

Acute dermal toxicity: The acute dermal toxicity was carried out in adult albino rats by “fix dose” method of OECD (Organisation for Economic Co-operation and Development) (Guideline No.434). The chloroform extract of S. asoca was applied topically at dose level 2000mg/kg and it showed no signs of erythema.

Drug formulations: The  chloroform  extract  of  S. asoca  was  formulated  as  5%w/w  and  10%w/w ointments  respectively. These  ointments  were  prepared  by  incorporating  5g  and  10g  of drug  respectively  into  100g  of  simple  ointment  base. The ointment base consists of glycol stearate, 1, 2-propylene glycol, liquid paraffin (3:6:1). This was achieved by melting together with glycol stearate, 1, 2 propylene glycol, and liquid paraffin on a hot plate/stirrer (at 45 ◦C). The chloroform extract of S. asoca was added to this molten base while stirring.

The entire mixture was stirred while cooling until a smooth ointment was obtained. The prepared ointments were stored at room temperature. The  standard  drug  used  for  wound  healing  activity  was  Nitrofurazone  0.2%w/w ⁹.

Animals: The animals were housed under controlled conditions (12 h light–dark cycle, 22–28 ◦C and 60–70% air humidity), fed with normal mice chow and water ad libitum. All animals were allowed to acclimate for at least 7 days prior to the first treatment. The rats were anesthetized before and during infliction of the experimental wounds. The surgical interventions were carried out under sterile conditions using light ether anesthesia. All animal experiments followed the guidelines for the care and use of animals established by Vignan Pharmacy College, Vadlamudi, and was approved by the Ethics Committee of Vignan Pharmacy College (7/IAEC/VPC/Pharma/RES/2011-12).

Experimental Protocol:

For each model; 4 groups were used. Each group consists of 6 animals.

Group 1, 2, 3, 4 – Served  as  control,  standard,  treatment  groups  receiving  5%  &  treatment groups   receiving  10%  ointment  respectively  for  Incision  wound  model.

Group 5,6,7,8 – Served  as  control,  standard,  treatment  groups  receiving  5%  &  treatment groups  receiving  10%  ointment  respectively  for  Excision  wound  model.

Group 9,10,11,12 – Served  as  control,  standard,  treatment  groups  receiving  5%  & treatment  groups   receiving  10%  ointment  respectively  for  Dead  space  wound  model.

Treatment Schedule: Chloroform  extracts  of  S. asoca  were  mixed  with  ointment  base  and applied  once  a  day  to  wounds of experimental  animals  until  they  were  cured.

Wound Models:

Incision Wound  Model: Two,  6 cms  long  paravertebral  incisions  were  made  through  the  full  thickness  of  the skin  on  either  side  of  the  vertebral  column  of  the  rat  ¹⁰.  Wounds were closed with interrupted sutures, 1 cm apart.  The sutures were removed on the seventh day. Wound-breaking  strength  was  measured  in  anesthetized  rats  on  the  tenth  day  after wounding.

Excision Wound Model: A rectangular skin  piece  of  full  thickness  (approximately  500 mm²)  was  removed  from  a predetermined  dorsal  area  ¹¹.  The  wounds  were  traced  on  1mm² graph  paper  on  the  day  of  wounding  and  subsequently  on  alternate  days  until  healing was  complete. The wound healing observations were shown in the figure 3.

Changes  in  the  wound  area  were  calculated,  giving  an  indication  of  the  rate  of  wound  contraction. The  number  of  days  required  for  falling  of  the  eschar without  any  residual  raw  wound  was  determined  as  the  period  of  epithelisation.

The degree of wound healing was calculated using the formula:

Percentage closure = 1-A_d/A₀ x 100

Where A_d  =  wound  area  on  corresponding  days, A₀  =  wound  area  on  zero  day

Histopathological examination: A specimen sample of skin tissues of each group of rats were taken out from the healed wounds of the animals in the excision wound model for histopathological examinations. The thin sections of the tissues were stained with Eosin I bluish solution and observed for the histological changes under microscope ¹² and shown in the figures 1 and 2.

Dead-Space Wound Model: Dead space wounds were created through a small tranverse incision made in lumbar region ¹³. A polypropylene tube (0.5 cm × 2.5 cm) was implanted subcutaneously beneath the dorsal paravertebral lumbar skin. The day of the wound creation was considered as day zero.  Granuloma tissue formed on the implanted tube was dissected out carefully on day 10.  The granulation tissue was dried in an oven at 60°C for 24hr and the dry weight was noted.  The acid hydrolysate of the dry tissue was used for the estimation of hydroxyproline content in the tissue.

Biochemical estimations: On the 2^nd day of wound induction and on complete healing blood was collected from retro orbital plexus and was estimated for the antioxidant activity ¹⁴.

Estimation of Superoxide Dismutase: To 3ml of packed blood cells equal volume of cold deionized water was added for the lysis of packed blood cells. Haemoglobin was then precipitated by the addition of chloroform and ethanol (1.5:1). This was then centrifuged for 15minutes at 3000rpm. To 100µl of supernatant 0.88ml of riboflavin and 60 µl of O-dianisidine was added and optical density was measured at 460nm ¹⁵.

Estimation of Catalase: To 0.1ml of serum 2.5ml of phosphate buffer was added and incubated for 30minutes. Later 650µl of hydrogen peroxide was added and measured at 240nm ¹⁶.

Estimation of Reduced Glutathione: To 0.5ml of citrated blood, 0.5ml of 5% trichloroacetic acid was added and centrifuged. To the supernatant1ml of sodium phosphate buffer and 0.5ml of DTNB reagent were added. The absorbance was measured at 412nm ¹⁷.

Estimation of Vitamin C: To 0.5ml of plasma, 6%TCA was added and centrifuged. 0.5ml of DNPH was added to supernatant and read at 530nm ¹⁸.

Estimation of Lipid Peroxidation: 0.1ml of plasma was treated with 2ml of TBA, HCL, TCA and placed in water bath for 15min, cooled centrifuged and the supernatant was measured at 535nm ¹⁹.

Statistical Analysis: All values were expressed as Mean ± SEM.  The  data  was  analysed  using  analysis of  variance  followed  by  student  T-test for reporting the p-value and significance with respect to the other groups.

RESULTS: Preliminary phytochemical screening of the plant showed the presence of carbohydrates, steroids, alkaloids, cardiac glycosides, anthraquinone glycosides. In incision wound model, a significant increase was observed in the skin tensile strength of 10% extract treated group when compared to control and standard (Table 1).

In studies using excision wound model the area of the wound was measured on 2, 4, 6, 8 and 10 days of post-surgery in all groups. The control group treated with simple ointment has shown little contraction compared with 10% treated group. A very rapid closure of the wound was observed on day 8 and 10 of post-surgery (p<0.0001) (Table 2).

The total wound closure was observed by day 10 of post wounding in 10% treated group and it was 20 days in the control group. Figure 2 represents the 10% treated group showing minimal inflammation with well-formed epidermis when compared to figure 1 as they contains irregularly arranged fibroblasts and macrophages. Hydroxyproline is not directly coded by DNA; however, Proline is hydroxylated to form hydroxyproline after protein synthesis. Hydroxyproline is a major component of the protein collagen.

FIGURE 1: CONTROL (H&E 400×) SHOWING WELL-FORMED BUT THICK GRANULAR CELL LAYER, THE UNDERLYING DERMIS CONTAINS DEPOSITED COLLAGEN FIBERS AND IRREGULARLY ARRANGED FIBROBLASTS AND MACROPHAGES

FIGURE 2: ANIMALS TREATED WITH S. ASOCA (H&E 400×) SHOWING THIN WELL-FORMED EPIDERMIS AND NO INFLAMMATORY CELLS IN A WELL-ORGANIZED DERMIS

FIGURE 3: APPEARANCE OF WOUND HEALING IN DAY 4, 8, 12 &16

TABLE 1: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON TENSILE STRENGTH IN INCISION WOUND MODEL

Values are expressed as Mean ± SEM (n=6); *(P<0.0001) Vs control group; +(P<0.0001) Vs NFZ group; x (P<0.0001) Vs SA 10%

Hydroxyproline and Proline play key roles for collagen stability. They permit the sharp twisting of the collagen helix. They help with providing stability to the triple-helical structure of collagen by forming hydrogen bonds. Hydroxyproline is found in few proteins other than collagen. The only other mammalian protein which includes hydroxyproline is elastin.

For this reason, hydroxyproline content has been used as an indicator to determine collagen content. Hydroxyproline contents were found to be increased significantly in the treated groups than the control group (Table 3), which implies more collagen deposition in the treated groups than the control group.

Studies on antioxidant enzymes revealed that the 10% treated group of S. asoca showed significant increase in the levels of superoxide dismutase, catalase, vitamin C, glutathione, the powerful antioxidant enzymes in the body that are known to quench superoxide radicals and there is a subsequent decrease in lipid peroxidation levels (Tables 4, 5, 6, 7, 8, 9).

TABLE 2: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON WOUND CLOSURE AND EPITHELIALIZATION IN EXCISION WOUND MODEL

Values are expressed as Mean ± SEM (n=6); ***(p<0.0001) Vs control group,**(p<0.0013) Vs control group,*(p<0.038) Vs control group; +++(p<0.0001) Vs NFZ group, ++(p<0.002) Vs NFZ group, +(p<0.0425) Vs NFZ group; xx (p<0.001) Vs SA 5%, x (p<0.045) Vs SA 5%

TABLE 3: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON HYDROXYPROLINE CONTENT IN DEAD SPACE WOUND MODEL

Valuesare expressed as Mean ± SEM (n=6); ***(p<0.0001) Vs control group; +++(p<0.0001) Vs NFZ group, ++(p<0.001) Vs NFZ group; xxx (p<0.0001) Vs SA 5%, xx (p<0.001) Vs SA 5%

TABLE 4 : EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON ENZYMATIC, NONENZYMATIC ANTIOXIDANTS AND ON LIPID PEROXIDATION IN INCISION WOUND MODEL ON SECOND DAY OF TREATMENT

Values are expressed as Mean ± SEM; n=6 in each group; *** Indicates (p<0.001) compared with control, ** Indicates (p<0.01) compared with control. +++ Indicates (p<0.001) compared with standard, + Indicates (p<0.05) compared with standard. xxx Indicates (p<0.001) compared with CE5%, xx Indicates (p<0.01) compared with CE5% .

TABLE 5: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON ENZYMATIC, NONENZYMATIC ANTIOXIDANTS AND ON LIPID PEROXIDATION IN INCISION WOUND MODEL ON SEVENTH DAY OF TREATMENT

Values are expressed as Mean ± SEM; n=6 in each group. *** Indicates (p<0.001) compared with control, ** Indicates (p<0.01) compared with control, *Indicates (p<0.05) compared with control. +++ Indicates (p<0.001) compared with standard, + Indicates (p<0.05) compared with standard. xxx Indicates (p<0.001) compared with CE5%, x Indicates (p<0.05) compared with CE5% .

TABLE 6: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON ENZYMATIC, NONENZYMATIC ANTIOXIDANTS AND ON LIPID PEROXIDATION IN EXCISION WOUND MODEL ON SECOND DAY OF TREATMENT

Values are expressed as Mean ± SEM; n=6 in each group; *** Indicates (p<0.001) compared with control, ** Indicates (p<0.01) compared with control, *Indicates (p<0.05) compared with control. +++ Indicates (p<0.001) compared with standard, + Indicates (p<0.05) compared with standard. xxx Indicates (p<0.001) compared with CE5%, xx Indicates (p<0.01) compared with CE5% .

TABLE 7: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON ENZYMATIC, NONENZYMATIC ANTIOXIDANTS AND ON LIPID PEROXIDATION IN EXCISION WOUND MODEL ON TENTH DAY OF TREATMENT

Values are expressed as Mean ± SEM; n=6 in each group; *** Indicates (p<0.001) compared with control, ** Indicates (p<0.01) compared with control, *Indicates (p<0.05) compared with control. +++ Indicates (p<0.001) compared with standard, ++ Indicates (p<0.01) compared with standard. xxx Indicates (p<0.001) compared with CE5%, x Indicates (p<0.05) compared with CE5% .

TABLE 8: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON ENZYMATIC, NONENZYMATIC ANTIOXIDANTS AND ON LIPID PEROXIDATION IN DEAD SPACE WOUND MODEL ON SECOND DAY OF TREATMENT

Values are expressed as Mean ± SEM; n=6 in each group; *** Indicates (p<0.001) compared with control, ** Indicates (p<0.01) compared with control. +++ Indicates (p<0.001) compared with standard, + Indicates (p<0.05) compared with standard. xx Indicates (p<0.01) compared with CE5% .

TABLE 9: EFFECT OF CHLOROFORM EXTRACT OF S. ASOCA ON ENZYMATIC, NONENZYMATIC ANTIOXIDANTS AND ON LIPID PEROXIDATION IN DEAD SPACE WOUND MODEL ON SEVENTH DAY OF TREATMENT

Values are expressed as Mean ± SEM; n=6 in each group; *** Indicates (p<0.001) compared with control, ** Indicates (p<0.01) compared with control. +++ Indicates (p<0.001) compared with standard. xxx Indicates (p<0.001) compared with CE5%, xx Indicates (p<0.01) compared with CE5% .

DISCUSSION: The  preliminary  Phyto-chemical  screening  of  the  chloroform  extract  of  S. asoca  showed the  presence  of  carbohydrates,  steroids,  alkaloids, glycosides. They are responsible for their wound healing and anti-oxidant property.

Wound  healing  is  a  step wise  process, which  consists  of  different  phases  such  as haemostasis,  inflammation,  proliferation,  remodelling  and  maturation  phase. The  three  different  models  were  used  in  the  present  study  to  assess  the wound  healing  activity  of  chloroform  extract  of  Saraca asoca.  The  standard   drug nitrofurazone  is  used  to  assess  the  healing  potency  of  crude  drugs . Plants are the storehouses for the variety of phytochemical constituents. The process  of  healing  is  promoted  by  various  active  principles  like  triterpenes,  alkaloids,  flavonoids  ²⁰.

Increase  in  breaking  strength  of  NFZ-ointment  treated  animals  showed  improved collagen  migration  by  increased  cross  linking. Wound  contraction  is  defined  as  the centripetal  movement  of  the  edges  of  a  full  thickness  wound  in  order  to  promote  seal of  the  defect. The  rate  of  wound  contraction  was  less  in  control  and standard  groups  when  compared  to  SA-ointment  treated  animals  increase  in  breaking strength  of  S. asoca  ointment  treated  animals  showed  improved  collagen  migration  by  cross linking.

Granulation  tissue  formed  in  the  final  part  of  the  proliferative  phase  is  primarily composed  of  fibroblasts,  collagen,  edema  &  new  small  blood  vessels. Collagen  is  a major  compound  that  strengthens  extracellular  tissue  and  is  composed  of  amino  acids, hydroxylproline, which is used  as  a  biomarker  for  tissue  collagen.  In  the present  study  hydroxyproline  estimation  is increased  levels  in  10%  treated groups  and  also  there  is  a  significant  increase  in  the  granulation  tissue.

The  wound  healing  activity  of  this  medicinal  plant  is  attributed  to  the  active constituents  present  in  it . Alkaloids, glycosides are responsible for their antimicrobial property. Glycosides are reported to be responsible for wound healing activity in Leucas hirta, Ocimmum sanctum, gratissimum. Proteins, carbohydrates provide essential nutrients for proper healing of wound ²¹.  Plant products  are  shown  to  possess  good  therapeutic  potential  as  anti-inflammatory  agents and  graduate  wound  healing, due  to  the  presence  of  active  terpenes, steroids, alkaloids  and flavanoids  ²². The cardiac glycosides exhibited antioxidant and antimicrobial properties in various plant studies ^{23, 24.} Steroids have anti-bacterial and antioxidant potential ^{25, 26}.

Antioxidant activity favours the regeneration of wounds, because the production of reactive oxygen species during the process of tissue injury aggravates the disorders in the tissue. As free radicals cause damage to membrane lipids, proteins, enzymes, nucleic acids hence, scavenging effect might be one of the essential components of wound healing. Enzymatic antioxidants (Superoxide dismutase (SOD), Catalase) and non-enzymatic antioxidants (Glutathione (GSH), Vitamin C) are known to quench radicals and thus prevent the damage of cells ²⁷.  Lipid peroxidation is a complex process occurring in aerobic cells and reflects the interaction between molecular oxygen and polyunsaturated fatty acids. By products of lipid peroxidation causes marked damage of cell structure and finally results in functional disruption. These byproducts formed under physiological and pathological conditions are scavenged by enzymatic and non-enzymatic antioxidants.

The steroids, alkaloids, cardiac and anthraquinone glycosides of  S. asoca  promotes  the  process  of  wound  healing  by  increasing  the viability and strength of  collagen  fibres,  either  by increasing  the  circulation  or  by  preventing  the  cell  damage  or  by  promoting  the  DNA synthesis.

CONCLUSION: Phytochemical  constituents  present  in  S. asoca  may be  responsible  for  the  wound  healing  activity. As S. asoca  is  having  antibacterial and anti-oxidant  property  it  could  be  advantageous  for  the  treatment  of  wound  infections.

This  study  shows  that  S. asoca  has  wound  healing  effect  when  formulated  as ointment  and  could  therefore  explain  the  success  sores,  boils  and  wounds.

AKNOWLEDGEMENT: The  authors  are  thankful  to  principal,  teaching  and  non-teaching  staff  of  Vignan Pharmacy  College, Vadlamudi, Guntur,  for  providing  all  the  necessary facilities and co-operation  for  conducting  research  work.

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    Bandarupalli D, Thaakur S, Puttugunta SB and Sasidhar RLC: Wound healing and antioxidant potential of chloroform extract of Saraca asoca (Roxb.). Int J Pharm Sci Res2014; 5(6): 2285-93.doi: 10.13040/IJPSR.0975-8232.5(6).2285-93

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