EVALUATION OF THE EFFICACY ACTIVITY OF HERBAL FORMULATIONS IN RAT MODEL OF EXCISION WOUND EXPOSED TO INFECTION OF STAPHYLOCOCCUS AUREUS1.0 TEST SYSTEM DETAILS:
Species : Rattus norvegicus (Rats)
Strain : Sprague Dawley or Wistar
Age : 6-7 weeks
Sex : Male
No. of animals: 8/Group
Total animals: 64
2.0 TEST ARTICLES DETAILS
HF1: Herbal Formulation
3.0 VEHICLE DETAILS
The test articles will be formulated by utilizing a carrier gel as the vehicle.
4.0 ALLOCATION OF GROUPS:
|
Group No. |
Group
Description |
Wound
Generation procedure |
Treatment
administered |
Quantity
and Route |
|
G1 |
Excision wound without Infection |
500 mm2 full-thickness skin will be exercised from the dorsal interscapular without Infection |
No Treatment |
10 mg/mm2, Topical application on the wound |
|
G2 |
Excision wound with Infection |
500 mm2 full-thickness skin will be exercised from the dorsal interscapular
region of rats and inoculated with the 0.1 ml S. aureus (approximately 109 CFU). |
No Treatment |
|
|
|
||||
|
G3 |
Excision wound with Infection vehicle control |
Treated with carrier gel |
||
|
G4 |
Reference Control |
Mupirocin ointment - once daily |
||
|
G5 |
Treated with low concentration of HF1 |
HF1:
1-3%, twice daily |
||
|
G6 |
Treated with intermediate dose 1 of |
HF1:
3-10%, twice daily |
||
|
G7 |
Treated with intermediate dose 2 of |
HF1:
10-30%, twice daily |
5.0 METHOD:
· Healthy animals will be selected for the study, randomized based on body weight, and will be assigned to 8 groups consisting of 8 animals each.
· Excision wounds without infection and with infection (animals assigned to group G1 and G2 respectively) will receive no treatment.
· Animals of group G3 will serve as Vehicle Control (Excision wounds without infection and with infection), receiving carrier gel used for formulating HF1.
· Animals of group G4 will be treated with reference formulation-Mupirocin ointment-10 mg/mm2, once daily, topically. Treatment will be initiated immediately after wound generation.
· Animals of group G5-G7 will be treated with HF1, at different incremental concentrations as outlined in Table, twice daily, topically. Treatment will be initiated immediately after wound generation.
· Animals allocated to groups G1-G7 will be anesthetized by the combination of (87.5 mg Ketamine + 12.5 mg Xylazine) by intraperitoneal route.
· The rats will be depilated on the back and a predetermined area of 500 mm2 full-thickness skins will be excised in the dorsal interscapular region and the created wound will be left undressed in the open environment.
· To confirm the infection, 0.1 ml of S. aureus (approximately 109 CFU) will be inoculated into the wound. After 24 hours, a bacteria sample will be collected using a sterile saline-moistened swab. The swab will be rolled around the infected wound area to ensure the establishment of S. aureus infection. The wound swab sample will then be inoculated onto nutrient agar for bacteriological examination. Cultures will be incubated at 37 degrees Celsius for up to 24 hours and checked for microbial growth.
· The formulation gels and standard topical preparation will be applied daily until complete healing is observed. In this model, wound contraction and epithelialization period will be monitored. Wound contraction will be measured as percent contraction every 3 days after wound formation using a vernier caliper. From the healed wound, a specimen sample of tissue of normal skin (Collected skin specimen on day 1st from the normal animals after their wound creation) and wounded (on the day of termination of study) site will be collected from each rat and will be fixed in 10% neutral buffered formalin for histopathological whereas some part of wounded skin will be stored at -80°C for the ensuing biochemical and molecular evaluations.
6.0 PARAMETERS TO BE EVALUATED:
· Body weight: Twice a week.
· Skin oxidative stress parameters: SOD, GSH: GSSG Ratio, MPO.
· Gene expression analysis in healed skin or normal skin (collected during wound generation) by Real-Time PCR: MMP-2, MMP-9, TGF-β1, Col1a1, NF-kB.
· Histological analysis of wounded skin (H & E and MT).
· Wound contraction TIME
· Epithelialization period
· Hydroxyproline estimation
· Estimation of microbial count every 4th day
· Protein analysis by zymography (matrix metalloproteinases (MMPs)) of wound tissue
· Histopathological studies
7.0 REFERENCE(S):
1. Hemant Kumar et al.. Pharmacological Investigation of the Wound Healing Activity of Cestrum nocturnum (L.) Ointment in Wistar Albino Rats. Hindawi Publishing Corporation Journal of Pharmaceutics Volume 2016, Article ID 9249040, 8 pages http://dx.doi.org/10.1155/2016/9249040.
2. Pawar RS et al. Wound healing activity of Sida cordifolia Linn. in rats. Indian J Pharmacol 2013;45:474-8
3. Saleh, M. A., Shabaan, A. A., May, M. & Ali, Y. M. Topical application of indigo-plant leaves extract enhances healing of skin lesion in an excision wound model in rats. J. Appl. Biomed. 20, 124–129 (2022). 4. Rajoo A, Ramanathan S, Mansor SM, Sasidharan S. Formulation and evaluation of wound healing activity of Elaeis guineensis Jacq leaves in a Staphylococcus aureus infected Sprague Dawley rat model. Journal of Ethnopharmacology. 2021 Feb 10;266:113414.
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