Evaluation of Cutaneous Wound Healing Activity of Citrus aurantium Fruit Peel Extract-based Ointment in Albino Rats


Sowmya Cherukuri1, Anitha Paramanayagam2*, Prabakaran R3, Mayakannan M3,

Lavakumar Vuppalapati3

1Assistant Professor, Dept of Pharmaceutics, Faculty of Pharmacy,

Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai – 600116, Tamilnadu, India.

2Associate Professor, Dept of Pharmacognosy, St. Mariam College of Pharmacy,

Tirunalveli - 627851, Tamilnadu, India.

3Dept of Pharmacology, Arulmigu Kalasalingam College of Pharmacy,

Anand Nagar, Srivilliputtur - 626126, Tamilnadu, India.

*Corresponding Author E-mail: csowmya05@gmail.com



The current study was aimed to evaluate the wound healing activity of ethyl acetate extract of fruit peels of Citrus aurantium. An ethyl acetate extract of fruit peel of Citrus aurantium was loaded into an ointment base with three different concentrations (0.25% w/w, 0.5% w/w and 1%w/w) and tested for wound healing activity using excision wound model by measuring the healing area and dermal toxicity. Wound healing activity and histopathology studies confirmed that all the tested three concentrations of extract accelerate the wound healing process by decreasing the surface area of the wound and increases the muscle tensile strength. Out of tested ointments, 5% w/w concentration showed comparative activity with the standard. Hence, natural Citrus aurantium peel-based ointment is an alternative ointment in wound healing.


KEYWORDS: Wound healing activity, Citrus aurantium, citrus peels.




Wound healing, is a specific process leading to the restoration of injured tissues. Wound healing process can be described as regeneration of the injured connective tissue of wounds in order to fill the wound gap followed by proliferation and migration of dermal and epidermal cells and matrix synthesis.1 Proliferation follows with the wound being rebuilt with new granulation tissue formed mostly by fibroblast and a new network of blood cells develop through the process of angiogenesis. Maturation involves remodeling and an improvement in collagen fiber components, leading to an increase in tensile strength. Parallel to this, scar formation occurs along with the accumulation of fibrous tissue at the edges of the wound, which eventually merges with surrounding tissue.2


Synthetic agents and natural wound healing agents act to accelerate and achieve the healing of wounds. Wound healing drugs are still unsatisfactory because of their low availability, high cost, and several detrimental side effects.3 For many years, plants have been used as the remedy for various skin and dermatological disorders especially cut, wounds, burns etc.4 Many plant drugs have been used in management and treatment of wounds over the years. Plants and their extracts have immense potential in the management and treatment of wounds. Therefore, medicinal plant derived drugs are under great demand due to common belief that they are safe, reliable, clinically effective, low cost and better tolerated by patients.5 Citrus aurantium (Rutaceae) tree bears dark green, leathery leaves and fruit of the bitter orange is very similar to the sweet orange.6 The fruit flesh taste is sour, while the peel and the skin surrounding the fruit the segment have a strong bitter taste. Bitter orange is used antiseptic, antioxidant, antispasmodic, aromatic, astringent, carminative, sedative, stimulant, stomachic, tonic and Wound healing.7 Fruit is used in constipation, dyspepsia and indigestion.8 In the present study, ethyl acetate extracts of fruit peels of Citrus aurantium was made in to a topical ointment and tested for wound healing activity in albino rats.  In addition, preliminary phytochemical study was performed to isolate the active constituents of the plant which might be responsible for the supposed healing activity.



2.1 Collection and Processing of Orange Peels:

The fresh fruit of Citrus aurantium was collected from Srivilliputhur fruit market in Virudhunagar district. After collection of fruits, they peeled out and dried them in shade for one week. Previously, Citrus aurantium fruits were identified by Botanist Dr. Stephen Ph.D., Professor, Department of Botany, Madurai.


2.2 Preparation of extract:

The powdered orange peel approximately weighing about 250g are extracted with 500ml of ethyl acetate using Soxhlet apparatus by using the heating mantle and the temperature was maintained at 700C. The extraction process was continued for 36 hours. After the completion of extraction, the solvent having the extract in the round bottomed flask was taken and concentrated by mild heating to evaporate the excess solvent. The extracts were placed in dark bottles and stored in refrigerator at 4°C until use.9,10


2.3 High Performance Thin Layer Chromatography:

The ethyl acetate extract of Citrus aurantium of was dissolved in ethanol to get a concentration of 10mg/mL and 2μL of this solution was used for taking HPTLC fingerprint. Aluminum sheets pre-coated with silica gel were used as the stationary phase. Toluene: Ethyl acetate: 100% formic acid (7: 3: 0.2) was used as the mobile phase for developing the chromatogram. The developed plates were examined at wavelength 254nm. The Rf values and finger print data were recorded by WIN CATS software.11-13


2.4 Preparation of ointment:

The three different concentration of 0.25% w/w, 0.5% w/w. 1%w/w of ethyl acetate extract of Citrus auratium ointment were prepared by mixing the 9.5 gm ointment base and 0.5gm of extract and the ointment was evaluated the physical parameters of colour, odour, pH, Consistency and Spreadablity.14,15


2.5 Testing of wound healing activity:

2.5.1 Selection of animals:

Wister albino rats weighing 140-180g were used for the study. The animals were housed individually in polypropylene cages in a well ventilated; temperature (30˚C) controlled rooms, 12 h light/dark cycle and fed on normal pellet diet and water ad libitum. The 92 animals were acclimatized to the laboratory conditions before the experiments were carried out.16 All animals were taken care of under ethical consideration as per the guidelines of CPCSEA with due approval from the Institutional Animal Ethics Committee (AKCP/IAEC/016/16-17)., Arulmigu Kalasalingam College of Pharmacy, Krishnankoil.


2.5.2 Dermal Toxicity Study:

The prepared ointments were tested for toxicity by topical application in Wister albino rats. Anesthetic ether was used as an anesthetic agent. The dorsal surface of the rats were depilated and the prepared ointments at 10% concentration were applied once daily on the depilated dorsal surface for 14 days. The skin was observed for erythema, edema and necrosis once a day for 14 days.17


2.5.3 Experimentally induced excision wound:

The dorsal surface of the skin of the rat was shaved. A predetermined area of 2.0- 2.5cm diameter of the skin in its full thickness was excised out under Ether anesthesia. (Group 1). The control group was treated with simple ointment base (Group 2). The standard group was treated with povidone iodine. (Group 3) The test groups were treated with ointments with different concentrations of extracts viz. 0. 25% (w/w), 0.5% (w/w) and 1% (w/w) incorporated in simple ointment base, in all the three models. The ointments were applied topically once daily for 14 days.18.19


2.5.4 Wound area Measurement:

The progressive changes in the wound area were monitored by tracing the wound on a tracing paper at every 4 days interval. The percentage reduction of the wound area was calculated with reference to control group periodically (Goyal et al., 2012). Wound contraction = healed area/total area × 100


2.6 Histological examination:

On 14 th day the regenerated tissues were removed for the histological examination. The sections of the tissues were stained with haematoxylin and eosin and evaluated for the extent of reepithelization, maturation and organization of the epidermal squamous cells, thickness of the granular cell layer and the degree of tissue formation.20



3.1 High Performance Thin Layer Chromatography:

In this study HPTLC finger printing of ethyl acetate extract of Citrus aurantium was showed several peaks. HPTLC profile under UV 254nm was recorded. Corresponding HPTLC chromatograms were presented 4 spots with Rf values in the range of 0.05 to 0.89. (Fig 1-1A,1B,1C)


Fig. 1A Visualization at 254nm                                                        Fig. 1B. Peak display of citrus aurantium at 254nm

Fig 1 HPTLC analysis of ethyl acetate extract of citrus aurantium peels


3.2 Wound healing activity:

From the study of 9th,12th, and 14th  day, the difference in the healing of wound area are distinctly observed that is the control groups has the wound area about 20 mm approximately, the standard drug treated groups has measured the wound area of 5-6 mm, the test drug ointment treated groups animals have wound area of minimum of 8-10 mm (0.25%w/w), 6-8 mm(0.5%w/w) and 7-10 mm (1%w/w) respectively. Hence from the above study, test ointment of 0.5% w/w concentration of ethyl acetate extract of Citrus aurantium was observed to have a better wound healing activity in treated animals (Fig 2).


Fig 2: wound healing activity of ethyl acetate extract of Citrus aurantium.

         A (Control)                               B (Standard)                   C Test (0.25%w/w)              D Test (0.5%w/w)                   E Test (1%w/w)

Fig 3 Histopathological studies of ethyl acetate extraxt of Citrus auratium peels



3.3 Histopathological studies:

Treatment with ethyl acetate extraxt of Citrus auratium (0.5%w/w) was showed a significant attenuation on histopathological alterations compared with that of standard drug. Wound healing involves four phases such as contraction, epithelization, granulation, collagenation, all stages of healing was shown in the fig.3.



In the present study of ethyl acetate extract of Citrus aurantium fruit peels was used to test the in vivo wound healing activity. The current studies have reported that, most plant derived bioactive compounds have anti oxidative, antimicrobial and anti-inflammatory properties which are the main mechanisms contributing to wound healing. In the present study, to avoid the disadvantages associated synthetic medicine of wound healing, a natural based ointment with fruit peel of Citrus aurantium was prepared and tested for wound healing activity in albino rats. Initially, ethyl acetate extracts of Citrus aurantium fruit peels were prepared. Later various concentrations of extracts were incorporated into ointment base to produce Citrus aurantium fruit peels ointment. HPTLC studies confirmed that the majority of fraction constitutes flavonoids in the extract. The pharmacological activity of Citrus aurantium peels was evaluated based on wound measurement and histopathological studies. The complete closure of the wound is an important parameter to assess the wound healing process. The result showed that control groups have the wound area about 20mm approximately, the standard drug treated groups has measured the wound area of 5-6mm, the test drug ointment treated groups animals have wound area of minimum of 8-10mm (0.25% w/w), 6-8mm (0.5 w/w) and 7-10mm ( 1%w/w) respectively. Hence from the above study, test ointment of 0.5% w/w concentration of ethyl acetate extract of Citrus auratium peels was observed to have a better wound healing activity in treated animals. It is less significant compared to that of the standard (povidone iodine 5% w/w) and more significant when compared to the activity of the negative control. The histopathological studies discovered that the wound healing occurs by decreasing the surface area of the wound with increases the tensile strength of the tissue. 21



The present study demonstrated that the ethyl acetate extract fruit peels of Citrus aurantium promotes wound healing activity in albino rats in the preclinical study. The ethyl acetate extract of Citrus aurantium showed remarkable wound healing activity and it may be suggested for treating various types of wounds and injuries in animals. The enhanced wound healing activity of ethyl acetate extract of Citrus aurantium could possibly be made use clinically in healing of open wounds. However, confirmation of the study has to be done through well designed clinical evaluation.



1.     Purohit SK. Solanki R. Mathur V. Mathur M Evaluation of Wound Healing Activity of Ethanolic Extract of Curcuma longa Rhizomes in Male Albino Rats. Asian Journal of Pharmaceutical Research. 2013;3(2):79-81. doi.10.5958/2231–5691.

2.     Shenoy Chitra. Patil MB. Kumar Rav. Antibacterial and Wound Healing Activity of the Leaves of Annona squamosa Linn. (Annonaceae). Research Journal Pharmacognosy and Phytochemistry. 2009; 1(1):44-50.

3.     Enoch S. Leaper DJ. Basic Science of Wound Healing. Surgery (Oxford). 2008;6(2):31-7. doi.org/10.1016/j.mpsur.2007.11.005.

4.     Purohit SK. Solanki R. Soni R. Mathur V. Evaluation of Wound Healing Activity of Ethanolic Extract of Azadirachta indica Leaves in Male Albino Rats. Asian J Pharm Techno 3(2): April-June. 2013; Page 73-75. doi: 10.5958/2231–5713

5.     Ghanshyam Dhalendra. Premlata Rathore. Trilochan Satapathy. Amit Roy. Pharmacognostical, Phytochemical and Pharmacological study of Calotropis procera: A Review. Research Journal of Pharmacy and Technology. 2014;7(3):346-351. doi.10.5958/0974-360X

6.     Sharma A. Khanna S. Kaur G. Inderbir S. Medicinal plants and their components for wound healing applications. Future Journal of Pharmaceutical Sciences. 2021; 7(53):1-13.doi.org/10.1186/s43094-021-00202-w.

7.     Borkar VS. Senthil Kumaran K. Senthil Kumar KL. Medicinal Plants with Potential Wound Healing Activity. Research Journal of Pharmacognosy & Phytochemistry. 2015;7(2):116-123.doi.10.5958/0975-4385.2015.00020.5.

8.     Munne SL. Parwate DV. Ingle VN. Preliminary Phytochemical Screening, Free radical Scavenging and Antimicrobial activities of Citrus auranticum fruit bio-mass. Research Journal of Pharmacy and Technology. 2009;3:607-608. DOI: 10.5958/0974-360X

9.     Sugandha DG. Dattatray GG. Yogendra TG. Ramiz MR. Azad YB. Et al. A Study of Chemical Compositional Characteristics of Citrus Fruits for D- Limonene, Organic acids, Minerals and Sugars. Asian journal of Research in Chemistry. 2021;5(2):299-304. Doi. 10.5958/0974-4150

10.  Suntar I. Khan H. Patel S. Celano R. Rastrelli L. An overview on Citrus aurantium L. Its functions as food ingredient and therapeutic agent. Oxidative medicine and Cellular Longevity. 2018;1-2.  doi: 10.1155/2018/7864269.

11.  Patil KM. Bodhankar SL. High-performance thin-layer chromatographic determination of lamotrigine in serum. Journal of Chromatography B. 2005;823(2):152-7. doi: 10.1016/j.jchromb.2005.06.029.

12.  Subramanian S. Ramakrishnan N. Chromatographic finger print analysis of Naringi crenulata by HPTLC technique. Asian Pacific Journal of Tropical Biomedicine 2011;1(2): S195-8. doi.org/10.1016/S2221-1691(11)60155-X

13.  Kaviarasan L. Sarojini S. Prakash Yoganandam G. Gopal V. Preparation and Standardization of Deepavali Avaleha - A Traditional Polyherbal Ayurvedic Formulation. Asian Journal of Pharmacy and Technology. 2016;6(4): 231-237. doi.10.5958/2231-5713.2016.00034.9

14.  Elzayat EM. Auda SH. Alanazi FK. Al-Agamy MH. Evaluation of wound healing activity of henna, pomegranate and myrrh herbal ointment blend. Saudi Pharmaceutical Journal. 2018;26(5):733-8. doi.org/10.1016/j.jsps.2018.02.016

15.  Krishna Murti. Vijay Lambole. Mayank Panchal. Megha Shah. Vipul Gajera. Evaluation of Wound Healing Activity of Polyherbal Formulation in Rats. Research Journal of Pharmacognosy and Phytochemistry 2011; 3(3):112-115 doi. 10.5958/0975-4385

16.  Sawant SE, Tajane MD. Formulation and evaluation of herbal ointment containing Neem and Turmeric extract. Journal of Scientific and Innovative Research. 2016;5(4):149-51.

17.  Perumal Samy R. Chow VT. Pilot Study with regard to the Wound Healing Activity of Protein from Calotropis procera (Ait.) R. Br. Evidence-based Complementary and Alternative Medicine. 2012; Article ID 6697174: 1-8. doi.org/10.1155/2012/294528.

18.  Rajesh Kumar Sharma, Rajni G.P., Deepak Nathiya, Ashish Kumar Sharma. Assessment of wound healing activity of roots of Bauhinia variegata Linn. by excision and incision model in Albino Rats. Asian Journal of Research in Pharmaceutical Sciences. 2015;5(3):145-152. doi.10.5958/2231-5659.2015.00023.5

19.  Goyal M. Nagori BP. Sasmal D. Wound healing activity of latex of Euphorbia caducifolia. Journal of Ethnopharmacology, 144(3);2012:786-90. doi: 10.1016/j.jep.2012.10.006

20.  Badmanaban R. Patel CN. Devi P. Meera R. Kameswari B. Eswarapriya B. Physio-Chemical investigation and Wound healing activity of Sesame oil and Formulated oil. Asian Journal of Research in Chemistry. 2009;2(4):501-505. doi.10.5958/0974-4150

21.  Ambrekar SN. Patil PA. Patil Suhas A. Wound Healing Activity of Root Extracts of Commelina benghalensis Linn. Research Journal of Pharmacy and Technology. 2011;4(11):1772-1776. doi.10.5958/0974-360X







Received on 22.10.2020             Modified on 21.07.2021

Accepted on 03.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):250-254.

DOI: 10.52711/0974-360X.2023.00046