Abstract
A topical antimicrobial ointment was developed from the fruit extract of Oroxylum indicum and was evaluated for its antibacterial and wound-healing effects and acute toxicity in animal models. O. indicum fruit and seed extracts exhibited antibacterial activities against clinically isolated bacteria and showed in vitro antioxidant activities. To develop a topical antimicrobial ointment from the fruit extract of O. indicum and evaluate for its antibacterial and wound-healing effects. O. indicum fruit extract ointment was prepared and qualitatively controlled. Acute toxicity of the extract was evaluated in the animal model. Antibacterial effects and healing effects of the ointment to the dog wound were investigated. The results are expressed as mean ± standard deviation. The ointment exhibited in vitro antibacterial effects. A single daily application of the ointment to a dog’s wound exhibited a wound-healing effect with complete epithelialization within 7 days while the wound was completely healed with the removal of the scabs, the size was decreased to 14% of the original size within 12 days. The ointment was found no acute toxicity in the animal model. O. indicum ointment promoted in vitro antibacterial activity and wound-healing effect in dogs with no acute toxicity.
Keywords: Antibacterial, ointment, Oroxylum indicum, wound healing
INTRODUCTION
Previous studies showed that the ethanol extracts of Oroxylum indicum showed antioxidant and antibacterial activities.[1,2] The aim of the study was to develop an ointment with the fruit extract of O. indicum and test for its in vitro antibacterial effects. Moreover, acute toxicity tests and the case report of this O. indicum ointment on a dog bite wound were also investigated.
MATERIALS AND METHODS
Preparation of plant material
O. indicum fruits were obtained from provinces in Thailand in 2019 and were identified according to Flora of Thailand.[3]
Preparation of plant extract
O. indicum fruits were extracted by maceration with 95% ethanol using an electric flask shaker. The extraction solutions were evaporated using a rotary evaporator and a water bath.
Quality control of plant extract
O. indicum fruit extract was quantitatively analyzed for total phenolic, total flavonoid, and flavone contents using previous reported methods.[2,4]
Acute toxicity test in animal model
To evaluate the safety of the extract, acute toxicity test was conducted in the animal model. According to OECD guidelines for testing of chemicals number 423, 2001,[5] 8-week-old female Sprague Dawley rats weighing 195–250 g were used.
Preparation of bacteria and culture media
Clinical isolates of selected bacteria including β-hemolytic Escherichia coli, Staphylococcus aureus, Staphylococcus intermedius, Pseudomonas aeruginosa, and Streptococcus suis were obtained from the Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand. S. Suis, P. aeruginosa, β-hemolytic E. coli, S. intermedius, and S. aureus were isolated and characterized using the method from Baron et al.[6]
In vitro antibacterial activity determination using a disc diffusion method
Preparation of Oroxylum indicum fruit extract ointment
The extract was incorporated into the ointment base using a fusion method to obtain a topical ointment containing 1%–10% w/w of O. indicum fruit extract. A physical stability study of the ointment was performed at 25°C, 75% relative humidity (RH), and 2°C–8°C, 75% RH for 6 weeks. The ointment formulation was stored at 4°C–8°C in refrigerator until it was used.
Case report: History and observation
An 8-year-old mixed breed male dog weighing 20 kg presented with a dog bite injury on the right foreleg was subjected to the experiment. The wound size was 1.4 cm × 1.4 cm with a depth of around 0.5 cm. The wound was generally cleaned and no bleeding remained. The dog was kept in a separate area in a standard environment. The dog was given free access to water and was given maintenance rations once daily. The treatment and observation were approved by the Kasetsart University Animal Committee (ID number: ACKU65-VET-063).
The entire surface of the wound was applied with the O. indicum fruit extract ointment (0.5 g) and covered with sterilized gauze and an adhesive medical bandage wrap. The ointment was applied and the sterilized gauze was changed once daily at 8.00 a.m. for 12 days. The wound characterization including the physical appearance and size was assessed each time. The percentages of wound size and wound contraction were calculated using the following equation;[9]
Percentage of wound size at day (x) = (Ax (mm2)/A0 (mm2)) × 100
Percentage of wound contraction at day (x) = 100−percentage of wound size at day (x)
Percentage of epithelialization at day (x) = (Ae (mm2)/Ax (mm2)) × 100
Where A0 = the original wound area, Ax = the wound area on the day of imaging, and Ae = the epithelialization area on the day of imaging.
Statistical analysis
All experiments were done in triplicate. The results are expressed as mean ± standard deviation.
RESULTS
Preparation and quality control of Oroxylum indicum extract
O. indicum fruit extract appeared as a dark brown semisolid. O. indicum fruit extract contained total phenolic and total flavonoid contents of 4.96 ± 0.37 g GAE/100 g extract and 5.16 ± 0.6 g QE/100 g extract, respectively. Baicalin, baicalein, and chrysin contents in O. indicum fruit extract analyzed by HPLC were found to be 0.40 ± 0.00, 0.36 ± 0.00, and 1.12 ± 0.00 g in 100 g extract, respectively.
Preparation of Oroxylum indicum fruit extract ointment
O. indicum fruit extract ointment appeared as brown semisolid ointment with a smooth texture and a specific odor. A physical stability study showed that after O. indicum fruit extract ointment was stored at 25°C, 75% RH, and 2°C–8°C, 75% RH for 6 weeks, no physical change in color, texture, and odor was found The results suggested the physical stability of the ointment for 6 weeks when it was kept at both room temperature and in a refrigerator.
Acute toxicity test in animal model
An acute toxicity test of O. indicum fruit extract or O. indicum fruit extract ointment in rats showed no toxicity (LD50 >5 g/kg rat body weight) by oral administration. No abnormal signs were found in visceral organs and there was no difference in average body weight. According to the Globally Harmonized System of Classification and Labeling of Chemicals, O. indicum fruit extract and O. indicum fruit extract ointment could be classified into category 5 or unclassified with an LD50 value >5 g/kg body weight.
In vitro antibacterial activity determination using a disc diffusion test
At a concentration of 1000 µg/disc, O. indicum extract showed antibacterial activities against all tested bacteria with the zone of inhibition in the range of 10–14 mm [Table 1]. O. indicum fruit extract ointment (concentration of extract = 500 µg/disc) also showed antibacterial activities against all tested bacteria, while the ointment base showed no activity.
Table 1:
Antibacterial effects of Oroxylum indicum fruit extract and ointment
| Sample | Zone of inhibition (mm) |
||||
|---|---|---|---|---|---|
| Staphylococcus aureus | Staphylococcus intermedius | Streptococcus suis | β - hemolytic Escherichia coli | Pseudomonas aeruginosa | |
| OIF* | 11.33±1.26 | 12.83±0.76 | 10.42±2.13 | 13.00±1.41 | 10.25±1.06 |
| OIF ointment** | 10.00±1.00 | 10.00±1.32 | 9.50±0.71 | 7.87±0.18 | 7.67±0.29 |
| Ointment base | 0 | 0 | 0 | 0 | 0 |
| AMC30 | 18.50±0.00 | 18.67±0.31 | 26.33±1.26 | 21.17±0.57 | 0 |
| DO30 | 16.67±0.58 | 15.83±0.76 | 17.17±1.61 | 24.33±1.04 | 14.83±0.76 |
| SXT25 | 0 | 0 | 0 | 27.50±0.87 | 15.13±1.15 |
*Concentration of extract 1000 μg/disc, **Concentration of extract 500 μg/disc. OIF: Oroxylum indicum fruit extract, AMC30: Amoxicillin/clavulanic acid 30 μg, DO30: Doxycycline 30 μg and SXT25: Sulfamethoxazole-trimethoprim 25 μg
Case report
Application of O. indicum fruit extract ointment promoted a wound-healing effect within 12 days. The original wound (day 0) was deep, pale white, and covered with white mucous. The wound area was 19,600 mm2, which was 100% of the wound size and no epithelialization was found. As shown in Table 2, the wound area continuously decreased after the application of the O. indicum fruit extract ointment. The wound contraction reached around 50% by day 5. The epithelialization also reached a high level on day 2, and the wound was completely healed (100% epithelialization) by day 7. On day 12, the wound was completely healed with the removal of the scab, and the size was decreased to 14% of the original size. No signs of infection such as swelling, redness, or pus forming were found during the observation. Signs of skin irritation including a rash, scabbing, red spots, dry or flaky skin were not found during the observation. The wound characteristics during the 12 days of observation after the application of O. indicum fruit extract ointment are shown in Figure 1.
Table 2:
Wound characteristics, wound contraction (%), and epithelialization (%) of case report after Oroxylum indicum ointment application
| Day | Wound characteristic | Wound size (mm2) | Percentage wound size | Percentage wound contraction | Epithelialization area (mm2) | Percentage epithelialization |
|---|---|---|---|---|---|---|
| 0 | Deep, pale white, covered with white mucous | 19,600.00 | 100.00 | 0 | 0 | 0 |
| 1 | Formation of red tissue in the middle of the wound, formation of white tissue at the edge of the wound | 19,600.00 | 100.00 | 0 | 6,600.00 | 33.67 |
| 2 | The wound looked shallower, forming more white tissue at the edge of the wound which had a yellow flesh color | 15,600.00 | 79.59 | 20.41 | 13,800.00 | 88.46 |
| 3 | The wound looked shallower, forming more yellow flesh-colored tissue at the edge of the wound with red tissue in the middle | 13,650.00 | 69.64 | 30.36 | 7,825.00 | 70.69 |
| 4 | The wound looked shallower, forming more yellow tissue at the edge of the wound, the red tissue in the middle looked narrower | 11,200.00 | 57.14 | 42.86 | 8,500.00 | 75.89 |
| 5 | The wound looked shallower, forming more yellow tissue at the edge of the wound, the red tissue in the middle looked even narrower | 9,428.57 | 48.10 | 51.90 | 8,628.00 | 91.51 |
| 6 | The wound looked shallower, the yellow tissue at the edge continued the healing effect toward the center of the wound | 7,700.00 | 39.29 | 60.71 | 7,010.00 | 91.03 |
| 7 | The wound was dry and the tissue was completely healed | 7,400.00 | 37.76 | 62.24 | 7,400.00 | 100.00 |
| 8 | The dried wound was scabbed showing the tissue was completely healed | 4,650.00 | 23.72 | 76.28 | 4,650.00 | 100.00 |
| 9 | The dry scabbed wound was narrowed and the tissue completely healed | 4,200.00 | 21.43 | 78.57 | 4,200.00 | 100.00 |
| 10 | The dried scabbed wound narrowed further and the tissue was completely healed | 4,000.00 | 20.41 | 79.59 | 4,000.00 | 100.00 |
| 11 | The dried scabbed wound was narrower still | 3,142.86 | 16.04 | 83.96 | 3,142.86 | 100.00 |
| 12 | All the scabs had fallen off | 2,750.00 | 14.03 | 85.97 | 2,750.00 | 100.00 |
Figure 1.
The wound characteristics in the case report. The different numbers after the letter ‘D’ indicate the day of observation after the application of the Oroxylum indicum fruit extract ointment
DISCUSSION
O. indicum ointment was applied to the bite wound in the dog case report once daily and exhibited wound-healing effects and the complete epithelialization of the wound within 7 days. Wound bites from animals and humans can lead to serious infections.[10,11,12] The biter’s oral cavity and the victim’s skin flora can originate the recovered microorganisms.[13] Various aerobic and anaerobic microorganisms or polymicrobial situations can be found in human and animal bite infections.[14] In human bites, Streptococcus spp. including Streptococcus pyogenes are usually found while Pasteurella spp. including Pasteurella multocida are the most common in dog and cat bites.[11,15] More specifically, in a dog bite wound, Capnocytophaga spp., Neisseria weaveri, Weeksella zoohelcum, Neisseria canis, and S. intermedius can be found.[14]
Extracts from various parts of O. indicum were reported to promote antibacterial effects.[2] In this study, O. indicum ointment also showed in vitro antibacterial effects. Previous mechanisms of action to inhibit bacteria were causing cell morphological damage and aggregation, cell disruption, and lysis.[2] The antibacterial effects of the ointment, therefore, should have a similar mechanism of action to the extract.
Dog bite wounds normally do not cause immediate symptoms aside from the tearing injury.[13] Signs and symptoms can be developed within 24–72 h due to the direct transfer of skin and oral microorganisms into the wound.[13] The indications of infection are such as swelling, redness, and discharge.[13] Moreover, the enlargement of adjacent lymph nodes could be found and the motion of a limb may be reduced.[13] Wound management depends on the stage of wound healing including irrigation, mechanical and chemical debridement, the use of antiseptics and antimicrobials, adherent and nonadherent dressings, and topical applications. The goal of a topical antimicrobial application in the treatment of wounds is to control microbial colonization and proliferation and promote the healing of the wounds.[16,17]
O. indicum ointment also showed a healing effect within 12 days and promoted the complete epithelialization of the tissue within 7 days in the dog wound case with no signs of infection or skin irritation. It was reported that flavones are the major compounds in fruit extract of O. indicum, in which baicalin, baicalein, and chrysin are most abundant.[2] Flavones exhibited antimicrobial, antioxidant, anti-inflammatory, and anticarcinogenic effects.[18] Moreover, baicalin was found to exhibit anti-inflammatory and antioxidative properties by modulating nuclear factor kappa-light-chain-enhancer of activated B cells, cyclooxygenase-1, and inducible nitric oxide synthase activity.[19] It also inhibited the ultraviolet-induced generation of reactive oxygen species in fibroblasts and prevented the activation of transcription factors responsible for collagen degradation.[20] The results suggest that O. indicum ointment could be used for the treatment of animal wounds for its antibacterial activity and wound-healing effects. Flavones, especially baicalin, baicalein, and chrysin, could be responsible for these effects.
CONCLUSION
It was the first time to demonstrate the in vitro antibacterial effects of O. indicum ointment. Application of the ointment once daily exhibited a wound-healing effect in the dog case. Further study in the animal model should be conducted to confirm the pharmacological activities along with the study of the mechanisms of action. Stability of the pharmaceutical formulation should be also performed.
Financial support and sponsorship
Biodiversity-Based Economy Development Office (Public Organization) and National Research Council of Thailand.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
The authors acknowledge the Biodiversity-Based Economy Development Office (Public Organization) and the National Research Council of Thailand for their support.
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