Evaluating the Wound Healing Activity of Tribulus terrestris L. leaf powder Ointment on Splinted excisional wounds in Balb/C mice

Racha Nedal Daiea¹, Jamal Abdul Naser Darwicha², Lina Ahmad Saker³,

Aziza Ibrahim Youssef³

¹Tishreen University, Syria, Latakia.

²Arab International University, Syria, Damascus.

³Tishreen University, Syria, Latakia.

*Corresponding Author E-mail: racha.daiea@tishreen.edu.sy, phjamal@aiu.edu.sy, lina.saker@tishreen.edu.sy, aziza.youssef@tishreen.edu.sy

ABSTRACT:

Background: Recent advancements in wound healing focus on botanically based medications with expedite the process with increased safety and fewer side effects. While Tribulus terrestris (T.T) L. has been acknowledged for its’ wound healing potential in vivo studies, particularly in murine models, faces challenges due to the role of the muscle layer Panniculus carnosus, leading to false positive results in clinical trials. Objective: This study aims to demonstrate the wound healing activity of powdered T.T leaves by employing a novel technique: splinted full-thickness excisional wounds in mice. Methods: Leaves of T.T were collected, grinded, sieved, and added to a hydrophilic ointment. The experiment was conducted on 50 Balb/C male mice, divided equally to 5 groups: Group I treated with 10% T.T, Group II treated with 15% T.T, Group III treated with MEBO, Group IV treated with ointment excipients, and Group Vwasn’t treated. Using isoflurane gas 5% in propylene glycol as anaesthesia, two excisional wounds were inflicted on the dorsal area on each mouse. Each group was treated with the appropriate treatment once daily and the wound areas were measured every two days for 14 days. Digimizer software was used formeasuring the wound areas then we calculated the wound healing percentages. SPSS software was used to analyse the wound healing percentages data, which they were demonstrated as mean±SD. The performed tests: One-way ANOVA test, Tukey’s test, Pearson’s correlation coefficient test, Dendrogram tree and proximity matrix. Results: A significant difference was obtained when comparing the groups treated with 10% and 15% powdered leaf T.T ointments with the negative control and positive control (MEBO) at P-value=0.000. Group II demonstrated the highest healing rate, followed by Group III then group I then group V, finally group IV. The healing increased with increasing the concentration of the powdered leaf, which was demonstrated by Group II in the first 7 days of the experiment. Pearson’s test proved there was a correlation between time and the healing rate. Dendrogram tree and proximity tree displayed a similarity among Groups I, II, and III, and between groups IV and V. T.T leaves proved their wound healing activity through the synergistic action of the present plentiful phytochemicals in leaves: phenolic acids, flavonoids, tannins, and ß-sitosterol. Conclusion: T.T leaves showed a good healing action in vivo and it was improved by increasing the powder’s concentration, therefore, it decreased the healing period.

KEYWORDS: Tribulus terrestris, powdered leaves, excisional wounds, splinted wounds, wound healing activity.

1. INTRODUCTION:

The skin is the primary mechanical barrier in the human body facing the external environment¹. When this barrier gets damaged or broken by physical, chemical, or biological agents, the affected tissue loses its continuity and functionality^2,3. However, any damage to this tissue triggers a wound healing response. Wound healing is an intricate recovery process which is composed of four phases:⁴hemostasis, acute and late inflammation, proliferation, and remodeling^1,5. Each phase occupies a certain time period within the healing phases. If all phases are completed within 30days in their hierarchically manner, then the wound is termed acute. However, if the process extends beyond 30 days without following the typical healing phases, then the wound is classified as chronic^1,6.

Throughout history, both Botanical medicines as well synthetic ones were used in the acceleration of the wound healing process. However, botanical medicines are preferred over synthetic agents for accelerating the wound healing process due to their safety, consistency, cost-effectiveness, and demonstrated therapeutic efficacy in clinical trials⁷.

Tribulus terrestris L. from the Zygophyllaceae family, is an annual herb predominantly found in tropical arid areas worldwide⁸. Since it is very well known in the Ayurveda medicine (Traditional Indian Medicine), it is used as an aphrodisiac, increasing hormone production both in males and females, tonic, diuretic and for urinary tract infections (UTI)^9,10,11. Moreover, the World Health Organization (WHO) has approved its’ treatment of mastitis, cough, headache, and male impotence¹². The phytochemicals that are responsible for the previous uses are: phenolic compounds, flavonoids, tannins, alkaloids, saponins, and glycosides^13,14. Recently, clinical studies have proved its’ wound healing activity by applying leaves or fruits extract on rat wounds^15,16,17.

Murine and human skin show several notable differences: first, the human skin is thicker than that inmurine. Second, the human skin is non-loose, unlike the loose skin in murine. Third, the human skin is composed of a greater number of cell layers morethan that inmurine. A fundamental distinction lies in the wound healing process, where murine models include a striated layer called Panniculus carnosus between the dermis and subcutaneous tissue responsible for wound contraction. Meanwhile ,in human models, wound closure occurs through the formation of granulation tissue, which becomes challenging in extrapolating findings from murine studies. To overcome this issue, it is suggested to add silicone or stainless-steel rings around excisional wounds, preventing contraction and allowing healing to occur in a similar manner as that inhumans.¹⁸

The aim of our research is to evaluate the wound healing activity of powdered leaves of Tribulus terrestris (T.T) on splinted excisional wounds in mice, and to compare the formulated ointment containing T.T powdered leaves with a well-known wound healing ointment (MEBO).

2. MATERIALS AND METHODS:

2.1 Conduct of research:

The research was conducted in the faculty of pharmacy laboratories in Tishreen University (Latakia, Syria), in collaboration with Arab International University (Damascus, Syria).

2.2 Plant collection:

T.T was collected from a field in the countryside of Hama in Syria (Qomhane) (figure 1), with the geographical coordination: Latitude: 35.211°, Longitude: 36.697°, Sea level altitude: 307.00 m. The leaves were cultivated and were air dried in the shade for 14 days.

Fig. 1: Image of the field and the leaves of T.T.

2.3 Ointment preparation:

Ointments (100g) containing 10% (W/W) and 15% (W/W) powdered T.T leaves were prepared using: beeswax (6.25%), cetyl alcohol (15.625%), lanolin (31.25%), jelly petroleum (31.25%), and sweet almond oil (15.625%). The leaves were crushed and pulverized using a 75 micron sieve, and the powder was collected, and added to the remaining ointment components, which was melted using a water bath at 70°C.

2.4 Evaluating the wound healing activity of T.T:

2.4.1. Animal selection:

A total number of 50 healthy, adult male Balb/C mice provided by the laboratory of pharmacology and Toxicology of Arab International University. The mice were about 8-11 weeks old, their weights were between 17-32g, and they were divided equally into five groups (10 mice per group). Mice were housed in pairs, acclimatized for one week, and provided water ad libitum and pellet food. Temperature was maintained at 23±2°C with a 12h light/dark cycle.

All procedures used in this experiment were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals(Care and Animals, 1986).

2.4.2. Splinted excisional wound procedure:

For anaesthesia, isoflurane gas in propylene glycol was used at 5% in the beginning of the procedure then it was reduced to 2% for maintenance¹⁹. After that,the dorsal hairwas removed by using a shaving razor and depilation cream. The skin was disinfected with liquid povidone (10%). A 5mm diameter biopsy skin punch was used to inflect the excisional wound (19.635mm² wound area), therefore, two wounds were created parallel to each other on the dorsal area. After the procedure, the wound was disinfected with povidone and was pressed with cotton to stop the bleeding.To avoid the contracting action of the muscle layer on wound healing process, we used sterilized silicone rings. The outer diameter of the silicone rings is 12mm and the inner diameter is 6mm. These rings were placed around the wound and were fixed to skin by using surgical nylon strings (diameter 0.3mm).

2.4.3. Animal groups:

· Group (I): Treated with T.T powdered leaves ointment (10% W/W)

· Group (II): Treated with T.T powdered leaves ointment (15% W/W)

· Group (III): Treated with MEBO® ointment (Julphar)

· Group (IV): Treated with prepared ointment excipients

· Group (V): Negative control

The proper medication for each groups was applied once daily for 14 days. To reduce pain, acetaminophen was injected (IP) once per day 3mg/0.3ml for 3 days after the surgicalprocedure²⁰.

2.4.4. Wound area measurement:

Using a digital camera (Fujifilm Finpix JZ500) and Digimizer software (Version 6.3), the wounds were photographed every two days (on day 0, day 2, day 4, day 6, day 8, day 10, day 12, and day 14) and the wound area was measured by placing a mm ruler next to the wound, then the measurement was read via Digimizer soft ware and calculated properly.

To calculate wound healing percentage, the following equation was used, which is adopted by Bagdad, Y.M, et al²¹:

Wound area on day 0 – Wound area on a respective day

Percentage of wound healing = --------------------------------------- × 100

Wound area on day 0

3: STATISTICAL ANALYSIS:

Statistical Package for Social Sciences (SPSS) software was used to analyse the obtained results (version 20).

· Primary data analysis:

First, data was scanned for outliers or false input on all variables of interest. Then, descriptive data analysis was conducted to obtain the average percentages and standard deviations of wound healing area by group.

· Secondary data analysis:

To assess the normality of the wound healing percentages data, Kolmogorov-Smirnov test was applied.

· One-Way ANOVA test was used to assess the presence of significant difference between means within different groups of wound healing percentages, significance was determined at a P-value≤0.05 with a 95% confidence interval. After that, Tukey’s HSD post hoc test was used to determine which specific group differed significantly from each other.

· Further to that, Pearson’s correlation coefficient was determined to measure the strength of the linear correlation between the day of the measurement (time) and the percentage of wound healing of each group. Dendrogram tree was drawn based on Chi-square distance test and a proximity matrix was also computed to show the hierarchical relationship between groups.

4. RESULTS:

Descriptive statistics of average wound healing percentage data are presented in Table 1.

· The Kolmogorov-Smirnov test: yielded a non-significant result(P-value>0.05) leading us to consider our wound healing percentage (WH%) data as conforming to a normal distribution. Consequently, we proceeded with parametric data analysis.

· One-Way ANOVA: table 1 presents the degrees of freedom (df), F-values, and significance levels for WH% across the five groups over the observation days. The consistently low P-value of 0.000 indicates statistical significant differences among the groups throughout the entire study. Upon examining individual groups:

· Group I: The means of the WH% of this group showed a significant statistical difference (P-value≤0.05) when compared with positive control (MEBO) and negative control groups. It showed a notable statistical significance when compared with positive control in the first 6 days. However after 6 days, there was no statistical significance which meant that the two groups have a similar phytochemical constituents, therefore, similar wound healing activity. In addition, this group showed a higher healing rate than negative control group (98.48%, 88.63% respectively). However, it showed less healing rate than positive control group (98.48%, 99.22% respectively).

· Group II: The means of the WH% of this group showed a significant statistical difference (P-value≤0.05) when compared with positive control (MEBO) and negative control groups. It showed a notable statistical significance when compared with positive control in the first 10 days. On day 14, comparing the groups treated with 10% and 15% T.T leaf powder ointments with positive control, all three groups showed almost full healing rate (100%). The healing rate of this group was higher than the positive control group (99.38%, 99.22% respectively), this high healing rate is reflected by the increasing of the T.T leaf powder concentration in the ointment, which means higher concentration of the phytochemicals, therefore, the activity was better than positive control. It also showed a notable higher healing rate than negative control (99.38%, 88.63% respectively).

· Group III: The means of the WH% of this group showed a significant statistical difference (P-value≤0.05) when compared with negative control, also, it showed a higher healing rate than the negative control (99.22%, 88.63% respectively).

· Group IV: The means of the WH% of this group showed a significant statistical difference (P-value≤0.05) when compared with negative control, with a lower healing rate than group V (80.71%, 88.63% respectively).

· Group V: The means of the WH% of this group showed significant differences (P-value≤0.05) compared to all groups, with a higher healing rate than group IV.

· The reason behind the notable higher healing rate demonstrated by group II comparing to group I resulted from the higher concentration of leaves powder in ointment. Consequently, the effect increased. This can also be seen in figure (2).

Table 1: Mean of wound healing percentages presented with ANOVA and Tukey HSD test results.

Days	df		F-value	Sig.	Group I		Group II		Group III		Group IV		Group V
Days	Within groups	Between groups	Between groups	Between groups	N.	Mean ±SD	N.	Mean ±SD	N.	Mean ±SD	N.	Mean ±SD	N.	Mean ±SD
2	4	95	60.659	0.000	20	9.16 ±4.58	20	18.33 ±7.09	20	4.48 ±1.85	20	2.59 ±0.98	20	1.48 ±1.53
4	4	89	234.701	0.000	20	26.22±7.13	20	43.20 ±4.25	18	17.26 ±5.44	20	4.71 ±1.57	16	3.63 ±2.54
6	4	89	274.200	0.000	20	37.83±5.26	20	46.75 ±3.66	18	22.65 ±4.26	20	10.37 ±2.76	16	10.96 ±5.15
8	4	89	305.890	0.000	20	51.55±5.52	20	64.4 ±7.27	18	55.59 ±6.75	18	13.97 ±3.11	16	15.51 ±5.86
10	4	87	187.096	0.000	20	72.19±8.61	20	83.73 ±8.42	18	77.44 ±9.35	18	19.54 ±5.25	16	50.73 ±8.70
12	4	87	140.870	0.000	20	95.74±3.10	20	98.00 ±2.69	18	97.48 ±3.04	18	58.43 ±9.55	16	69.81 ±11
14	4	87	82.803	0.000	20	98.84±1.68	20	99.38 ±1.53	18	99.22 ±1.27	18	80.71 ±7.94	16	88.63 ±8.47

· Pearson’s correlation test: Table 2 displays the results of Pearson’s correlation test, revealing a strong and statistically significant correlation between time and healing percentages. Group I showed the highest correlation coefficient, followed by groups II, III, IV, and V, respectively. These findings affirm a significant and strong correlation between time and the rate of wound healing across the various groups.

Table 2: Results of Pearson’s correlation test

Group	Correlation coefficient	P-value
I	0.992	0.000**
II	0.983	0.000**
III	0.980	0.000**
IV	0.897	0.006**
V	0.953	0.001**

** P-value <0.05

· Dendrogram tree: The calculated Chi-square distance helped with drawing thedendrogram tree of the five groups with consideration of the time factor (days) (fig.2). The clustering increases when the groups are homogenous among each other and vice versa. The tree is branched to two axes.

First axis: groups IV and V showed high clusteration. Second axis: groups I, II, and III presented high clusteration among each other, however groups I and III were in closerproximity to each other. Whereas, group II presented clustering alone which shows it has the highest healing percentage and lowest healing time.

· Proximity matrix: As shown in table (3): firstly, we noticed thatthe highest similarity (1.000) was within the group itself. Secondly, a high similarity of (0.991) was also seen between groups I and II because both groups were treated with the same treatment, which differs only in concentration. Thirdly, a high similarity of(0.988) was seen between groups I and III, additionally we noticed that the closest proximity value to the previous one (0.988) was (0.986) and it was seen when we compared between group II and group III. These results could be explained by the fact that all these groups (I,II,III) were treated with active ingredients having wound healing activities.

Finally, the poor proximity values were shown when we compared the first three groups (I, II, and III) with the last two groups (IV, V). We can justify this reduced proximity values with the presence of active ingredients in the treatments applicated on the first three groups, while in groups IV and V there were no application of any active ingredients.

· In addition, the figure (fig.3) shows that the highest similarity was between groupsI and III, moreover, they were both similar to group II.

· Groups IV and V were also similar, sincethey were both untreated with active ingredients.

Fig. 3: Dendrogram tree

· Wound healing percentages (WH%): Figure (4) shows the WH% through-out the selected days for all groups.Group IIshowed the highest WH% from day 2 to day 6. However, from day 8 to day 14 the significance has been reduced when compared to groups I and III.

Fig. 4: Wound healing percentages as mean±SD through-out the days for all groups.

X: Groups, Y: Wound Healing% (WH%).

5. DISCUSSION:

Medicinal plants are known for their abundant and biologically effective secondary metabolites which are widely distributed in every part of the plant.

Moreover, when it comes to accelerating the wound healing process, consumers prefer using topical pharmaceutical dosage forms containing medicinal plant extracts due to their safety and therapeutic effects²⁰.

Most notably, phenolic acids and flavonoids are best known for their antioxidant, antinflammatory, and antimicrobial effects during the healing process due to their unique structures. The hydroxyl group²²found in phenolic acids and 4-oxo, 5-hydorxyl groups that are found between the heterocyclic ring and ring A in the flavonoid structure, both serve as chelators for metallic ions and directly scavenge Reactive Oxygen Species (ROS). When ROS exceed the average levels during the typical healing response, they destruct cells and DNA and therefore delays the healing process, the wound is then termed a chronic wound.Both of phenolic acids and flavonoids have an antinflammatory potential, through inhibiting the cyclooxygenase (COX) enzyme. Tannins are known for their astringent properties which induce blood vessels contraction in the wound area, also tannins have antioxidant effects^{23,24,25,26,27}.

A conclusion comes from the previous results that the T.T powdered leaves have significant wound healing activity in both concentrations (10% and 15%), however, the wounds treated with 15% T.T showed higher healing rates than those treated with 10%. A comparison between wounds treated with T.T powdered leaves (groups I and II) and the wounds treated with MEBO (group III) showed similar wound healing rates after day 10. Results might be justified with the presence of phenolic acids, flavonoids, and tannins in powdered leaves, which can be very effective through synergistic action especially when using leaf powder containing all active phytochemical compounds leading to greater effects rather than using extracts containing just a few phytochemical compounds.

Despite the presence of previous secondary metabolites in T.T, it also contains ß-sitosterol: a plant steroid and that mainly stimulates epithelization and retains the moisture inside the wound. ß-sitosterol is the main ingredient of MEBO ointment (0.25%). This may explain the similar healing rate between T.T and MEBO from day 10 to day 14²⁸.

The viewed healing process was mainly due to the action of the secondary metabolites in the T.T through promoting epithelization, formation of granulation tissue and increase the formation of fibroblasts (the normal healing process in humans). Allowing a healing process to occur with similarity to that in humans, this process was realized and maintained by the addition of silicone rings, preventing the contraction action of the muscle layer in mice.

In 2009, Wesley, J.J²⁹and his colleagues evaluated the wound healing activity of 5% aqueous leaf extract gel of T.T in rats. The results of their research showed a reduction of the healing period from 21 days to 17 days. On the other hand, our research showed thatthe application of a 15% leaf powder ointment resulted in a reduction of the haling period from 14 to 12 days. The disparity in outcomes can be attributed to the comprehensive presence of phytochemical compounds in the powder, whereas the aqueous extract comprises solely polar compounds. The research by Wesley, J.J and his colleagues proved that the T.T aqueous leaf extract have a wound healing activity in vivo. On the other hand, we used in our research the silicone rings to stop the contracting action of the muscle layer, by that way T.T powdered leaves presented in vivo wound healing action which is similar to healing processes in humans and thus it could have promising results in clinical trials.

6. CONCLUSION:

Tribulus terrestris L. have an excellent wound healing activity in-vivo when using 10% and 15% concentrations of powdered leaf ointment, showcasing a higher healing rate in the latter. The current literature suggests the healing mechanism is attributed to the antioxidant, antinflammatory ,antimicrobial, re-epithelization effects of T.T due to the existence of many phytochemical compounds (exclusively phenolic acids, flavonoids, tannins, and ß-sitosterol).

Overall, this research highlights the importance of including botanical medicines in topical dosage forms rather than using harmful and expensive synthetic compounds.

7. ACKNOWLEDGMENTS:

The authors acknowledge the effort and help of Prof. Assistant. Tamim Hammad, Head of the Industrial Pharmacy Department of Tishreen University.

8. CONFLICT OF INTEREST:

Authors have no conflict of interest to declare.

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Received on 27.12.2023 Modified on 26.01.2024

Research J. Pharm. and Tech 2024; 17(7):3346-3352.

DOI: 10.52711/0974-360X.2024.00523

Groups	Correlation between vectors of values
Groups	1:Group I	2:Group II	3:Group III	4:Group IV	5:Group V
1:Group I	1.000	0.991	0.988	0.898	0.960
2:Group II	0.991	1.000	0.986	0.845	0.929
3:Group III	0.988	0.986	1.000	0.864	0.947
4:Group IV	0.898	0.845	0.864	1.000	0.948
5:Group V	0.960	0.929	0.947	0.948	1.000