Wound healing activity of Euphorbia antiquorum stem extract on rats

Introduction

Wound is defined as disruption of the cellular and anatomical continuity at a tissue. It may be produced by physical, chemical, thermal and microbial damage to tissue. A large number of herbs are used as a folklore medicine for the treatment of cuts, wounds, and burns. In India there is a surplus number of variety of species of plants present which can be used in the treatment of various wounds (Biswas et al., 2003).  

The wound healing process consists of three phases. First phase includes homeostasis which often occurs immediately after the wound occurs with vascular constriction and aggregation of platelets. Second phase is inflammatory phase characterized by the five common signs i.e redness, pain, heat, swelling and loss of function. All the inflammatory mediators are produced in this phase cause chemotaxis of monocytes, lymphocytes and initiates epithilisation process.  The last phase is remodeling phase characterized by increasing the strength of scar and cause complete reepithilisation (Mutsaers et al., 1997). Various factors like oxygen, age, sex hormones, stress, will affect the wound healing process (Sabale et al., 2012).

The Euphorbia antiquorum stem consists of various phytochemical constituents that may responsible for wound healing. This plant is topical plant widely distributed over the world ranging from annual weeds to trees. The plant contains latex, green stem, ribs are prominent generally three. The characteristic feature is triangular in shape. Flowers are cyathia yellowish green to pinkish in colour (Wine et al., 2011). Male flowers with one stamen, female flower lies at the center of cyathium. Fruits are yellowish to orange in colour. It contains various chemical constituents like flavonoids, di & tri terpenes which shows a significant activity of antimicrobial, anti HIV and cytotoxic properties (Sivaraj et al., 2011; Sumathi et al., 2012). The latex also having the lavicidal activity and mosquitocidal (Vimal  et al., 2014; Vimal  et al., 2015).

Materials and Methods

Plant material and collection

Stem of Euphorbia antiquorum was collected around Machilipatnam, Krishna District and authenticated by Botanist.

Extraction

Euphorbia antiquorum stems were collected washed thoroughly, then cut into small pieces. The pieces were dried under the shade for 3-4 days. The dried stem material was pulverized into a coarse powder. The powder was weighed and debris is removed. About 150gm of powder was weighed and subjected to maceration process with various solvents depends up on the polarity. The mixture is occasionally stirred vigorously and kept for 72 hrs. Finally the marc was filtered through a muslin cloth and the filtrate was concentrated by using rotary evaporator. The obtained filtrate was subjected to phytochemical screening for the identification of chemical constituents (Kumar  et al., 2016).

Preparation of ointment

The water soluble base was prepared by triturating the PEG – 400 & PEG – 4000 in a motor. 2.5% and 5% ethanolic extract containing ointment was prepared (Esimone  et al., 2006;  Jyothi  et al.,  2014).

Screening of wound healing activity

The study was approved by the Institutional Animal Ethics Committee with Ref no: IAEC/XI/3/BCOP/2017. Adult wistar rats weighing 200-250 gm of either sex was selected for the study and maintained under standard experimental conditions as per CPCSEA guidelines. Animals are divided into 4 groups containing six animals each. Group – 1 was treated as control, Group -2 was treated as standard (Povidine Iodine 5%), Group – 3 was treated with 2.5% Euphorbia antiquorum ethanolic extract ointment, Group – 4 treated with 5% Euphorbia antiquorum ethanolic extract ointment. Excision wound was made on the back of rats near the neck region. The region was fully shaved and made a incision about 2mm diameter. The wound was left undressed to the open environment (James et al., 2010). To the standard and test group animals drugs was topically applied for 24 days. The wound area was observed at 5, 10, 15, 20^th day and the parameters like wound contraction period and percentage of inhibition were monitored (Mathew et al., 2004; Umachigi et al.,  2008). 

Biochemical estimation

To estimate collagen and hexosamine, the tissue samples were defatted in chloroform methanol (2:1) and dried in acetone before use. Collagen, hexosamine and uronic acid was estimated by the method of Anusha (Bhaskar et al., 2012).

Results and discussion

The phytochemical screening of various solvent extracts of Euphorbia antiquorum was carried out and the results are given in the table 1. The ethanol extract was selected for further pharmacological screening due to quantity and the presence of flavonoids, tannins and saponins.

The formulation (2.5% and 5%) was subjected to different tests like pH, skin irritation test, colour, odour and their results is shown in table 2.

For the determination of wound healing activity the wound size for all animals groups was measured in mm². The percentage wound contraction of the control changed from 5^th to 20^th day is   21.22% ± 6.54 to 92.86% ± 1.30. For the standard it is from 54.85% ± 3.08 to 99.6% ± 0.10. The Euphorbia antiquorum formulation (2.5%) showed a change from 45.76% ± 3.07 to 98.05% ± 0.24 from 5^th to 20^thday. Euphorbia antiquorum formulation (5%) showed an increased percentage wound contraction from 72.45% ± 3.43 to 99.13% ± 0.25 from 5 to 20^th day.

The effect of Euphorbia antiquorum on the reduction of the wound area (in mm²) for 0 to 20^th day is given in the figure 1 & 2. The 5% ointment showed significant decrease in the wound size when compared with control group. Biochemical changes were evaluated on 19th day and the results are tabulated as (Table 3).

Table 1.  Phytochemical analysis of Euphorbia antiquorum extract

Table 2. Properties of the ointment

Table 3. Biochemical parameters

Figure 1. Percentage Wound Contraction on Day 5

Figure 2. Percentage Wound Contraction on Day 20

Conclusion

The present study evaluated the wound healing activity of Euphorbia antiquorum ethanol extract using excision wound model in rats. The regenerated skin in the 5% ointment treated rats showed the best healing features compared to the control and 2.5% ointment treatment groups. Therefore it is concluded that ethanol stem extracts of Euphorbia antiquorum possesses wound healing activity.

Conflicts of interest: Nil

References

Bhaskar A,  Nithya V. 2012. Evaluation of the wound-healing activity of Hibiscus rosa sinensis L (Malvaceae) in Wistar albino rats. Indian Journal of Pharmacology 44(6): 694-698.

Biswas TK,  Mukherjee B. 2003. Plant medicines of Indian origin for wound healing activity: a review. The International Journal of Lower Extremity Wounds  2(1): 25-39.

Esimone CO, Ibezim EC, Chah KF. 2006. The wound healing effect of herbal ointments formulated with Napoleona imperialis. Journal of Pharmaceutical and Allied Sciences 3(1): 294-299.

Farahpour MR,  Habibi M. 2012. Evaluation of the wound healing activity of an ethanolic extract of Ceylon cinnamon in mice. Journal of Veterinary Medicine  57(1): 53-57.

 James O, Victoria  IA. 2010. Excision and incision wound healing potential of Saba florida (Benth) leaf extract in Rattus novergicus. International Journal of Pharmaceutical and Biomedical Research (IJPBR)  1(4): 101-7.

 Jyothi D,  Koland M,  Priya S. 2014. Investigation of anti-inflammatory activity of ointments containing fenugreek extract. Asian Journal of Pharmaceutical and Clinical Research (AJPCR)  7(2) : 66-69.

Kumar A, Saikia D. 2016. Euphorbia Antiquorum Linn: A Comprehensive Review of Ethnobotany, Phytochemistry and Pharmacology. Journal of Analytical & Pharmaceutical Research  2 (4): 1-5.

Mathew A, Taranalli AD, Torgal SS. 2004. Evaluation of anti-inflammatory and wound healing activity of Gentiana lutea rhizome extracts in animals. Pharmaceutical Biology 42(1): 8-12.

Mutsaers SE, Bishop JE, McGrouther G, Laurent GJ. 1997. Mechanisms of tissue repair: from wound healing to fibrosis. The International Journal of Biochemistry & Cell Biology 29(1): 5-17.

Mwine JT, Van Damme P. 2011. Why do Euphorbiaceae tick as medicinal plants? A review of Euphorbiaceae family and its medicinal features. Journal of Medicinal Plants Research 5(5): 652-662.

Sabale P, Bhimani B,  Prajapati C, Sabale V. 2012. An overview of medicinal plants as wound healers. Journal of Applied Pharmaceutical Science  2(11): 143-150.

Sivaraj R, Balakrishnan A, Thenmozhi M, Venckatesh R. 2011. Preliminary phytochemical analysis of Aegle marmelos, Ruta graveolens, Opuntia dellini, Euphorbia royleana and Euphorbia antiquorum. International Journal of Pharmaceutical Sciences and Research  2(1): 132.

Sumathi S, Malathy N,  Dharani B, Sivaprabha J,  Hamsa D,  Radha P, Padma PR. 2011. Cytotoxic studies of latex of Euphorbia antiquorum in in vitro models. Journal of Medicinal Plants Research  5(19): 4715-4720.

Umachigi SP, Jayaveera KN, Kumar CA, Kumar GS, Kumar DK. 2008. Studies on wound healing properties of Quercus infectoria. Tropical Journal of Pharmaceutical Research 7(1): 913-919.

Vimal  JB, Das M. 2014. Euphorbia antiquorum latex and its mosquitocidal potency against Aedes aegypti. Journal of Entomology and Zoology Studies (2): 267-269.

Vimal JB, Das M. 2015. Toxicity of Euphorbia antiquorum latex extract to fresh water fish Poecilia reticulata. International Journal for Fisheries and Aquatic Studies  2(3): 214-216.