Excision Wound Healing and Antioxidant Activity of Different Root Extract of  Plumbago indica

Sujin Jebakumar Kumar. T.<sup>1,2</sup>; Balavigneswaran C.K.<sup>1</sup>; Prakash S.<sup>1</sup>; Natheer Hassan.Y.<sup>2</sup>; Srinivasakumar K.P.<sup>2</sup>

Excision Wound Healing and Antioxidant Activity of Different Root Extract of Plumbago indica

Sujin Jebakumar Kumar. T.^1,2

, Balavigneswaran C.K.¹

, Prakash S.¹

, Natheer Hassan.Y.²

, Srinivasakumar K.P.²

1. Department of Biotechnology, Udaya School of Engineering, K.K. District, Tamil Nadu, India
2. Inbiotics, Parvathipuram, Nagercoil-3, Kanyakumari District, Tamil Nadu, India

Author

Correspondence author
Medicinal Plant Research, 2013, Vol. 3, No. 3 doi: 10.5376/mpr.2013.03.0003
Received: 08 Jan., 2013 Accepted: 15 Jan., 2013 Published: 23 Jan., 2013

This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Preferred citation for this article:

Sujin J.K.T., et al., 2012, Excision wound healing and antioxidant activity of different root extract of Plumbago indica, Medicinal Plant Research, Vol.3, No.3 13-19 (doi: 10.5376/mpr.2013.03.0003)

Abstract

The present study was to evaluate wound healing activity of ethanolic, chloroform and hexane extracts of root of Plumbago indica using excision wound model and also in its antioxidant activity. The tested animals were divided into five groups with five in each group 10% w/v of each extract was prepared in normal saline with 0.1% propylene glycol. The anti-oxidant activities of different root extracts were investigated including scavenging activity of hydrogen peroxide, DPPH and free radicals of superoxide anion in vitro. Ethanol extract of Plumbago indica were found to possess better wound healing activity than the chloroform and hexane extract, and it was more effective than the commercial wound healing agent Burnol tried in experiments with complete wound healing on the 9^th day. It showed rapid epithelization, better wound closure and reduction of infection. Hexane extract also had good wound healing capacity while the chloroform extract was found to be ineffective as a wound healing agent. Biochemical analysis post wound healing showed normal counts while the differential WBC counts was the higher in the mice treated with chloroform extract and it was even higher in the untreated mice. The results showed that the ethanolic extract has powerful antioxidant potential and hexane extract showed some antioxidant activity while chloroform extract was ineffective as an antioxidant. The ethanolic extract of Plumbago indica had good wound healing capacity and also showed good antioxidant property than the other extracts.

Keywords

Plumbago indica; Excision; Epithelization; DPPH; Hydrogen peroxide; Superoxide anion

Plumbago indica (family Plumaginaceae), a dicoty- lendonous plant, is well known for its ethno medicinal values. P. indica is a rich source of therapeutically active, root specific, natural napthoquinone plumbagin. Plumbagin has been reported to possess filaricidal (Mathew et al., 2002) anticancer (Kuo et al., 2006), cardiotonic (Itoigawai, et al., 1991), antimalarial (Likhitwitayawuid et al., 1998), antimicrobial (Didry et al., 1994) and anti-fertility (Bhargava., 1984) activities. Among six different species of Plumbago, P.indica is the richest source of plumbagin (Mallavadhani et al., 2002).

Wound healing process involves several steps, which involves coagulation, inflammation, formation of granulation tissue, matrix formation, remodeling of connective tissue, collagenization and acquisition of wound strength. It was observed that Aloe vera increased the collagen content of the granulation tissue as well as its degree of cross-linking as seen by increased aldehyde content and decreased acid solubility (Kaufman et al., 1988). A. vera, Channa striatus, were also thought to have positive influence on the synthesis of glycosaminoglycan and by beneficially modulate wound healing (Chitra et al., 1998). By topical application of 0.2% solution of asiaticoside isolated from Centella asiatica produced 56% increase collagen content and better epitheli- sation (Shukla et al., 1999), but Curcuma longa aqueous drops definitely delayed healing of superficial corneal wounds and delayed healing of penetrating corneal wounds also markedly reduced the tensile strength of corneal wounds (Mehra et al., 1984).

Reactive oxygen species (ROS) including superoxide radicals, hydroxyl radicals, singlet oxygen and hydrogen peroxide are often generated as byproducts of biological reaction or from exogenous factors. (Kikuzaki et al., 1993). A potent broad spectrum scavenger of these species may serve as a possible preventive intervention for free radical mediated cellular damage and diseases (Ahmad et al., 1993). Recent studies have shown that a number of plant products including polyphenols, terpenes and various plant extracts exerted an antioxidant action (Zho et al., 1991; Bang et al., 2001).

Our present study deals about the wound healing of different root extracts of Plumbago indica and its antioxidant activity.

1 Results
1.1 Wound healing activity
Complete wound healing of different root extracts of Plumbago indica were studied by counting the number of days, the behavior of mice to different treatments and the overall process of epithelization were observed day by day (Table 1; Table 2).

Table 1 Phytochemical screening of different root extracts of Plumbago indica

Table 2 Effect of different root extracts of Plumbago indica on wound healing (concentration of extracts 200 g/mL glycerin)

Topical applications of 10 % w/v of ethanolic extract of Plumbago indica increase the percentage of wound contraction, reduced infections and rapid epitheli- zation and collagenization made complete wound contraction by the 9^th day. Rapid bio-synthetic activities in topically treated mice during phase of granulation and in remodeling phase were observed. Topically 10% application of chloroform extract showed slow and bio-synthetic activity and also restlessness were observed in mice. Topical application of hexane extract showed normal wound healing activity and better resistivity to infection when compared to the chloroform extract and in the remodeling phase, maturation of collagen takes place by the formation of inter and intra-molecular cross links. The infection causing organisms was found to be Staphylococcus species and Escherichia coli. The colony count was high in the untreated wound and the count was very low in ethanolic and Burnol treatment. Biochemical analysis showed that all the functions of mice, post wound treatment were normal as shown in Table 3. WBC counts showed that the infection was more in the hexane extract. Complete wound contraction using a treatment of Burnol, a commercial wound healing agent acquired on the 10^th day (P< 0.01 as compared with control group).

Table 3 Biochemical analysis on mice models post wound treatment

1.2 DPPH radical scavenging activity
When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance. The antioxidant activity of the extracts is shown in the Figure 1. The experimental data reveals that the Ethanolic extract have more scavenging activity than the chloroform and hexane extracts. At the maximum concentration the scavenging activity of the free radicals are Ethanol extract > Hexane extract > Chloroform extract. We also observed the activity of ethanolic extract is fully dose dependent and the other extracts saturated at the concentration of 0.2 mg/mL.

Figure 1 The scavenging effect of different root extracts of Plumbago indidca on DPPH radicals

1.3 Superoxide Scavenging Activity
Among the three extracts ethanol extract found to be an efficient scavenger of super oxide radical generated in riboflavin–NBT–light system in vitro and the other extracts showed little activity like 20% (Figure 2). From this experiment using ethanol extract of P.indica, it is noted that the inhibition of the formation of blue formazan and the percentage inhibition are dose dependent, other extracts inhibition was not considered since there were much less formation of the formazon products.

Figure 2 The scavenging effect of different root extracts of Plumbago indidca on superoxide radicals

1.4 Hydroxyl radical scavenging assay
The ability of the extracts of P.indica to scavenge hydrogen peroxide is determined. The scavenging ability of various extracts with hydrogen peroxide is shown in Figure 3. It was observed that the ethanol extract showed highest scavenging activity as likeother DPPH and Superoxide scavenging activity and the other extracts activity is not that good as we expected. And the activity of the extracts is concentration dependent. At the highest concentration 5mg/ml the scavenging activity of the extracts as follows Ethanol extract (86.68) % > Hexane extract (45.18%) >Chloroform extract (21.39 %).

Figure 3 The scavenging effect of different root extracts of Plumbago indidca on hydroxyl radicals

2 Discussions

2.1 Excision wound healing

Wound healing is a complex phenomenon involving a number of well constructive processes. The processes include regeneration of parenchymal cells migration and proliferation of both parenchymal and connective tissue cells. Further processing includes synthesis of extra cellular matrix protein, remodeling of connective tissues parenchymal components collagenization and acquisition of wound strength. In this study, excision wound model were used to establish the healing potentials of ethanolic, chloroform, hexane extract of Plumbago indica. From the results, it is evident that the ethanolic and hexane extract possess a definite potential healing action. It showed significant percentage closure by enhanced epithelialization which may be due to collagen synthesis. Few studies have shown that phytochemical constituents like flavanoids, alkaloids, saponins and tannins are known to promote the wound-healing process (Tuschiya et al., 1996; Mukherjee, 2002; Madhura et al., 2002; Ansel, 2006). Therefore, the study reveals that both ethanolic and hexane extract possess wound healing property which may be attributed to the individual or combined action of phytochemical constituents like flavanoids, alkaloids, saponins and tannins present in it. From the experimental results it was clear that ethanolic extract had better activity on wound healing compared to hexane extract and Burnol, a commercial wound healing agent used as standard. Chloroform extract was ineffective as a wound healing agent. Bioche- mical analysis showed that all functions of mice were normal, post wound healing and this indicates the extract was non toxic when applied topically and it was confirmed that it won’t interfere with normal body function. There was significance difference in the WBC counts which was as a result of infection caused in the wound in case of untreated mice and also to mice treated with chloroform extract. Ethanol extract significantly reduces infection and compared to hexane extract which made ethanolic extract of Plumbago indica with its better wound closure time as a better would healing alternative even when compared to commercial wound healing agent like Burnol (aminacrine hydrochloride, cetrimide IP).

There is plenty of evidence to suggest that increased production of reactive oxygen species, lipid peroxi- dation and ineffective scavenging play a crucial role in various skin lesions and in modulation of fibroblast proliferation (Murrel et al., 1990). Cutaneous wounding causes a depression in the overall antioxidant status making it more vulnerable to oxygen radical attract (Shukla et al., 1997). All these findings indicate that in wound healing, antioxidants may play an important role.

2.2 DPPH radical scavenging activity

The DPPH antioxidant assay is based on the ability of DPPH a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 490 nm and also for deep purple color (Luo et al., 2010). The involvement of free radicals, used to treat human diseases, including cardiovascular disease and cancer (Sreejayan et al., 1996). It has been found that cysteine, glutathione, ascorbic acid, to copherol, flavonoids, tannins, and aromatic amines (p-phenylene diamine, p-aminophenol, etc.), reduce and decolourise DPPH by their hydrogen donating ability (Deighton et al., 2000). We are assuming that the phenolic compounds present in the ethanolic extracts of P.indica probably involved in their enhanced antioxidant/irradical activity.

2.3 Superoxide scavenging activity

Superoxide anion radical is one of the strongest reactive oxygen species among the free radicals that are generated (Blois, 1958). It is known to be a very harmful species to cellular components as a precursor of more reactive oxygen species (Garrat, 1964). The superoxide radical is known to be produced in vivo and can result in the formation of H₂O₂ via dismutation reaction. Moreover, the conversion of superoxide and H₂O₂ into more reactive species, e.g., the hydroxyl radical, has been thought to be one of the unfavourable effects caused by superoxide radicals (Halliwell, 1991). Photochemical reduction of flavonoids generates O₂, which reduces NBT, resulting in the formation of blue formazan (Nakayama, 1994), this indicates that the flavonoids in the ethanolic extract of Plumbago indica was responsible for such a reaction.

2.4 Hydroxyl radical scavenging assay

Ferrous salts can react with hydrogen peroxide and hydroxyl radicals by Fenton’s reaction. The required iron taken from either pool of iron or the heme containing proteins. Hydrogen peroxide itself is not very reactive, but sometimes is toxic to cell because it may give rise to hydroxyl radical in the cells (Murrel et al., 1990). Therefore, removing of H₂O₂ is very important for antioxidant defence in cell or food systems. Dietary polyphenols have also been shown to protect mammalian and bacterial cells from cytotoxicity induced by hydrogen peroxide, especially compounds with the orthodihydroxy phenolic structure quercetin, catechin, gallic acid ester, caffeic acid ester (Shukla, 1997). Therefore the phenolic compounds of the ethanolic extract of P.indica extracts may probably be involved in removing the H₂O₂.

3 Materials and Methods

3.1 Preparation of different root extracts

The roots of the plant Plumbago indica was collected washed thoroughly shade dried and powdered. 100 gm of the powdered mass obtained was stored in a clean sterile bottle at room temperature and used for various extractions. The powdered material was extracted using petroleum ether chloroform, ethanol and water for 72 h each in a soxhlet apparatus. The extracts were evaporated under reduced pressure to obtain solid masses and the percentage yield of the extracts was found to be 2.32%, 2.05%, 4.57% and 16.6%, respectively.

3.2 Phytochemical screening

In order to determine the presence of alkaloids, glycosides, flavones, tannins, terpenes, sterols, saponins, fats and sugars, a preliminary phytoche- mical study (color reactions) with different root extracts was performed (Khandelwal, 2005)

3.3 Animals and grouping

All animals were housed in standard laboratory conditions [temperature (22±2)â„ƒand humidity 45%±5% with 12 h day: 12 h night cycle] and standard laboratory diet was provided to the animals. Studies carried out after the approval of Institutional Animal Ethics Committee in accordance with institutional ethical guidelines for the care of laboratory animals as per Approval number inbiotics/IEC/217-11/2011.

Swiss albino mice of either sex, weighing 30 g±5 g were selected. Ten percent w/v of all the extracts were prepared in normal saline consisting of 0.1% propylene glycol. The mice were divided into five groups consisting of five in each group where group 1 was untreated mice (control), group 2 animals was treated with Burnol, a commercial wound healing agent (ingredients aminacrine hydrochloride, cetrimide IP). Group 3 animals were topically applied with 10 % w/v ethanolic root extract, group 4 animals were applied with 10% w/v of chloroform extract and group 5 animals with n-hexane root extract of Plumbago indica topically.

3.4 Wound healing model

An impression was made on the dorsal thoracic region 2 mm away from the vertebral column and 1 cm away from the year using a round seal of 5 mm diameter on the anaesthetized rat. The skin of the impressed was excised to full thickness to obtain a wound area of 100 mm square. Homeostasis was achieved by blotting the wound with cotton swab soaked in normal saline. Mechanisms which results for wound closure in first week were studied by tracing the raw wound. The whole process of wound healing on various batches of mice was studied thoroughly by careful observation and the physical state and also the behavioral changes of the mice were studied.The degree of wound healing = 1 (wound area on corresponding day/wound area on zero day)×100%

3.5 Biochemical analysis

The blood was drawn from mice using cardiac puncture through anterior thoracic aperture. Then they were analyzed using semi auto analyzer Rayto 1904C, Product code 11 and the results were tabulated in Table 3.

3.6 Analysis of antioxidant activity

3.6.1 DPPH radicals scavenging assay

The antioxidant activity of the plant extract was assessed on the basis of the radical scavenging effect of the stable DPPH free radical (Bang et al., 2001). The ethanolic extract (0.2~5 mg/mL) was added to 200 µL of DPPH in methanol solution (100 µM) in a 96-well microtitre plate. After incubation at 37â„ƒ for 30 min, the absorbance of each solution was determined at 490 nm using Elisa microtitre plate reader (Bio Rad Laboratories Inc., CA; model 550). The corresponding blank readings were also taken and the remaining DPPH was calculated. IC₅₀ value is the concentration of sample required to scavenge 50% DPPH free radical. DPPH scavenging effect (%) = [A_o-(A₁)]/ A_o ×100, where A₀ is the absorbance of the control, A₁ is the absorbance of the extracts

3.6.2 Superoxide radicals scavenging activity

The assay was based on the capacity of the extracts of P.indica to inhibit formazan formation by scavenging the superoxide radicals generated in riboflavin– light–NBT system (Beauchamp et al., 1971). Each 3ml reaction mixture contained 50mM sodium phosphate buffer (pH 7.6), 20 mg riboflavin, and 12 mM EDTA, and 0.1 mg NBT and 1ml sample solution. Reaction was started by illuminating the reaction mixture with different concentrations of sample extract (25~100 mg/mL) for 90 s. Immediately, after illumination, the absorbance was measured at 590 nm. The entire reaction assembly was enclosed in a box lined with aluminium foil. Identical tubes with reaction mixture were kept in the dark and served as blanks. The percentage inhibition of superoxide anion generation was calculated using the following formula: Superoxide Scavenging Activity (%) = [A_o-A₁]/ A_o × 100, where A₀ is the absorbance of the control, and A₁ is the absorbance of the extracts.

3.6.3 Hydroxyl radical scavenging assay

Hydroxyl radical scavenging activity of ethanol, chloroform and hexane root extracts of Plumbago indica was determined according to the method used in (Beauchamp, et al., 1971; Wang et al., 2008) with a few modifications. Reaction mixture contained 1.0 mL of 9 mL FeSO₄. 1.0 mL of sample solutions in water at different concentrations (0~10 mg/mL), 1.0 mL of 8.8 mN H₂O₂ was added finally, and then the reaction mixture was incubated at ambient temperature for 30 min. The absorbance was recorded at 510 nm, and the scavenging of the different root extracts of Plumbago indica were calculated according to the following equation: Hydroxyl radical scavenging effect (%) = [(Ao-A₁)/A₀]×100, where A₀ is the absorbance of the control, A₁ is the absorbance of the extracts

3.7 Statistical analysis

Results are expressed as mean S.D. The differences between experimental groups were compared by one way ANOVA–Keuls test and were considered statistically significant when p<0.01.

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