Invitro Antioxidant and Wound Healing Activity of Pun Purai Ennai, A Polyherbal Siddha Formulation

Abstract

Pun Purai Ennai, a traditional Siddha formulation from Ethiopia used for wound treatment, lacks scientific validation. This study assessed its in vitro antioxidant and wound-healing activities. Sourced from the government Siddha College in Chennai, the study employed the DPPH radical scavenging and Chick Chorioallantoic Membrane (CAM) assays. The DPPH assay gauged antioxidant potential by measuring free radical neutralization. The CAM assay evaluated wound healing through angiogenesis. Results unveiled, In the DPPH assay, Pun Purai Ennai exhibited substantial, concentration-dependent DPPH radical inhibition, with 79.547% inhibition at 500 ?g/ml, akin to ascorbic acid's 81.3387%. The IC50 value of 60.94 ?g/ml for Pun Purai Ennai mirrored ascorbic acid's 61.01 ?g/ml, indicating comparable antioxidant efficacy. In the CAM assay, Pun Purai Ennai enhanced angiogenesis, evidenced by 21 blood vessels (versus the control's 17), akin to the standard (Regen D) at 23 vessels. These findings underscore the formulation's potential as a wound-healing agent, stimulating blood vessel formation akin to the standard. In conclusion, Pun Purai Ennai exhibited potent antioxidant activity akin to ascorbic acid and significant wound-healing potential, evident by proangiogenic effects. These insights propose its application as a natural remedy for oxidative stress and wound healing, necessitating further mechanistic exploration and clinical investigation.

Keywords

Introduction

       
           
       
Fig.1: DPPH radical scavenging activity of pun purai ennai ^[2]

Material Required

The IC50 value of the tested sample (PPE) was determined to be 60.94 ?g/ml. The normal range for the IC50 value of ascorbic acid is 61.01 ?g/ml. In comparison, the tested sample, PPE, demonstrated a slightly lower IC50 value compared to the IC50 value of ascorbic acid (61.01 ?g/ml). This indicates that PPE possesses a similar potency to ascorbic acid in terms of inhibiting the target or producing the desired effect. The results of the DPPH radical scavenging activity of pun purai ennai display the tested sample concentrations of PPE in ?g/ml, the corresponding OD values at 517 nm, and the percentage of inhibition. The PPE samples demonstrated dose-dependent inhibition of DPPH radicals, with higher concentrations exhibiting greater inhibition. The highest inhibition was observed at 500 ?g/ml with a value of 79.547%. Ascorbic acid, used as a reference compound, showed an inhibition of 81.3387%. The IC50 value, representing the concentration at which 50% inhibition is achieved, was determined to be 60.94 ?g/ml for PPE, slightly lower than the IC50 value of ascorbic acid (61.01 ?g/ml). These findings indicate that PPE exhibits significant DPPH radical scavenging activity, comparable to the reference compound ascorbic acid, emphasizing its potential as an antioxidant. The results suggest that PPE may possess beneficial properties related to its ability to scavenge free radicals, which are implicated in various pathological conditions.

       
           
       
Fig.4: CAM control

       
           
       
Fig.5: CAM with standard (regen D gel) (recombinant human epidermal growth factor gel)

Fig.6: CAM with 100 ?g/ml of sample PPE

Table No 3: Chick Chorioallantoic Membrane (Cam) Assay Of Pun Purai Ennai

These results suggest that PPE demonstrated a higher capacity to stimulate the formation of blood vessels, indicating its potential as a proangiogenic compound. This effect was comparable to that of the standard (recombinant human epidermal growth factor gel), surpassing the normal healing observed in the control group. The Chick Chorioallantoic Membrane (CAM) assay of Pun Purai Ennai revealed its ability to stimulate blood vessel formation, indicating its proangiogenic properties. The group treated with 100 ?g/ml of PPE showed an increase in the number of blood vessels from 17 (control group) to 21, demonstrating its proangiogenic effect. This effect was comparable to the group treated with the standard gel (regen D gel) containing recombinant human epidermal growth factor, which had 23 blood vessels. The mechanism behind PPE's proangiogenic activity involves the activation of signaling pathways that promote angiogenesis. Specific bioactive compounds present in PPE may interact with endothelial cells, stimulating their proliferation, migration, and differentiation. This, in turn, leads to the formation of new blood vessels. The proangiogenic properties of PPE are crucial for wound healing, as they facilitate improved oxygen and nutrient supply to the injured area, enhance waste removal, and promote the migration of cells involved in tissue repair.  EGF binds to its receptor on endothelial cells, initiating a signaling cascade that triggers the activation of various intracellular pathways, including the Ras-Raf-MEK-ERK and PI3K- Akt pathways. These pathways are involved in promoting endothelial cell proliferation, migration, and survival, leading to the formation of new blood vessels. Similarly, PPE may contain bioactive compounds that interact with endothelial cells and activate similar signaling pathways. Activation of these pathways promotes endothelial cell proliferation, migration, and differentiation, ultimately resulting in increased blood vessel formation. While the specific bioactive compounds in PPE responsible for its proangiogenic activity are yet to be identified, their ability to mimic the proangiogenic effects of EGF suggests that PPE may engage similar molecular mechanisms involved in endothelial cell growth and blood vessel development.

CONCLUSION

The study highlights Pun Purai Ennai (PPE), a polyherbal Siddha formulation, as a promising agent for wound healing and antioxidant therapy. In vitro analyses using the DPPH and CAM assays demonstrated PPE's strong free radical scavenging and proangiogenic activities, comparable to standard treatments. PPE's IC50 value for antioxidant activity closely matched that of ascorbic acid, indicating its potential to mitigate oxidative stress in wound healing. Additionally, PPE stimulates blood vessel formation, enhancing oxygen and nutrient delivery to wounds. These findings support the efficacy of plant-based therapies, aligning with natural healing mechanisms. Further research is needed to identify PPE's bioactive compounds and evaluate its clinical applications, reinforcing its potential as a safe, effective wound care solution in modern healthcare.

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