Exploring the Therapeutic Potential of Cissampelos pareira in Wound Healing: A Comprehensive Investigation into its Anti-Inflammatory and Antioxidant Properties

 

Mrutyunjaya Satpathy, Vipin Saini*

1MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University),

Mullana-Ambala, Haryana, 133207, India.

*Corresponding Author E-mail:

 

ABSTRACT:

Aim: This study analyses the potential action of Cissampelos pareira in amelioration of wound healing. Materials and methods: A Carrageenan-induced acute inflammatory model was used for the induction of inflammation and excision wound model for creation of wounds in rats. The commencement of the chronic administration of different extracts of Cissampelos pareira CIAQ (Cissampelos pareira aqueous extract 200, 400 mg/kg), CIME (Cissampelos pareira methyl extract 200,400 mg/kg) for a period of 14 days after wound creation. Results: Administration of different extracts of Cissampelos pareira was found to have a significant ameliorating effect on wound healing, and a moderating effect on oxidative stress and inflammation. Conclusion: Cissampelos pareira may be helpful in treatment of chronic wound by reducing oxidative stress and inflammatory cytokines using Carrageenan-induced acute inflammatory and excision wound model.

 

KEYWORDS: Anti-inflammatory, Antioxidant, Wound, Healing, Cissampelos pareira, Oxidative stress.

 

 


INTRODUCTION: 

Wound healing is an intricate biological process involving a harmonious interplay of cellular and molecular events aimed at restoring tissue integrity. In recent years, there has been a resurgence of interest in botanical remedies for wound management, with a particular focus on traditional medicinal plants1-4. Among these, Cissampelos pareira, a plant deeply entrenched in traditional medicine systems across the globe, has emerged as a captivating candidate for its potential in wound healing, inflammation modulation, and antioxidant properties5. Cissampelos pareira, commonly known as "Abuta" or "Velvetleaf," has a rich ethnopharmacological history, with diverse cultures harnessing its therapeutic properties for centuries. This research article embarks on an in-depth exploration of Cissampelos pareira, aiming to unravel its multifaceted impact on wound healing, with a specific emphasis on its anti-inflammatory and antioxidant attributes1-8.

 

Chronic wounds and delayed healing often stem from dysregulated inflammatory responses9-10. The anti-inflammatory properties of cissampelos pareira have attracted interest because to the abundance of bioactive chemicals found in the plant, including polyphenols, alkaloids, and flavonoids. These compounds are believed to intervene in key inflammatory pathways, regulating the expression of cytokines, chemokines, and other inflammatory mediators11-15. Through this modulation, Cissampelos pareira may offer a balanced and controlled inflammatory environment conducive to optimal wound repair. Moreover, the plant's influence on immune cell activity, particularly macrophages, presents an intriguing facet of its anti-inflammatory potential. Macrophages play a pivotal role in orchestrating the immune response during wound healing, and Cissampelos pareira may hold the key to fine-tuning their functions to promote effective tissue repair. Oxidative stress is a critical factor in impaired wound healing, characterized by an imbalance between reactive oxygen species (ROS) and antioxidant defenses. Cissampelos pareira, with its rich repertoire of antioxidants, including flavonoids and polyphenols, emerges as a potential scavenger of ROS. By neutralizing free radicals, the plant may mitigate oxidative stress, thereby creating a conducive environment for optimal cellular regeneration and tissue repair. Experimental studies utilizing in vitro and in vivo models are underway to substantiate the traditional claims surrounding Cissampelos pareira16-19. As we delve into the therapeutic tapestry of Cissampelos pareira, the potential for integrating this botanical remedy into mainstream wound care strategies becomes increasingly apparent. However, a comprehensive understanding of its optimal dosage, potential side effects, and formulation considerations is crucial for its successful translation into clinical practice. In conclusion, this research endeavors to shed light on the untapped potential of Cissampelos pareira in the realm of wound healing. By bridging traditional wisdom with contemporary scientific investigation, we aspire to unlock the secrets encoded in this botanical treasure, paving the way for a future where Cissampelos pareira stands as a testament to the enduring synergy between nature and medicine.

 

MATERIALS AND METHODS:

Plant material and Preparation of Herbal extract

Cissampelos pareira leaves were collected and authenticated by Dr S. Shweta and Dr. Ashwini Kumar Dixit of Guru Ghasidas Vishwavidhyalya with authentication no. Bot/GGV/2022/20. The first step was to wash the leaves with clean water to remove any dirt or other debris that might have stuck, and then to dry them in an oven set at 35 to 40 degrees Celsius. The powder was made by crushing and grinding the dried leaves, and then the powder was weighed. After that, a solution of ethanol and water was used in a sequential fashion for extraction. After collecting and distilling the mensturm under vacuum, it was air dried in an evaporating dish until it reached a consistent weight.

 

Chemicals/Animals

The following items were acquired: carrageenan, diagnostic kits, and diclofenac sodium (a standard anti-inflammatory medicine), cytokine assay kits.  All the chemicals were of analytical grade.

 

The protocol number was used to obtain approval from the Institutional Animal's Ethics Committee for the experimental protocol 1355/PO/Re/S/10/CPCSEA. Animals were maintained in accordance with the recommendations made by the CPCSE. Wistar rats (male) weight about 180-220 g were housed in a standard habitat. These parameters comprised a temperature of 23 ± 2 degrees Celsius and a light/dark cycle of 12 hours. The rats were provided with regular rodent feed and free access to clean water.

 

Carrageenan-Induced Acute Inflammatory Model

A test for carrageenan-induced paw edema in rats was used to measure its anti-inflammatory efficacy. Each rat in each group (excluding Group I) had 100 μl of a freshly produced solution of carrageenan in distilled water injected subplantarly into its right hind paw to cause edema. Thirty minutes before injecting carrageenan, animals in Group II received a single dosage of 20 mg/kg diclofenac sodium, while animals in Groups III–VII received a single dose of plant extract, respectively. At "0 hour," shortly before the carrageenan injection, we measured the paws' diameter and volume. We repeated the measurements at 1, 2, 4, and 8 hours following the injection. To calculate the increase in paw thickness, the thickness at "0 hour" was deducted from the thickness at each hour.

 

Experimental Design

The experimental design for carrageenan induced Inflammatory Model is given in (Table 1).

 

Table 1 Grouping of animals for carrageenan induced Inflammatory Model:

Group No

Group

Group I

Normal control (0.5% carboxymethyl cellulose)

Group II

Carrageenan control

Group III

Carrageenan + Standard (Diclofenac Sodium 20mg/kg)

Group IV

Carrageenan + CIAQ (Cissampelos pareira aqueous extract 200 mg/kg)

Group V

Carrageenan + CIAQ (Cissampelos pareira aqueous extract 400 mg/kg)

Group VI

Carrageenan + CIME (Cissampelos pareira methanol extract 200 mg/kg)

Group VII

Carrageenan + CIME (Cissampelos pareira methanol extract 400 mg/kg)

 

Digital Plethysmometer/Paw volume:

The paw volume was measured before (0 h) and after carrageenan injection at 1, 2, 4, and 8 h by digital plethysmometer.

 

Paw diameter:

Using a vernier calliper, the paw diameter was measured both before and after injecting the animals with carrageenan and medications at 0, 1, 2, 4, and 8 hours respectively. We calculated the percentage inhibition of paw diameter using the following formula, after comparing the control group's foot swelling to that of the test and standard groups:

 

Percentage Inhibition = [(V1 − V2)/(V1)] × 100; V1 = paw diameter before carrageenan injection and V2 = paw diameter after treatment.

 

Measurement of excision wound healing

The centre portion of the dorsal thoracic region of the anaesthetized rats had their hairs plucked. The entire designated area was surgically removed to create a wound that is approximately 300 mm2 in size. A cotton swab dipped in alcohol was used to clean the wound. The formula was used to calculate the percentage of wound closure:

 

% Wound closure = 100 × (Initial wound area) ‐ (Nth day wound area) /(Initial wound area).

A regression analysis was conducted, and the results were plotted against the time it took for the wound to close, depicting the percentage of closure (in days). The CT50 is the half-life of a wound, where X is the initial measurement and Y is the final measurement.

 

Experimental Design

The experimental design for excision wound model (Table 2).

 

Table 2 Grouping of animals for excision induced model:

Group No

Group

Group I

Wound control

Group II

Wound + Standard

Group III

Wound + CIAQ (Cissampelos pareira aqueous extract 200 mg/kg)

Group IV

Wound + CIAQ (Cissampelos pareira aqueous extract 400 mg/kg)

Group V

Wound + CIME (Cissampelos pareira methanol extract 200 mg/kg)

Group VI

Wound + CIME (Cissampelos pareira methanol extract 400 mg/kg)

 

Homogenizing tissue

Collection, rinsing with saline, and homogenization of tissues from various groups took place at 4°C in a 0.1 M Tris-HCl buffer with a pH of 7.4. Following that, homogenates were centrifuged (3000 RPM) for the time period of 10 min. at 4°C. After that, aliquots were obtained for further analyses. The BSA technique was used to measure the amount of protein in the tissue homogenate20.

 

Catalase:

The 10% tissue homogenate was diluted with phosphate buffer pH-7 after adding a detergent, such as 1% Triton X-100, to the pH 7 mixture (1:100). The method for estimating catalase was reported by21.

 

GSH estimation:

Mixing 2.5 millilitres of 5% TCA with 0.5 gramme of skin tissue yielded a tissue homogenate. For 10 minutes, the protein that had precipitated was spun in a centrifuge at 1000 rpm. The GSH concentration was estimated using the supernatant (0.1 ml) 22.

 

Lipid peroxidation assay (MDA): 

The TCAe-TBA HCl was mixed with 1 ml of tissue homogenate, and then each tube was vortexed for a few seconds in a water bath that was boiling. When the tubes reached room temperature, centrifugation was applied for a duration of fifteen minutes. Before measuring the optical density (OD) at 535 nm relative to a blank, the supernatants were pipetted into a cuvette23.

 

Biochemical analysis:

Estimation of Hydroxyproline:

Tissue that had healed was removed and placed in glass vials to dry in an oven set at 110 °C for 48 hours. To quantify the amount of hydroxyproline, 5 mg of lyophilized material and 5 ml of 6 N HCl were hydrolyzed in a sealed tube at 110 °C for 18e20 hours, following the process that was detailed24.

 

Estimation of collagen:

Simply multiplying the hydroxyproline content by 7.46 will provide collagen of the same molecular weight24.

 

Estimation of hexosamine:

The procedure was heated to 110 0C for 6-7 hours with 5 ml of 2 N HCl and 5 mg of lyophilized tissue. The residue was dissolved in a specific volume of water after it had evaporated. The solution was heated to boiling for fifteen minutes after adding one millilitre of freshly manufactured 2% acetyl acetone in half a millimolar of sodium carbonate. Once cooled, one millilitre of Ehrlich's reagent was mixed with five millilitres of 95% ethanol. The evolution of the purple-red hue was revealed by a spectrophotometric measurement performed at 530 nm for 30 minutes later25.

 

Statistical Analysis:

To compare the groups, the mean ± SEM of the edema volume is shown, and then ANOVA is used, followed by Tukey's post hoc test.

(a*): Normal control vs Disease control

(b*): Disease control vs different doses of plants extract

(***): p<0.001, (**): p<0.01, (*): p<0.005

 

RESULTS:

Carrageenan-Induced Acute Inflammatory Model:

Effect of Cissampelos pareira extracts on paw volume:

After getting subplantar carrageenan injected for 1 hour, the control rats' paw volume significantly increased (P < 0.001) compared to normal rats. The paw volume of rats administered Cissampelos pareira at dosages of showed a substantial and dose-dependent decrease 200, 400 mg/kg. Moreover, with diclofenac (20 mg/kg) significantly decreased (P < 0.001) paw volume from 2hr to 8hr as compared to control rats (Figure 1a). The control rats demonstrated a notable rise (P < 0.001) in paw volume following 1 hour of subplantar carrageenan injection, in contrast to normal rats. Rats treated with Cissampelos pareira (CIAQ, CIME 200 and 400 mg/kg) showed significant and dose-dependent attenuation (P < 0.001 respectively) in paw diameter. Moreover, with diclofenac (20 mg/kg) significantly decreased (P < 0.001) paw diameter from 2hr to 8hr as compared to control rats (Figure 1b). Experimental wounds treated with extracts healed more quickly than control and conventional wounds in this study. The average percentage of wound contraction on day 7 was significantly different between the control and extracts treated groups. This difference persisted all the way up to day 14, following wound development (Figure 1c) (Figure 2).

 

 

(A)

 

 

(B)

 

 

(C)

Figure 1 Effect of Cissampelos pareira on paw volume, diameter, wound contraction

 

 

Figure 2 Visual depiction of the rate of contraction illustrating the percentage of wound area under contraction on various days

 

Antioxidant activity in wound healing:

The levels of glutathione and catalase were considerably greater in the group that received the formulation compared to the control group. Control group showed 1.77 ± 0.05 U/gm, animals that were usually given drugs demonstrated 2.6 ± 0.15 U/gm in CIME 200 mg/kg and CIME 400 mg/kg-treated animals showed 3.65 ± 0.10 U/gm of catalase in dry tissue. GSH results measured in control group, standard drug group, CIAQ 200 mg/kg, CIAQ 400 mg/kg, CIME 200 mg/kg and CIME 400 mg/kg groups were found to be 168.3 ± 3.05 ml/gm, 329.16 ± 5.52 mol/gm, 251 ± 6.38, 321.1 ± 4.35, 258.33 ± 4.33 and 323.85 ± 9.93 mol/gm respectively. In contrast, the control group had higher tissue MDA values 246.83 ± 5.76 U/gm, but standard drug-treated group showed 104.66 ± 3.21 U/gm as well as the CIAQ 200 mg/kg, CIAQ 400 mg/kg, CIME 200 mg/kg and CIME 400 mg/kg 65.16 ± 2.33, 95 ± 3.37, 64.83 ± 6.0, 97.16 ± 3.06 U/gm, which showed the considerable decrease in MDA levels that was observed when formulation was used (p < 0.001) (Figure 3).

 

 

Figure 3 Antioxidant activity in wound healing

 

Biochemistry of wound healing:

The methyl extract group demonstrated better wound healing capacities than the control group, as seen by higher levels of hydroxyproline, collagen, and hexosamine per gramme of dried regenerated tissue. The control animals' hydroxyproline levels were 33.16 ± 1.84 mg/g, standard drug treated animals showed 55.33 ± 3.5 mg/ g and CIAQ 200 mg/kg, CIAQ 400 mg/kg, CIME 200 mg/kg and CIME 400 mg/kg treated animals showed 37.33 ± 0.99, 51.16 ± 1.84, 40.5 ± 1.77 and 53.83 ± 2.27 mg/g of tissue. There was a detection of tissue hexosamine was 6.5 ± 0.4 mg/gm in control group, 22.33 ± 1.06 mg/gm in standard drug-treated group and CIAQ 200 mg/kg, CIAQ 400 mg/kg, CIME 200 mg/kg and CIME 400 mg/kg treated animals showed 15.33 ± 1.83, 19.83 ± 1.36, 15 ± 1.76 and 21.16 ± 1.45 mg/gm in test drug-treated animals. There is a one-to-one relationship between hexosamine and collagen formation; the control group demonstrated 243.1 ± 4.31mg/gm of tissue, standard drug-treated animals showed 370.16 ± 2.58 mg/gm whereas 281.5 ± 1.49, 355.66 ± 6.4, 299.5 ± 9.18, 356.33 ± 10.41 mg/g of tissue collagen in CIAQ 200 mg/kg, CIAQ 400 mg/kg, CIME 200 mg/kg and CIME 400 mg/kg treated group was observed (Figure 4).

 

Figure 4 Biochemistry of wound healing: Hydroxyproline, collagen, and hexosamine concentrations

 

DISCUSSION AND CONCLUSION:

There are three stages to the healing process of a wound: inflammation, angiogenesis, and collagen deposition. When compared to the control group that did not receive any treatment, the Achyranthes aspera extracts demonstrated a statistically significant reduction in wound size after 15 days. Collagen plays a crucial role in the extracellular matrix, which aids in wound healing. There is a one-to-one relationship between hydroxyproline and collagen production. Compared to the control group, animals treated with a polyherbal formulation had considerably higher levels of hydroxyproline in their newly created tissue. Traditional methods for evaluating plant-based wound healing agents rely on invasive and distressing procedures carried out on animal models. The in vitro tests outlined here allow us to screen a plethora of plant compounds for the angiogenic, antioxidant, and cell mobilisation characteristics that are crucial to wound healing. Earlier, it was mentioned that certain plant items include both wound healing and antioxidant characteristics. In most cases, antioxidants help speed up the healing process of wounds. This article outlines in vitro assays that can be used to screen herbal preparations for wound healing qualities. By doing so, we can avoid utilising animals in experiments that aren't necessary.

 

According to the results of our research, several extracts (CIAQ 200 mg/kg, CIAQ 400 mg/kg, CIME 200 mg/kg and CIAE 400 mg/kg) of Cissampelos pareira speeds up the healing process of wounds by encouraging angiogenesis and fibroblast and keratinocyte mobilisation.

 

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Received on 28.02.2024            Modified on 18.03.2024

Accepted on 30.03.2024           © RJPT All right reserved

Research J. Pharm. and Tech. 2024; 17(3):1336-1341.

DOI: 10.52711/0974-360X.2024.00210