In-vitro Antioxidant and In-vitro Anti-inflammatory activities of Ethanolic leaves extract of Ormocarpum Cochinchinense

Gayathri Somashekar; Uma Sudhakar; Poonudurai Sameul Gnana Prakash; Snophia Suresh; Seshadri Srividya; Sumathi Hanumantha Rao

doi:10.4103/jofs.jofs_253_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 14 | Issue : 2 | Page : 134-140

In-vitro Antioxidant and In-vitro Anti-inflammatory activities of Ethanolic leaves extract of Ormocarpum Cochinchinense

Gayathri Somashekar¹, Uma Sudhakar², Poonudurai Sameul Gnana Prakash³, Snophia Suresh², Seshadri Srividya⁴, Sumathi Hanumantha Rao⁴
¹ Dr.M.G.R. Educational and Research Institute, Chennai, India
² Thai Moogambigai Dental College and Hospital, Chennai, India
³ SRM Dental College and Hospital, Chennai, India
⁴ Sathyabama Dental College and Hospital, Chennai, India

Date of Submission	25-Oct-2022
Date of Decision	10-Nov-2022
Date of Acceptance	15-Nov-2022
Date of Web Publication	10-Jan-2023

Correspondence Address:
Dr. Gayathri Somashekar
Research Scholar, Department of Periodontics, Dr. M.G.R Educational and Research Institute, Chennai 600095
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jofs.jofs_253_22

Abstract

Introduction: Periodontitis, a chronic inflammatory disease with microbial etiology, is mediated by multiple inflammatory processes and oxidative stress is now well recognized as a part of periodontal pathogenesis. A balance between reactive oxygen species and antioxidants is required to maintain periodontal health. Medicinal herbs with bioactive phytocompounds have rich source of antioxidants and anti-inflammatory compounds. Ormocarpum cochinchinense is a medicinal herb with antioxidants and anti-inflammatory phytocompounds. The phytocompounds activities of the herb are not much explored. This study is focused on the In-vitro antioxidant and anti-inflammatory activities of the ethanolic extract of leaves of O. cochinchinense. To assess the In-vitro antioxidant and In-vitro anti-inflammatory activities of ethanolic extracts of O. cochinchinense. Materials and Methods: The leaves of O. cochinchinense were collected, air dried in the shade, and then powdered in an electric blender. The preparation of ethanolic extract was carried out. In-vitro antioxidant studies using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Nitric Oxide (NO) assays along with anti-inflammatory activity by protein denaturation inhibition and membrane stabilization method were studied. Descriptive statistics were used for continuous variables and expressed in mean and standard deviation. One way ANOVA with post-hoc tukey test or Kruskal–Wallis test, Post-hoc Mann–Whitney U test was used according to the normal distribution of the sample. To compare the individual study group against their standard group, independent t test, and Mann–Whitney U test have been used. P < 0.05 was considered significant. Results: O. cochinchinense had significant antioxidant and anti-inflammatory activities. The ethanolic extract showed dose-dependant activity in all analyses performed (P < 0.05). NO inhibition assay showed 95% of antioxidant activity and 80% of anti-inflammatory activity in the Human Red Blood Cell (HRBC) Membrane Stabilization assay. Conclusions: O. cochinchinense could be used as an adjuvant supplement to conventional therapy in the treatment of chronic inflammatory diseases.

Keywords: Anti-inflammatory, Antioxidant, O. cochinchinense, Periodontitis

How to cite this article:
Somashekar G, Sudhakar U, Prakash PS, Suresh S, Srividya S, Rao SH. In-vitro Antioxidant and In-vitro Anti-inflammatory activities of Ethanolic leaves extract of Ormocarpum Cochinchinense. J Orofac Sci 2022;14:134-40

How to cite this URL:
Somashekar G, Sudhakar U, Prakash PS, Suresh S, Srividya S, Rao SH. In-vitro Antioxidant and In-vitro Anti-inflammatory activities of Ethanolic leaves extract of Ormocarpum Cochinchinense. J Orofac Sci [serial online] 2022 [cited 2023 Jun 9];14:134-40. Available from: https://www.jofs.in/text.asp?2022/14/2/134/367445

Introduction

Chronic inflammatory diseases and conditions like cancer, diabetes, neurodegenerative diseases, cardiovascular diseases, and periodontitis all have oxidative stress as a major contributor to their etiology. Long-term exposure to elevated pro-oxidant levels can lead to structural flaws in mitochondrial deoxyribonucleic acid (DNA) as well as modifications in enzymes and cellular components that can induce anomalies in gene expression.^[1] Oxidative stress is a process causing damage to the physiological and biochemical milieu of various tissues. It is also believed to be a crucial physiological process where some amount of oxidative stress known as oxidative eustress helps the defense system to cope with microbial attack and intracellular cell signaling.^[2] Damage from oxidative stress occurs when free radicals and antioxidant defenses are out of equilibrium.^[3] Periodontitis primarily is a disease of microbial etiology which causes alteration in host response and ultimately leads to periodontal tissue destruction. Studies have shown an inverse relationship exists between periodontal disease severity and systemic antioxidant concentration.^[4] Oxidative damage in periodontitis is due to overproduction of reactive oxygen species (ROS).^[5] Free radicals or reactive oxygen species are generated during normal cell metabolism. During inflammation, innate immune system cells, such as neutrophils and macrophages, boost ROS production significantly through the metabolic pathway known as the "respiratory burst.”^[6] This overproduction of ROS is physiologically balanced by adequate quantity of antioxidants and prevents ROS-induced tissue damage. When the inflammation progresses, the antioxidant defense system cannot balance the excess release of ROS and oxidative stress occurs.^[7] Oxidative stress may further lead to cascading effects of inflammation and destruction on host tissues. This suggests that the conventional methods for treating and preventing periodontal disease, which concentrate on managing bacterial pathogens alone is appeared to be insufficient, and therefore promising preventive and therapeutic adjuncts in the form of oxidative stress-reduction regimens using antioxidant supplementation and control of inflammation in the disease process is necessary.^[8]

Antioxidant substances are available in food, plants, tea, vitamins, minerals, etc.^[9] Herbal-based medicine has flourished in India over centuries. Both indigenous recordings and modern studies exist on antioxidant and anti-inflammatory properties of medicinal herbs.^[10] Currently used modern anti-inflammatory agents have wider range of action but also exhibit various side effects. Literature suggests that herbal extracts might have less side effects due to their natural origin.^[11] Inflammatory process is associated with oxidative stress, and it is therefore advantageous to have both anti-inflammatory and antioxidant property in the same herbal extract. In other words, if a herb possesses both antioxidant and anti-inflammatory properties, it would be advantageous in the treatment of chronic inflammatory diseases.^[12]

Ormocarpum cochinchinense (OC), a Fabaceae family member, has been documented as a potent bone healer.^[13] This herb, also known as bone-knit, Elumbotti, or Kattumuringai, is found in the forests and hills of India’s Coromandal region.^[14] It is also found in tropical and southern Africa, Madagascar, Southern Asia, Northern Australia, and Pacific Ocean islands. It has traditionally been used for fracture repair.^[13] The documentation with respect to pharmacological activities of OC is still in its nascent stages and needs more studies to know about the phytochemical benefits of the herb. This In-vitro study aims at analyzing the antioxidative and anti-inflammatory properties of OC ethanolic extract.

Materials and Methods

The leaves of O. cochinchinense were collected from the hills of Hosur, Tamil Nadu in the period between December and February 2020. This study was approved (protocol no. SU/CLATR/IAEC/XVIII/190/2021) by Institutional ethics committee, Sathyabama Institute of Science and Technology, Chennai 600119 on 09 October 2021. The leaves were washed in distilled water and dried under shade and then powdered in an electric blender. Until further investigation, this powder was stored in an airtight container.

Ethanolic extract preparation of leaves of Ormocarpum Cochinchinense (OC)

100 g of leaf powder was added to 99.9% of ethanol and placed in rotary apparatus for 3 days. The supernatant was filtered with Whatmann filter paper (no. 1). The extract was further concentrated using rotary evaporator and stored at 4°C for further analysis. 1 g/mL of extract was submitted for Gas Chromatography–Mass spectroscopy analysis (JEOL GCMATE II GCMS) to identify the phytocompounds. Compounds were identified by comparing the chemical names of the compounds in the database of National Institute of Standards and Technology (NIST).

In-vitro antioxidant studies

2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity

The total free radical scavenging capability of OC extract was calculated using the previously described methodology.^[15] DPPH solution of 1.0 mL was added to 1.0 mL of different concentrations of plant extracts (100, 200, 300, 400, and 500 μg/mL). The mixture was kept at room temperature for 50 minutes, and the antioxidant activity was measured by a spectrophotometer at 517 nm. Ascorbic acid at various concentrations (100, 200, 300, 400, and 500 μg/mL) was used as standard. The percentage of free radical inhibition was calculated as half minimal inhibitory concentration (IC₅₀). IC₅₀ denotes the concentration of the sample required to scavenge 50% of DPPH free radical. The capability of plant extract to scavenge the DPPH radical was calculated using the following formula:

Nitric oxide (NO) radical scavenging activity

Inflammatory diseases have high production of NO. Plant extracts which can scavenge or inhibit the production of NO are known to have antioxidant properties.^[16],[17] The methodology described by Marcocci et al.^[18] was followed for the NO assay. The reaction mixture (3 mL) containing sodium nitroprusside (10 mM, 2 mL), phosphate buffer saline (0.5 mL), and different concentrations (100, 200, 300, 400, and 500 μg) of extracts (0.5 mL) were incubated at 25°C for 150 minutes. 0.5 mL of the reaction mixture containing nitrite was pipetted out and mixed with 1 mL of sulfanilic acid reagent (0.33% in 20% acetic acid) and allowed to stand for 5 minutes for completing diazotization. 1 mL of naphthyl ethylene diamine dihydrochloride was added, mixed, and allowed to stand for 30 minutes at 25°C. A pink colored chromophore was formed in diffused light. Ascorbic acid at various concentrations (100, 200, 300, 400, and 500 μg) was used as standard. The activity was measured at 550 nm and the results were expressed as percentage (%) of scavenging using the following formula:

Protein denaturation inhibition assay for Anti-inflammatory activity

The Williams et al.^[19] technique was followed while performing the protein denaturation assay. Reaction mixtures were heated in a water bath to 70°C for 5 minutes. The reaction mixture was then allowed 15 minutes to cool at room temperature. Absorbance of reaction mixture before and after denaturation was measured for each concentration (1000, 100, 10, 1, 0.1, and 0.01 μg/mL) at 680 nm using a colorimeter. Each test was repeated thrice, and the mean absorbance was recorded. The percentage of inhibition of protein was determined on a percentage basis with respect to control (Diclofenac sodium 100 mg) using the following formula:

Anti-inflammatory activity by Human Red Blood Cell (HRBC) Membrane Stabilizzation Method

Gandhidasan’s et al.^[20] HRBC membrane stabilization approach is used to investigate the extract’s In-vitro anti-inflammatory effects. Lysosomes undergo lysis during inflammation, and release specific enzymes into circulation leading to inflammatory diseases. Non-steroidal anti-inflammatory medicines (NSAIDs) work to reduce inflammation by either preventing the release of lysosomal enzymes or stabilizing the membranes around them.^[20] In this assay nonsteroidal anti-inflammatory drug, Diclofenac sodium of about 100 mg was used as standard positive control. Red blood cells (RBCs) undergo membrane lysis, and hemoglobin oxidation when exposed to harmful agents including hypotonic medium, heat, methyl salicylate, or phenylhydrazine. The suppression of hypotonicity and heat-induced red blood cell (RBC) membrane lysis was taken as a measure of the mechanism of anti-inflammatory effect of OC extract because HRBC membranes are comparable to lysosomal membrane components. The percentage of RBC lysis was used to measure anti-inflammatory activity.^[21] If the HRBC membrane stabilized because of the presence of herbal extract OC, the lysosomal membrane will also similarly stabilize. The amount of hemoglobin in the suspension was estimated using a spectrophotometer operating in the 560 nm region. The test solution was prepared by adding 1 mL of phosphate buffer, 2 mL of hypotonic saline, 0.5 mL of plant extract of various concentration (100, 200, 300, 400, and 500 µg/mL), and 0.5 mL of 10% weight per volume (w/v) HRBCs. The test control solution was prepared by adding 1 mL of phosphate buffer, 2 mL of water, and 0.5 mL of 10% w/v HRBCs in isotonic saline. The standard solution was prepared by adding 1 mL of phosphate buffer, 2 mL of hypotonic saline, 0.5 mL of plant extract of various concentration (100, 200, 300, 400, and 500 µg/mL), and 0.5 mL of 10% w/v human red blood cells. All the assay mixtures were incubated at 37°C for 30 minutes and centrifuged at 3000 rpm. The percentage of hemolysis was estimated by assuming the hemolysis produced in the content as 100%. The percentage of HRBC membrane stabilization or protection was calculated by using the following formula:

Data analysis

The data were analyzed using IBM SPSS version 21 (SPSS version 21.0; IBM corporation, Armonk, NY, USA) statistical software. Statistical significance was set at 0.05 levels. Descriptive statistics were used for continuous variables and expressed in mean and standard deviation. Test of normality was applied.

To understand the effects of antioxidants of nitric oxide group and DPPH group as well as anti-inflammatory effects of the protein denaturation group and HRBC study group at different concentration levels of the extract individually, one way Analysis of variance (ANOVA) and Kruskal–Wallis test had been used. Shapiro-Wilk test was used to test the normality.

Results

One-way ANOVA was used to determine the effects of Nitric Oxide group and DPPH group against the standard group across various concentration levels. The standard used was Ascorbic acid. [Table 1] shows significant differences (P-value < 0.05) between Nitric Oxide group and standard group across all the concentration levels. In DPPH and standard group, it shows significant differences (P-value < 0.05) across 200, 300, and 400 µL concentration levels. Graphs 1 and 2 show that as the concentration increases, the percentage inhibition also increases.

Table 1 Antioxidant capacity of various concentrations of OC extract by Nitric Oxide and DPPH assays compared to Standard

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One-way ANOVA was also used to determine the effects of protein denaturation group against standard group across various concentration levels. The standard used was Diclofenac sodium 100 mg. [Table 2] shows significant differences (P-value < 0.05) between protein denaturation group and standard group across 100, 200, and 500 µL concentration levels. Graph 3 shows as the concentration increases, the percentage inhibition also increases.

Table 2 Anti-inflammatory capacity of various concentrations OC extract by Protein denaturation inhibition and HRBC assays compared to Standard

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Since Shapiro-Wilk test (test of normality) for HRBC shows significance (P-value < 0.05), so ANOVA test is not applicable. Therefore, Kruskal–Wallis test was performed to find significance between HRBC group and standard group for determining the anti-inflammatory effect. [Table 2] shows that there were significant differences seen between HRBC group and standard group across all the concentration levels. Graph 4 shows as the concentration increases the percentage inhibition also increases.

Discussion

The present study aimed to analyze the In-vitro antioxidant and anti-inflammatory activities of the ethanolic extract of O. cochinchinense. Scientific literature has documented the use of antioxidants in the treatment of various inflammatory diseases.^[22] Antioxidants have been used as co-adjuvants in traditional therapy to counteract oxidative stress.^[23]

Plants are the primary source of natural antioxidants. However, it should be noted that antioxidant and anti-inflammatory activities vary when the herb is collected from different geographical locations. Furthermore, the activity of natural products is controlled by a variety of other elements, such as meteorological and soil conditions as well as harvest time. Further, there is a dearth of literature where both these antioxidant and anti-inflammatory properties are described in a single herb like OC.

Extraction of herbal plants is a procedure by which active ingredients like plant phytocompounds are separated from the plant using solvents like ethanol, acetone, methanol, etc.^[24] Based on the solubility of the active compound in the solvent, different phytocompounds are obtained. The composition of active compounds, recovered from a wide range of plant materials (herbs, vegetables, berries, and fruits), is influenced by the extraction processes used. Varied extraction procedures result in different extraction yields on the same plant material.^[25]

Ethanolic extract of OC extract in the study conducted by Hepsibah and Jyothi showed 92.87% antioxidant capacity done with DPPH assay.^[26] In this study, the ethanolic extract was used to analyze antioxidant and anti-inflammatory activities.

Study done by Pazhanisamy on In-vitro antioxidant capacity of OC herb reported 73.4% activity by the DPPH method.^[27] Nitric oxide antioxidant group in our study showed highest percentage of 95% antioxidant activity compared to standard group. Among the anti-inflammatory groups, HRBC membrane stabilization assay showed 80% of the anti-inflammatory activity compared to the standard. Natural antioxidants are increasingly being utilized as adjuvant for the treatment of chronic inflammatory diseases.^[28] Therefore, herbal actives having antioxidant and anti-inflammatory activities can potentially modify the progression of periodontal disease.

OC extract has shown significant antioxidant and anti-inflammatory activities with regard to NO scavenging, DPPH, protein denaturation inhibition, and HRBC assays ([Table 1], [Table 2]).

Under normal physiological conditions, NO is required for the control of many physiological functions, including blood pressure, immunological response and neuronal transmission.^[29] Overproduction of NO, on the other hand, can cause tissue damage and is linked to inflammatory illnesses such as atherosclerosis and hypertension. As a result, researchers have focused their efforts on identifying natural antioxidants that may act as effective inhibitors of NO generation in the treatment of chronic inflammatory diseases.^[30] Dose dependent inhibitory activity of OC makes it a putative anti-inflammatory and antioxidant drug awaiting clinical research in future.

OC was able to successfully scavenge free radicals at various concentrations. One widely established model against lipid oxidation was DPPH free radical scavenging activity. The ability of antioxidants to donate hydrogen was assumed to be responsible for their influence on the DPPH radical scavenging. The OC demonstrated strong antioxidant activity. Superoxide is widely recognized as a highly reactive radical that can be transformed into more reactive species, such as hydroxyl radical or peroxynitrite, contributing to tissue damage and disease. The inhibitory effect of superoxide production in the reaction mixture could explain the OC’s scavenging function.^[31] The In-vitro anti-inflammatory efficacy of the OC was investigated using easy and feasible protein denaturation and the HRBC membrane stabilization procedures. Denaturation of tissue proteins is well known to cause inflammatory and arthritic diseases.^[32] Natural compounds that can prevent protein denaturation would thus be advantageous for the development of anti-inflammatory medication therapy.^[33] In this investigation, protein denaturation inhibition assay demonstrated that OC is potentially effective anti-inflammatory herb.

Since the erythrocyte membrane is similar to the lysosomal membrane, the extract may also stabilize lysosomal membranes.^{[34],[35],[36]} The OC reduced hypotonicity-induced lysis of the erythrocyte membrane, had a membrane stability impact on the lysosymal membrane, and hence had a strong anti-inflammatory effect. This study is the first to examine the ethanolic extract of the OC plant’s combined antioxidant and anti-inflammatory activities.

Conclusion

The present study showed that ethanolic extract of O. cochinchinense has potential antioxidant and anti-inflammatory activities. Incorporation of the phytocompounds of the OC herb could be considered in future as an adjuvant therapy in the treatment of oxidative stress and inflammatory diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

Authors declare no conflicts of interest.

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