INTRODUCTION:

The usage of herbal plants in the field of medicine has a long history.^1,2 The current medications being used have shown cytotoxic and deleterious effects to human health^3,4,5. Embelia ribes is a therapeutic woody climber, belongingto Myrsinaceae family⁶. It is commonly recognized as false black pepper or vidanga. The pharmacological activity of Embelia ribes is known to exhibit anthelmintic⁷, antioxidant,⁸ antiprotozoal, abdominal disorders, lung disease, fungal infection, analgesic, hepatoprotective², anti-inflammatory, wound healing^9,10and various more.⁹

Owing to its significant contribution in the field of medicine, The Medicinal Board of Government of India, New Delhi has recognized Embelia ribes as crucial one out of 32 medicinal plants for its large-scale cultivation and is labelled as Agro-techniques of Selected Medicinal Plants. Embelia ribeshave array of 75 Ayurvedic preparations with its leaves, seed, fruit and roots. The significant chemical bioactive constituent, embelin,^9,11 has therapeutic and medicinal ability to be converted to innovative drugs, for which it has gained cogitation recognition. The gaining impetus in the use of nanoparticles is due to increased efficacy in reaching minute areas,¹²which is inaccessible for the conventional medication.¹³

Green synthesized Silver Nanoparticles are more biocompatible and effective with least cytotoxicity.^14,15 The primary objective of the current study is to discover the antioxidant activity of seeds of Embelia ribes-mediated silver nanoparticles using DPPH, H202 and Frap assay.¹⁶The seeds leaves and stems of Embelia ribes is proven to have free radical scavenging and inhibitory action on DPPH radical and also been shown to inhibit hydroxyl radicals¹⁷. As the alcoholic and epithelial extracts were found to be effective in treating ailments along with epithelialization combined with a significant increase in the pace of wound healing, an antioxidant measure of the crude extract was conducted in this study.^10,16

MATERIALS AND METHODS:

The aim of this study is to examine the antioxidant effect of green synthesized Embelia ribes-mediated silver nanoparticles using DPPH and hydrogen peroxide assay. The methodology of the present study was determined using the following studies.^18,19

Preparation of Plant Extract:

The seeds of Embelia ribes were procured online from an herbal company. The seeds procured were refined to powder form.1 gram of E.ribes (fig. 1 )was mixed with 100ml distilled water and added to beaker and boiled for 10-20 minutes in a heating mantle. Whatman Filter paper No 1 was used to filter the plant extract.²⁰

Silver Nanoparticles Synthesis:

A solution of 1mMol of silver nitrate in 90ml was utilized to synthesize nanoparticles. To this solution 10 ml of filtered E.ribes extract is added and kept on the orbital shaker with a magnetic stirrer for 2hours and Color change was observed every 24hours for 3 days.(Fig.2a and b) The synthesized nanoparticles was analyzed using UV visible spectroscopy.^18,21 For the final step, the formulated solution was centrifuged at 8000rpm (Fig 3) to prepare nanoparticles pellet and is followed by drying at 80 degrees Celsius in hot air oven. The dried powder is sent for characterization. The solution left was used to compute the antioxidant activity.^22,23

Fig . 1: Powdered form of Embelia ribes seed.

Fig 2 a: Embelia ribes derived silver nanoparticles on Day 1 2b: Color change noted in the third day

Fig. 3: Centrifugation done at 8000rpm to obtain the nanoparticles.

Antioxidant Activity:

DPPH Method:

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) the assay was used to evaluate the efficacy of antioxidant activity of green synthesized silver nanoparticles.²⁴ Assorted concentrations (10-50μm/ml) of Embelia ribes-mediated silver nanoparticles was mixed with 1ml of 0.1mM DPPH within methanol. 450 microliters of 50mM Tris HCL buffer (ph 7.4) and incubated for 30 minutes. The reduction in the quantity of DPPH free radicals was evaluated as dependent on the absorbance at 517nm. Ascorbic Acid (Vitamin C) was designated as Control. The percentage of inhibition was derived from the following equation.²⁵ (Results noted in Graph 1)

Percentage of inhibition = (Absorbance of control - Absorbance of the test sample x 100)/Absorbance of control.

Hydroxyl Radical Scavenging Assay:

The foregoing mentioned solution 1.0ml of reaction mixture 100 microliters of 28mM of 2-deoxy 2-ribose was dissolved in a phosphate buffer of ph 7.4. To this solution, differing concentrations of E.ribes mediated silver nanoparticles were added in varying concentrations as noted in graph 1. After an incubation period of 1hour at 37°C the extent of deoxyribose degradation at about 532nm against the blank solution. Ascorbic Acid is used as the standard control. (Results noted in graph 2)

Frap Assay:

Ferric-Reducing Antioxidant Power assay measures the reduction of ferric ion ligand complex in the presence of oxidation properties. Here, similarly, small samples of the E.ribes mediated silver nanoparticles were added in varying concentrations . Vitamin E is usedas control. (results noted in graph 3)

RESULTS AND DISCUSSION:

Graph 1: DPPH Assay Done in Different Concentrations With Control To Assess The Antioxidant Activity

Inference: X-axis depicts the concentration used and Y- axis shows the percentage of antioxidant activity in μL. With increase in the concentration of the Embelia ribesmediated silver nanoparticle, increased antioxidant effect was observed. Maximum effect was noted at 84% at 50μL concentration for DPPH Assay. Ascorbic acid was used as the standard.

Graph 2: H2o2 Assay Done in Different Concentrations with Control to Assess the Antioxidant Activity

Inference: X-axis depicts the concentration used and Y- axis depicts the percentage of antioxidant activity in μL. With increase in the concentration of the Embelia ribes mediated silver nanoparticle, an increased antioxidant effect was observed. Maximum effect was noted at 82% at 50µl concentration for H202 Assay. Ascorbic acid was used as the standard.

Graph 3: Frap Assay Done in Different Concentrations with Control to Assess theAntioxidant Activity

Inference: X-axis depicts the concentration used and Y- axis gives the percentage of antioxidant activity in μL. With increase in the concentration of the Embelia ribes mediated silver nanoparticle, an increased antioxidant activity was observed. Maximum effect was noted at 80% at 50µl concentration for FRAP Assay. VitaminE was used here as the positive control.

This study shows that Embelia ribes mediated silver nanoparticles exhibited broad antioxidant activity against different reactive oxygen species (ROS). (Graph 1).

The working mechanism of action of silver nanoparticles is contributed to the nature of silver which can exist in two oxidation states depending upon the reaction condition.(Ag+ and Ag2+).²⁶ Plant-derived nanoparticles confers to a complex mechanism of action in biological realms.²⁷ AgNPs have a yellowish to dark brown color attributed to its Surface Plasmon Resonance, based on its size and shape , change in color was considered as secondary factor for identification of the nanoparticles formation.²⁸ (Fig.2a and b) Mainly the change noted in the color of the solution of Embelia ribes mixed with silver nitrate is ruling factor for formation of silver nanoparticles. On UV Spectroscopy, the colloidal solution of Embelia ribes mediated silver nanoparticle exhibited sharp bands of silver colloids.²⁹

Antioxidants are chemical substances that neutralize free radicals by donating an electron or hydrogen atom, therefore preventing the cells and tissues from oxidative damage.³⁰ The potential of antioxidants to convert stable radical DPPH to a non-radical state is supervised in this study using the DPPH test. The freshly synthesized Embelia ribes-AgNPs were shown to have high antioxidant properties. DPPH is a steady radical with peak absorption at 520nm. The E.ribes-mediated silver nanoparticles were measured at the dosage of 10, 20, 30, 40 and 50uL/mL and a control group was kept. The antioxidant activity increased with increased activity and was noted at maximum with 50µl concentration as noted in Graph 1 and Graph 2 and Graph 3.

The oxidation property of the body is very essential for a disease-freelife. Cytotoxic damage often results in genotoxicity with several anomalies. Use of synthetic antioxidants such as butylated hydroxytoluene has inevitable side effects with accumulated toxicity. Embelin in the major constituent in Embelia ribes which has antioxidant and antitumor properties.³¹ Embelin has a unique structure due to presence of both phenolic and quinone group in the same ring.³²

Embelia ribes are shown to have good antioxidant properties due to its free radical scavenging properties. Prospects can have studiesbased to discover various other attributes of Embelia ribes and its application in the field of medicine.³³

CONCLUSION:

This current study enlightens the antioxidant potential of Embelia ribes seed extract using silver nanoparticles, which was analyzed using DPPH, H202 and FRAP scavenging assay. Further study can define its uses in the field of medical sciences ,especially in the field of cancer.

ACKNOWLEDGMENTS:

The author is grateful to Saveetha Dental college for the platform provided to conduct the study.

CONFLICTS OF INTEREST:

The authour declares no conflict of Interest.

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Received on 08.12.2023 Modified on 21.05.2024

Research J. Pharm. and Tech 2024; 17(9):4199-4202.

DOI: 10.52711/0974-360X.2024.00649