Biocompatibility Stainless Steel Brackets with Nickel and Chromium Ions: A Scoping Review

 

Ari Triwardhani*, Yashica Alifia Amanda, Thalca Hamid, Alexander Patera Nugraha,

Louis Steventhie, Intan Vallentien Dwi Hariati

Department of Orthodontics, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

*Corresponding Author E-mail: ari-t@fkg.unair.ac.id

 

ABSTRACT:

Background: The use of stainless steel brackets in the oral cavity for a long time will definitely experience a corrosion reaction. The corrosion process is always followed by the release of ions from metal elements. The release of nickel and chromium ions is the most common and studied because of its negative effects on the human body. Objective: to review the biocompatibility and release effect of nickel and chromium ions from stainless steel brackets on the human body. Methods: The data source of this study was obtained through literature search through a database with descriptions related to the biocompatibility of the use of stainless steel brackets with the release of nickel and chromium ions. Results: The results of research and literature studies show that the corrosion results obtained from orthodontic appliances do not cause DNA damage and low genotoxicity, toxicity and cytotoxicity. Conclusion: Biocompatibility of dental materials is the basic point of the success of these materials to be used in the oral cavity. The biocompatibility of stainless steel brackets is good due to the low level of toxicity, cytotoxicity and genotoxicity in the body.

 

KEYWORDS: Stainless steel bracket, Biocompatibility, Ions release, Medicine, Non-communicable disease.

 

 


INTRODUCTION: 

The orthodontic treatment process has a relatively long treatment time, this is an obstacle in treatment so that a safe, comfortable and durable appliance component is needed in the oral cavity1.  Stainless steel brackets are one of the orthodontic material that are widely used today because they have advantages such as good mechanical properties, strength, and biocompatibility2. Most of the brackets are orthodontic made of stainless steel AISI 316L which contains several metals including 16-18% chromium, 12-15% nickel, 2-3% molybdenum and a small amount of manganese and silicon3. The use of stainless steel brackets in the oral cavity tends to be used for a long time and can be sure to experience a reaction and release ions from the reaction. The reaction that often occurs is the corrosion process of stainless steel brackets.2 There are several factors in the oral cavity that can increase the risk of corrosion in brackets such as temperature, pH of saliva, fluoride, bacteria, enzyme activity and protein4.

 

The corrosion process of stainless steel bracket in the oral cavity can release ions such as nickel, manganese, iron, chromium, and copper. Among other corrosion products, the release of nickel and chromium ions is the most frequent and studied due to there are negative effects on the human body, such as allergies, dermatitis, gingivitis, enamel discoloration, and it has been found that other effects are mutagenic, carcinogenic, cytotoxic that can induce DNA damage2. Biocompatibility of dental materials is the basic point of the success of these materials to be used in the oral cavity. Biocompatibility is very important for further analysis because it has elements of safety, responsibility and to keep the body's immune system capable of reacting normally to dental materials5. Nickel is also frequently used in orthodontics in the form of NiTi, or nickel titanium. One of niti’s features is superelasticity, the ability to return to its original form after deformations with relatively low tensions.6 Knowing the frequent use of orthodontic brackets and wires containing nickel and chromium, the aim of this scoping review is to describe biocompatibility of stainless steel brackets and release of nickel and chromium ions.

 

MATERIAL AND METHODS:

This study is a narrative review to provide biocompatibility of stainless steel brackets through 3 main steps to formulate the result studies. First, we identify and select related research about our topic. Second, we assess the retrieved research that meets all eligibility criteria. Third, we made the review and data extraction for each study.  The literature sources used in the writing of this article are Scopus and PubMed databases, by the following keywords: Stainless steel bracket, biocompatibility, ion release and corrosion. The limitation of the search process is not only related to the theme, but also the year of the article was published within the last 15 years. A total of 110 articles were found related to the keywords and the publication year limits, yet only 10 articles were selected after the author read the whole contents of the articles based on relevant topic, inclusion and exclusion criteria (Figure 1).

 

Figure 1. PRISMA diagram of retrieved studies

 


RESULTS:

Table 1: Summarizes the descriptive Component of the Studies.

No

Title

Author Publisher

Purpose

Methods

Result

1

Nickel And Chromium Ion Release from Stainless Steel Brackets Immersed In Carbonated Drinks

Sumule, et al.

 (2015)1.

E-GIGI

To know the amount of nickel and chromium ion released from the stainless steel bracket immersed in carbonated drinks

-    8 samples in the form of a solution were divided into two groups each with four samples

-    Test with UV -visible spectrophotometry to determine the release of nickel and chromium ions from stainless steel brackets immersed in saliva without carbonated drinks and saliva with carbonated drinks for 312 minutes

-       The release of nickel and chromium ions in the test group was greater with the average release of nickel ions in the control group of 1.556 ppm and in the treatment group of 2.624 ppm, while the average release of chromium ions in the control group was 0.038 ppm and in the treatment group 0.109 ppm

2

Nickel and chromium ion release from stainless steel bracket on immersion various types of mouthwashes

Mihardjanti, et al. 

(2017)2.

Journal of Physics: Conference Series

To determine the nickel and chromium ions released from the stainless steel bracket when immersed in mouthwash and distilled water

 

-     17 maxillary premolar brackets with 0.022 slots

-     Each group was immersed in different mouthwash and distilled water and incubated at 37 ° C for 30 days

-     For statistical analysis, both the Kruskal-Wallis and Mann-Whitney tests were used

-         The difference between the four groups in the released nickel ion (p <0.05) and the chromium ion released (p <0.5)

-         The ions released as a result of soaking mouthwash have a small value that is below the daily intake limit recommended by the World Health Organization

3

Corrosion of Metal Orthodontic Brackets and Archwires Caused by Fluoride-Containing Products: Cytotoxicity, Metal Ion Release and Surface Roughness

Yanisarapan, et al.

(2018)4.

Orthodontic Waves

To determine the cytotoxicity, release of metal ions and surface roughness of metal orthodontic appliances after immersion in different fluoride product solutions

 

-    12 sets of 20 brackets and four tubes are attached with three types of arch wires: stainless steel, nickel-titanium, and beta-titanium

-    Samples in each arch wire group were divided into three subgroups and immersed in fluoride toothpaste solution, 1.23% acidulated phosphate fluoride (APF), or artificial saliva without fluoride as a control group

-       In the APF group, the four metal ion levels and the surface roughness of the bracket and arch wires were significantly increased, while the cell viability decreased significantly

 

 

4

Effect of fixed orthodontic treatment on salivary nickel and chromium levels: A systematic review and meta-analysis of observational studies

Imani et al. (2019)7.

Dentistry Journal

To know the ratio of nickel and chromium concentrations in oral mucosal cells of patients with fixed orthodontic appliances

-    60 patients were included in this study

-    Determination of metal content was carried out using atomic absorption spectrophotometry with a graphite furnace

-       According to spectrophotometric analysis, no significant difference in the chromium (p = 0.09) and cobalt (p = 0.10) content of oral mucosal cells was found between the test and control samples

-        The nickel content in mucosal samples was significantly higher (p = 0.003) in orthodontic patients compared to controls

-       The mean nickel levels in the control and orthodontic groups were 12.26 and 21.74 ng ⁄ ml

5

The Release of Total Metal Ion and Genotoxicity of Stainless Steel Brackets: Experimental Study Using Micronucleus Assay

Karlina et al.  (2016)8.

The Indonesian Biomedical Journal

 

To know the difference and correlation of metal ion release and genotoxic activity between three brands of stainless steel bracket

-    3 brackets were immersed in artificial saliva for 672 hours and examined for the release of chromium, nickel and iron ions

-    Micronucleus block cytokinesis (CBMN) tests using lymphocytes were also performed

-       The highest metal releases were nickel, chromium and iron (30.5, 27.2, 23.4 ppb)

-      Genotoxicity was significantly correlated with the release of chromium (p = 0.03) and nickel (p = 0.01)

-       The genotoxicity of the stainless steel bracket was influenced by the immersion time but not by the brackets brand

6

Cytotoxic effects of the nickel release from the stainless steel brackets: An in vitro study

Pillai, et al.  (2013)9.

Dental Science

To determine the value released from stainless steel brackets has a cytotoxic effect on gingival fibroblasts

-     2 complete sets of 20 brackets in each set manufactured by 3M Gemini Metal Brackets, American Orthodontics, Morelli Orthodontics Brazil, with 0.022 inch (0.56 mm) with standard edgewise slots, were selected for this study

-     Six groups of material (20 brackets each) were immersed in a sterile conical flask containing 50 ml of 0.9% w / v normal saline solution and maintained at 37 ° C for 1 month

-      The results showed that a certain amount of nickel can damage fibroblasts

-      Nickel solution at a minimum concentration of 1.18 μg can damage human gingival fibroblasts

7

In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells

Faccioni, et al.

(2003)10.

 

To know the biocompatibility in vivo of fixed orthodontic appliances, evaluating the presence of metal ions in the oral mucosal cells, their cytotoxicity, and possible genotoxic effects

-    Mucosal sampling was performed by gently rubbing the inside of the right and left cheeks of 55 orthodontic patients and 30 control subjects who did not receive orthodontic treatment. The cells were immediately prepared for cell survival and comet assays. The cellular contents of nickel and cobalt were measured by inductively coupled plasma mass spectrometry (ICP-MS)

-     Cells from patients with high nickel or cobalt levels show high DNA damage and low viability, even though the nickel and cobalt content is different

8

Cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: A longitudinal in-vivo study

Hafez, et al. (2011)11.

American Journal of Orthodontics and Dentofacial Orthopedics

To determine the cytotoxicity, genotoxicity, and release of metal ions in patients using fixed orthodontic appliances

-    18 control subjects and 28 treated subjects were evaluated longitudinally. Four combinations of brackets and archwires were tested. Buccal mucosal cell samples were collected before treatment, and 3 and 6 months after appliance placement. These cells are processed for cytotoxicity, genotoxicity, and nickel and chromium content

-     Significant changes occurred in orthodontic patients who were treated with fixed appliances over a 6 months

-     Changes include increased cellular nickel and chromium content, decreased cellular viability, and evidence of DNA damage

9

Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro

Angelieri et al. (2011)12.

American Journal of Orthodontics and Dentofacial Orthopedics

To evaluate the corrosion elongation obtained from commercially available orthodontic brackets was capable of inducing genetic defects in vitro

-    Genotoxicity was assessed by single cell gel (comet) test using chinese hamster ovary cells (CHO)

-    Orthodontic bracket Morelli, Abzil, Dentubum, 3M unitek was put into a solution containing acetic acid and sodium chloride in the same amount with a concentration of 0.1M for 1, 3, 7, 14, 21, 35, and 70 days

-     The results of this study indicate that corrosion obtained from orthodontic appliances does not cause DNA damage as illustrated by the single cell gel (comet) test

-     The results of this study indicate potential awareness of the correct evaluation of the potential health risks associated with exposure to this compound

10

Biocompatibility Test on Orthopedic Implants Between Imported Implants, Local Implants from Imported Materials, and 316L Stainless Steel Prototypes from Local Materials

 

Ferdiansyah, et al.

 (2019)13.

Qanun Medika - Medical Journal Faculty of Medicine Muhammadiyah Surabaya

To know the discovery of the role of basophils as antigen-presenting cells in experimental animals raises big questions regarding the same role in humans

-    In the test in in vitro, cytotoxicity tests were carried out by culturing human mesenchymal cells using the MTT method Assay. Cell viability was calculated based on changes in formazan color

-    In the test in in vivo, the implant biocompatibility test consisted of an irritation test, an acute systemic toxicity and pyrogenicity test, and an implantation test conducted on male rabbits in healthy condition (6 animals per group)

-     Based on ISO (International Organization for Standardization), prototype stainless steel 316 L has safe biocompatibility results

 


Orthodontic treatment using stainless steel brackets can be said to have good biocompatibility13. Although some of the side effects associated with the release of nickel ions have been demonstrated in several studies. The orthodontic treatment using stainless steel brackets has the potential for corrosion in the oral cavity2. The composition contained in the stainless-steel bracket is 70-72% ferrum or iron (Fe), 18-19% chromium (Cr), 8-9% nickel (Ni), and a carbon content (C) of less than 0.2%.5 The austenitic stainless steel used in orthodontic braces contains 18% chrome, 0.15% carbon and 8% nickel14.

 

Nickel is a nutritionally essential trace element for humans and animals at a low concentration15. On the other hand, at a high concentration, nickel is a toxic element. In humans, nickel can cause damage to the kidney, liver, brain, spleen and tissue. Nickel is also known for causing allergic contact dermatitis. Aside from that, nickel is often used because of its ability to protect metals from corrosion and wear and its plating is an effective method due to their good corrosion resistance and high wear resistance16.

 

Chromium is one of the heavy metals that occur naturally in earth crust in small quantities associated with other metals. It may be found naturally in plants, rocks, soil, and animals, including people. Cr(III) is most common from naturally occurring chromium. Under strong oxidizing condition, chromium is present in the Cr(VI) state and persist in anionic form as chromate17. Corrosion is the gradual loss of materials from surface by electrochemical cause when a metallic material is places in fluid environmental of oral cavity18. The use of stainless steel brackets in the oral cavity tends to be used for a long time and can be sure to experience a reaction and release ions from the reaction. The reaction that often occurs is the process of corrosion of stainless steel brackets. There are several factors in the oral cavity that can increase the risk of corrosion in brackets such as temperature, pH of saliva, fluoride, bacteria, enzyme activity and protein4. In addition, when masticatory forces are combined with the salivary environment of the oral cavity, it could also lead to the synergistic interaction between wear and corrosion that can increase the unbeneficial effect19.

 

Nickel release in vitro from orthodontic appliances is reported to be 22-40µg / day, chromium release in vitro from orthodontic appliances was reported to be 36 µg / day. The daily intake of nickel from food is estimated at 200-300 to 600 µg / day. The average dietary intake of chromium is estimated at 50-200 µg / day. The release of nickel and chromium ions in vitro is lower than the estimated nickel and chromium ions that enter food intake6. The normal amount of nickel ions in blood is 5 mg / L blood while the normal amount of chromium ions in blood is 0.28 g / L blood. Nickel ions with high concentrations will cause toxicity and are carcinogenic. However, excessive release of chromium ions can cause toxicity which affects cell viability and impairs DNA synthesis7. Although the result of the release of nickel and chromium ions obtained are still at normal limits or below the daily intake, it should be noted that 20-30% of allergy incidents can occur due to nickel ions8. The allergic response to nickel is a type IV hypersensitivity reaction that is mediated by T-lymphocytes. It was explained that long-term exposure to nickel ions to the body can have an adverse impact on monocyte cells and human oral mucosal cells. Nickel is known to be a common cause of hypersensitivity reaction and contact allergy. The signs and symptoms are burning sensation, gingival hyperplasia and numbness on the side of the tongue. It is confirmed by using 5% nickel sulphate in petroleum jelly (patch test)20.

 

The amount of nickel and chromium released from orthodontic appliances was below the average intake of nickel and chromium consumed in food. But this amount is enough to cause an allergic reaction in those who are sensitive to nickel. Although the level of nickel concentration is minimal, it is sufficient to induce important biological changes in the oral mucosal cells. Regarding the cytotoxic effect of the solution on gingival fibroblasts, there was a statistically significant difference between each control and test sample after 72 hours exposure periods. Group comparisons showed that very low cell counts were reported at a concentration of 1.18 µg compared with other specimens. Nickel solution at a minimum concentration of 1.18 μg can damage human gingival fibroblasts9.

 

Nickel and chromium ions released from orthodontic appliances can act as allergens or have serious negative biological effects. In addition, the release of nickel and chromium ions has cytotoxic, mutagenic, and carcinogenic properties in the human body in small amounts in the nanogram range21. Nickel and chromium ions can cause dermatitis, hypersensitivity reactions and asthma in the human body22. The majority of investigations have found that nickel sensitive patients are able to tolerate steel without any noticeable reaction13. On the other hand, increased intake of chromium can cause damage in the organs. Research by Abbas Hadi and Dohan in mice shows that excess chromium in the body leads to oxidative damage in the ovarian tissues by increasing the number of free radicals and or reducing antioxidant levels23. Similar result is found with nickel. The increased nickel level in patients with Polycystic Ovary Syndrome, stating that nickel possibly has a role in disrupting physiological homeostasis by triggering glucose deregulation24. The toxicological effects of nickel and cobalt ions have been shown to produce an effect on DNA damage in oral mucosal cells. In general, normal cells can repair lesions from these detrimental effects, but loss of repair capacity due to decreased detection of damage or enzymatic deficiency in the repair process may be an early occurrence of adverse biological effects9,10.

 

The choice of dental material to be used in the oral cavity needs to consider both the effect on the surrounding tissue and the toxicity effect if the material is ingested. Biocompatibility of dental materials is the basic point of the success of these materials to be used in the oral cavity. Biocompatibility is very important for further analysis because it has elements of safety, responsibility and to keep the body's immune system capable of reacting normally to dental materials5. The biocompatibility of stainless steel brackets can be studied by conducting cytotoxicity and genotoxicity tests. The biocompatibility of stainless steel brackets is the basis of successful orthodontic treatment in the oral cavity. The biocompatibility of stainless steel brackets can be studied by conducting cytotoxicity and genotoxicity tests. The genotoxicity test is a test related to cells that are genetically altered by exposure to metal ions released by orthodontic devices. Genotoxicity tests can be defined as tests in vitro and in vivo designed to detect compounds that cause genetic damage, including DNA damage, gene mutations, chromosomal damage, altered DNA repair capacity, and cell transformation. In recent decades, genotoxicity testing has been widely accepted as an important indicator of carcinogenicity7. Corrosion obtained from orthodontic appliances did not cause DNA damage as illustrated by the single cell gel or comet test11.

 

Biocompatibility test conducted the International Stainless-steel Forum and the European Confederation of Iron and Steel Producers against stainless steel both in vitro and in vivo, stainless steel has a very low level of toxicity12. Stainless steel is a safe material to use for all intra oral orthodontic component for nickel sensitive patient13. The use of stainless steel brackets in the oral cavity for a long time will definitely experience a corrosion reaction. The corrosion process is always followed by the release of ions from metal elements. The release of nickel and chromium ions is the most common and can have negative effects on the body such as allergies, contact dermatitis, hypersensitivity, cytotoxicity, and DNA damage. Biocompatibility is very important for further analysis because it has elements of safety, responsibility and to keep the body's immune system capable of reacting normally to dental materials.

 

CONCLUSION:

Based on this review, biocompatibility of dental materials is the basic point of the success of these materials to be used in the oral cavity. The biocompatibility of stainless steel brackets is good due to it has low level of toxicity, cytotoxicity and genotoxicity in the body. 

 

ACKNOWLEDGEMENT:

The authors especially grateful to the Faculty of Dental Medicine for warm support, inspiration, and thoughtful guidance.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Sumule I. Anindita PS. Waworuntu OA. Pelepasan Ion Nikel Dan Kromium Braket Stainless Steel Yang Direndam Dalam Minuman Berkarbonasi. e-GIGI. 2015;3(2):1–6.

2.      Mihardjanti M. Ismah N. Purwanegara MK. Nickel and chromium ion release from stainless steel bracket on immersion various types of mouthwashes. J Phys Conf Ser. 2017;(1): 884.

3.      Zhang X. Song Y. Wang X. Microstructure and Corrosion Properties of Orthodontic Brackets by Laser Treatment. 2017;12:32–9.

4.      Yanisarapan T, Thunyakitpisal P, Chantarawaratit P on. Corrosion of Metal Orthodontic Brackets and Archwires Caused by Fluoride-Containing Products: Cytotoxicity, Metal Ion Release and Surface Roughness. Orthod Waves. 2018;77(2):79–89.

5.      Chaturvedi TP. Upadhayay SN. An overview of orthodontic material degradation in oral cavity. Indian J Dent Res. 2010;21(2):275–84.

6.      Priya B. George A. Niti Self Expandable Surgical Splints. Research Journal of Pharmacy and Technology. 2019;12(1):443-444.

7.      Imani MM. Mozaffari HR. Ramezani M. Sadeghi M. Effect of fixed orthodontic treatment on salivary nickel and chromium levels: A systematic review and meta-analysis of observational studies. Dent J. 2019;7:1.

8.      Karlina I. Amtha R. Roeslan BO. Zen Y. The Release of Total Metal Ion and Genotoxicity of Stainless Steel Brackets: Experimental Study Using Micronucleus Assay. Indones Biomed J. 2016;8(2):97.

9.      Pillai AR. Gangadharan A. Gangadharan J. Kumar NV. Cytotoxic effects of the nickel release from the stainless steel brackets: An in vitro study. J Pharm Bioallied Sci2013;5:1–4.

10.   Halkai S. Mangalgi A. Shah K. Paramshivam G. Mallasure D. Comparison of metal release from fixed orthodontic appliances in oral mucosa cells in patients with and without fixed orthodontic appliances - An in vivo study. J Dent Spec. 2017;2(1):142–7.

11.   Faccioni F. Franceschetti P. Carpelloni MT. Fracasso MM. In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells. Am J Orthod Dentofacial Orthop. 2003;124(6):687-93.

12.   Hafez H. Selim EM. Eid FH. Tawfik W. Al-Ashkar E. Mostafa Y. Cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: A longitudinal in-vivo study. Am J Orthod Dentofacial Orthop. 2011;140(3):298-308.

13.   Angelieri F. Marcondes JP. Almeida DC. Salvadori DM. Ribeiro D. Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro. Am J Orthod Dentofac Orthop [Internet]. 2011;139(4):504–9.

14.   Ferdiansyah. Waskita H. Utomo D. Suroto H. Martanto T. Uji Biokompabilitas pada Implan Orthopedi Antara Implan Impor, Implan Lokal dari Material Impor, dan Prototipe Stainless Steel 316L dari Material Lokal. Qanun Med - Med J Fac Med Muhammadiyah Surabaya. 2019;3(1):7.

15.   Dav HP. Abilasha R. Allergic Reaction and Nickel - Free Braces: A Review. Research J. Pharm. and Tech. 2016; 9(9):1516-18

16.   Brrow F. Bargiul S. Estimation of Nickel Level in Blood Serum among Hemodialysis Patients in Syria. Research J. Pharm. and Tech. 2021; 14(3):1507-10.

17.   Gushcha D. Parii V. Tyshko D. Mykhailov A. Shpak D. The Study of Ni-Cr, Co-Cr Dental Alloys and Ceramics Chemical Indifference using ZrO2-HfO2-Y2O3 System in Vitro. Research Journal of Pharmacy and Technology. 2019; 12(9):4085-4089.

18.   Gupta AK. Ganjewala D. Goel N. Khurana N. Ghosh S. Saxena A. Bioremediation of tannery chromium: A microbial approach. Research J.Pharm. and Tech. 2014; 7(1):118-22.

19.   Jani GH. Abdulsahib AJ. Fatalla AA. Corrosion behavior of implant coated with different biocompatible material. Research J. Pharm. and Tech. 2020; 13(2):810-814.

20.   Arockiasamy PG. Thenmozhi R. Santhi J. Evaluation of Corrosion Resistance of Electroless Ni-P/Ni-W-P Double Layer Coatings on 6061 Aluminium Alloy. Asian J. Research Chem. 2014;7(6): 551-557.

21.   Varshita A, Nasim I. Allergic reaction to dental materials - A review. Research J.Pharm. and Tech. 2016; 9(10):1819-22.

22.   Genchi G, Carocci A, Lauria G, Sinicropi MS. Nickel : Human Health and Environmental Toxicology. 2020;3(17):679.

23.   Achmad TR, Budiawan, Auerkari IE. Effects of Chromium on Human Body Effects of Chromium on Human Body. 2017:7

24.   Hadi A, Dohan K. Effect of Potassium Dichromate on Ovary and Uterus in Female Mice. Research Journal of Pharmacy and Technology. 2018;11(2):429.

25.   Guzar S, Jawad E, Altahan M, Hameed N. The Effects of Trace Element levels on Poly cystic Ovary Syndrome in human female Sat Thi-Qar governorate Iraq. Research Journal of Pharmacy and Technology. 2019;12(9):4447-4453.

 

 

 

Received on 06.05.2021           Modified on 10.04.2022

Accepted on 06.06.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3251-3256.

DOI: 10.52711/0974-360X.2023.00534