The Potential of Lactobacillus casei on TNF-alpha and IL-1beta Levels Type 2 Diabetes Mellitus

 

Retno Pudji Rahayu¹*, Bambang Sumaryono¹, Nadia Farikha Sandi²

¹Department of Oral and Maxillofacial Pathology, Faculty of Dental Medicine,

Universitas Airlangga, Surabaya, Indonesia.

²Undergraduate Student, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

 

ABSTRACT:

Type 2 diabetes mellitus is a disease caused by insulin resistance which will cause blood glucose levels to increase. That factor can result in an increase of proinflammatory cytokines such as TNF-alpha dan IL-1 beta which will cause the severity of the disease gets worse. Intraoral antidiabetic drugs have several side effects; therefore, it is necessary to provide probiotic supplements such as Lactobacillus casei to reduce levels of TNF- alpha dan IL-1 beta so that type 2 diabetes mellitus doesn’t get worse. These bacteria can also increase levels of anti-inflammatory cytokines such as IL-10. This study aims to learn and understand more deeply about the potential of Lactobacillus casei on TNF- alpha and IL-1 beta levels in type 2 diabetes mellitus. Diabetes mellitus type 2 is a chronic disease by the pancreas not being able to produce the hormone insulin in large quantities due to insulin resistance caused by impaired insulin secretion in pancreas. TNF- alpha and IL-1 beta is a cytokines that plays role in the body’s defense function, if cytokines produce in excess amount it will increase the inflammatory process. Lactobacillus casei is a probiotic bacterium that can be found in yoghurt, cheese, raw meat, and other food ingredients. Lactobacillus casei has the potential to reduce TNF- alpha dan IL-1 beta levels in type 2 diabetes mellitus.

 

 

 

INTRODUCTION:

Along with the times, there have been changes in lifestyles and patterns of life. One of the most frequent changes is consuming unhealthy foods and drinks such as fast food and drinks containing soda, which can affect blood sugar levels in the body. This causes the emergence of dangerous diseases such as diabetes mellitus. One of the signs of diabetes mellitus is that the body is unable to metabolize carbohydrates, fats, and proteins, causing high blood glucose levels (hyperglycemia)^1,2. The most common type of diabetes is type 2 diabetes mellitus. Approximately 90% of the incidence of diabetes mellitus in the world is type 2 diabetes mellitus³.

 

Type 2 diabetes mellitus is caused by insulin resistance so that it is unable to maintain glucose homeostasis in the body⁴.

 

 

This insulin resistance can be caused by an increase in TNF-alpha cytokine levels in patients with type 2 diabetes mellitus⁵. Besides being caused by insulin resistance, type 2 diabetes mellitus can also be caused by a decrease in pancreatic beta cells in secreting insulin⁶. The decrease in the amount of insulin secretion is caused by an increase in the levels of the proinflammatory cytokine, namely IL-1beta in people with diabetes mellitus⁷.

 

In type 2 diabetes mellitus, there is an increase in proinflammatory cytokines such as TNF-alpha and IL-1beta which is caused by an increase in blood glucose (hyperglycemia) thus it can trigger the emergence of reactive oxygen species (ROS) and advanced glycation end products (AGEs). If the levels of ROS and AGEs increase, it will cause an inflammatory process or inflammation. In this disease, an inflammation can occur which will result in stimulation of a non-specific immune response and will activate macrophages to secrete proinflammatory cytokines⁸.

 

Various oral antidiabetic drugs such as alpha-glucosidase inhibitors, sulfonylureas, meglitinides, and others cause side effects such as diarrhea and flatulence. Therefore, it is necessary to take a supplement that can cause a decrease in blood sugar in people with type 2 diabetes mellitus. Several studies have shown that probiotic bacteria can cause a decrease in blood sugar levels in people with type 2 diabetes mellitus without causing side effects^9,10. Research shows that probiotic bacteria such as Lactobacillus casei can reduce levels of proinflammatory cytokines such as TNF-alpha and IL-6 in rheumatoid arthritis patients¹¹. Besides that, other studies have also shown that Lactobacillus casei has been shown to reduce other proinflammatory cytokines such as IL-1beta and IL-17 in rheumatoid arthritis patients¹². If the levels of these cytokines are lowered, the inflammatory process in inflammatory diseases such as diabetes mellitus can be inhibited^13,14.

 

This review article aims to study and understand more about the potential of the probiotic bacteria Lactobacillus casei on TNF- and IL-1beta levels in patients with type 2 diabetes mellitus.

 

REVIEWS:

Type 2 Diabetes Mellitus:

Chronic disease caused by the pancreas not being able to produce the hormone insulin was not produced in large quantities due to insulin resistance caused by impaired insulin secretion in the pancreas¹⁶. This disease was related to genetics or can be passed down from the family tree. In addition, this disease was also associated with obesity or overweight^12,17.

 

Inflammation in Type 2 Diabetes Mellitus:

Hyperglycemia experienced by people with diabetes mellitus can cause an increase in reactive oxygen species (ROS). ROS generated during physiological activities can affect cell function^18,19. Increased levels of glucose in cells will cause damage to mitochondrial DNA. Damage to mitochondrial DNA will activate an enzyme that played a role in DNA repair, namely poly ADP-ribose polymerase (PARP). In addition, activation of the PARP enzyme was able to inhibit the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)^18,20–22. Inhibition of GAPDH leads to increased activation of the polyol and hexosamine pathways. Increased activation of the polyol and hexosamine pathways will lead to an increase in non-enzymatic glycation reactions, oxidative stress, diacylglycerol synthesis and excessive AGEs^19,23. The increase of these reactions and compounds will increase protein kinase C (PKC). An increase in PKC will result in changes in vascular cell function thus it can damage blood vessels. An increase in PKC activity will lead to an increase in NFkB. NFkB was a transcription factor capable of causing activation of proinflammatory genes in blood vessels (proinflammatory gene expression). If NFkB was activated, there will be an increase in the number of proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, IL-8, IL-12, and IFN-g. Increased AGEs will also cause an increase in proinflammatory cytokines because these compounds will bind to macrophages so that they can produce these cytokines²⁵.

 

Tumor Necrosis Factor Alpha (TNF-alpha):

TNF-alpha was a mediator of the acute inflammatory response to pathogens. These cytokines were produced by macrophages, T lymphocytes and NK cells and have a pleiotropic effect on normal and malignant cells^26,27. TNF-alpha not only plays a role in the inflammatory process, but is also involved in apoptosis, cell differentiation and recruitment. The proinflammatory activity carried out by TNF-alpha involved the formation of prostaglandins (proinflammatory factors) and the induction of COX-2^28,29.

 

Interleukin - 1beta (IL-1beta):

IL-1beta cytokine was a major cytokine that plays a role in monocyte activation and activation of proinflammatory signaling pathways in peripheral tissues and muscles³⁰. These cytokines are produced by monocytes, microglial cells, astrocytes and cerebral endothelial cells³¹. Interleukin-1beta which was a proinflammatory cytokine with pleiotropic effects on the immune response of a number of diseases related to the immune system such as multiple sclerosis. In addition, these cytokines are also involved in chronic inflammatory diseases such as rheumatoid arthritis, cardiovascular disease and type 2 diabetes mellitus^29,31,32.

 

Management of Diabetes Mellitus:

The management of diabetes mellitus was to lead a healthy lifestyle and take antihyperglycemic drugs orally or by injection. Pharmacological therapy given to people with diabetes mellitus was divided into two, namely oral and injection drugs, for instance, sulfonylureas, metformin, glinid, and others. Therefore, it was necessary to regulate food for people with diabetes mellitus, namely eating a healthy and balanced diet^3,5,33.

 

Lactobacillus casei:

Lactobacillus casei was a gram-positive bacterium that has a rod shape with a width of 0.7-1.1m and a length of 2.0-4.0m. These bacteria can be seen singly, in pairs, or in colonies. These bacteria did not form spores and have no means of locomotion. Lactobacillus casei was a probiotic bacteria that can be found in fermented milk drinks (yogurt), cheese, raw meat, and other food ingredients^23,34–37.

 

DISCUSSION:

In type 2 diabetes mellitus, there is an increase in proinflammatory cytokines that can cause an inflammatory process in the sufferer's body, one of which is in the oral cavity. Increased proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and others lead to inflammation of the periodontal tissues, pocket formation, alveolar bone loss, and loss of periodontal attachment³⁸. Proinflammatory cytokines (especially IL-1beta, IL-6, and TNF-alpha) which are increased in periodontitis will trigger the synthesis and activate enzymes that are responsible for destroying connective tissue, killing fibroblasts and osteoblasts, and increasing osteoclasts^21,39.

 

Probiotics have had various benefits such as increasing the production of peripheral immunoglobulin levels, stimulating the formation of IgA cytokines, and decreasing the production of proinflammatory cytokines (IL-1beta, TNF-alpha, IL-6, IL-8, and others)^40,41. These bacteria can also reduce streptozocin levels in diabetic rats. High levels of streptozocin can cause oxidative damage to the pancreatic tissue of rats^42,43. In a study conducted by Wang et al. (2017) and Li et al. (2017), a group of diabetic rats inoculated intragastrically using Lactobacillus casei showed significantly decreased levels of TNF-alpha. Lactobacillus casei bacteria can be beneficial when given orally if the levels of these bacteria are around 10⁹ CFU^44,45.

 

The results of the research conducted by Chen et al. (2014) also found that diabetic rats given Lactobacillus casei for 12 weeks experienced a decrease in fasting blood glucose levels and 2 hours PP blood glucose. Increased levels of TNF-alpha in type 2 diabetes mellitus play an important role in the occurrence of insulin resistance, namely by inhibiting the activity of the tyrosine kinase enzyme at the insulin receptor which can exacerbate the development of the disease ⁷. According to Gurung et al. (2020), administration of Lactobacillus casei bacteria can reduce IL-1α and TNF-β cytokines. This component of the bacterial wall is able to reduce the phosphorylation of NF-kB, so that the levels of proinflammatory cytokines resulting from the activation of this factor will also decrease⁴⁶.

 

Lactobacillus casei bacteria are able to bind molecules on the surface so that it will suppress signaling pathways from the production of proinflammatory cytokines. Anti-inflammatory cytokines such as IL-10 increased after administration of Lactobacillus casei, an increase in these cytokines would cause a decrease in macrophage activation. If macrophages are inactivated, the synthesis of cytokines such as TNF-alpha, IL-1beta, IL-6, and IL-8 will also be inhibited^8,47. In the group of rats with type 2 diabetes mellitus, the administration of Lactobacillus bacteria could reduce the levels of advanced glycation end products (AGEs). AGES can play a role in increasing levels of proinflammatory cytokines which will exacerbate the occurrence of type 2 diabetes mellitus^4,36

 

However, in a study conducted by Tripolt et al. (2013) showed that there was no significant change in TNF-alpha cytokine levels in patients with type 2 diabetes mellitus who had been given Lactobacillus casei at a dose of 10⁸ cells/mL. There are several weaknesses in the study using patients with type 2 diabetes mellitus compared to diabetic rats, including the pathophysiology of insulin and different inflammatory reactions between humans and mice, as well as the exact dose administered to patients in this study⁴⁶.

 

CONCLUSION:

Insulin resistance causes type 2 diabetes mellitus, which results in a rise in blood glucose levels. This factor can trigger an increase in proinflammatory cytokines like TNF-alpha and IL-1beta, making the condition worse. Lactobacillus casei is a probiotic bacteria that has the potential to reduce TNF-alpha and IL-1beta levels in type 2 diabetes mellitus.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

There is no acknowledgments

 

REFERENCES:

1.      Zamaa MS. Sainudin S. Hubungan Kepatuhan Pengobatan dengan Kadar Gula Darah Sewaktu pada Pasien Diabetes Melitus Tipe II. JAmbura Nursing Journal. 2019; 1(1):11–18.doi:10.37311/jnj.v1i1.2057.

2.      Rao USM. Zin T. Kyi Kyi WRN. Subramaniam SA/L. Shan TB. Mogan KA/P. Ismail ASB. Cross-Sectional Study on Knowledge, Attitude and Practice Regarding Diabetes Mellitus among Medical and Non-Medical Students. Research Journal of Pharmacy and Technology. 2018; 11(11):4837–4841.doi:10.5958/0974-360X.2018.00879.X.

3.      Internasional Diabetes Federation. Managing Type 2 Diabetes in Primary Care. 2020.

4.      Shita, ADP. Perubahan Level TNF-α Dan IL-1 pada Kondisi Diabetes Mellitus. Pros Dent Sci Meet II, Fakultas Kedokteran Gigi, Universitas Jember.2015; 1:11–7.

5.      Fatimah RN. Diabetes Melitus Tipe 2. Jurnal Majority.2015; 4 (5):93-101

6.      Zhao G. Dharmadhikari G. Maedler K. Meyer-Hermann M. Possible Role of Interleukin-1β in Type 2 Diabetes Onset and Implications for Anti-Inflammatory Therapy Strategies. Public Library of Science. 2014; 10(8):e1003798.doi:10.1371/journal.pcbi.1003798.

7.      Chen P. Zhang Q. Dang H. Liu X. Tian F. Zhao J. et al. Antidiabetic Effect of Lactobacillus casei CCFM0412 on Mice with Type 2 Diabetes Induced by a High-Fat Diet and Streptozotocin. Nutrition. 2014; 30(9):1061–1068.doi:10.1016/j.nut.2014.03.022.

8.      Amdekar DS. Singh V. Singh DR. Sharma DP. Keshav P. Kumar A. Lactobacillus casei Reduces the Inflammatory Joint Damage Associated with Collagen-Induced Arthritis (CIA) by Reducing the Pro-Inflammatory Cytokines. Journal of Clinical Immunology. 2011; 31:147–154.doi:10.1007/s10875-010-9457-7.

9.      Amdekar S. Singh V. Kumar A. Sharma P. Singh R. Lactobacillus casei and Lactobacillus acidophilus Regulate Inflammatory Pathway and Improve Antioxidant Status in Collagen-Induced Arthritic Rats. Journal of Interferon and Cytokine Research: The Official Journal of the International Society for Interferon and Cytokine Research. 2013; 33(1):1–8.doi:10.1089/jir.2012.0034.

10.   Preethikaa S. Brundha MP. Awareness of Diabetes Mellitus among General Population. Research Journal of Pharmacy and Technology. 2018; 11(5):1825–1829.doi:10.5958/0974-360X.2018.00339.6.

11.   Aydoğdu S. Karamese M. Altoparlak Ü. Karameşe S. The Protective Effects of Long-Term Probiotic Application on Experimental Sepsis-Dependent Inflammation Process. Bezmialem Science. 2019; 7:180–185.doi:10.14235/bas.galenos.2018.2643.

12.   Azrimaidaliza A. Asupan Zat Gizi Dan Penyakit Diabetes Mellitus. Jurnal Kesehatan Masyarakat Andalas. 2011; 6(1):36–41.doi:10.24893/jkma.v6i1.86.

13.   Ali S. Kadhum. Jwad ZA. Raheem HN. Noor H. Yaseen. Ali YK. Comparing The Levels of Trace Elements in Females with Diabetes Mellitus. Research Journal of Pharmacy and Technology. 2019; 12(12):6119–6123.doi:10.5958/0974-360X.2019.01063.1.

14.   Vanmathi S. Star M. Jishala MI. Sunadaram R. A Pathophysiological Approach of Macrovascular Complication in Diabetes Mellitus with Hypertension: A Systematic Review. Research Journal of Pharmacy and Technology. 2019; 12(2):901–906.doi:10.5958/0974-360X.2019.00154.9.

15.   Sutawardana, JH. Yulia. Waluyo, A. Studi Fenomenologi Pengalaman Penyandang Diabetes Melitus yang Pernah Mengalami Episode Hipoglikemia. 2016; 1(1):159–175.

16.   Varghese L. Murthi K. Pandey K. Teneligliptin as an Add on Therapy to Oral Anti Diabetic Agents in Type 2 Diabetes Mellitus. Research Journal of Pharmacy and Technology. 2020; 13(5):2310–2314.doi:10.5958/0974-360X.2020.00416.3.

17.   Alza Y. Hubungan Hiperglikemia dengan Kadar Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) pada Penderita Diabetes Mellitus Tipe 2. Photon: Jurnal Sain dan Kesehatan. 2013; 4(1):19–26.doi:10.37859/jp.v4i1.165.

18.   Yerizel E. Hendra P. Edwar Z. Bachtiar H. Pengaruh Hiperglikemia Terhadap High Sensitive C Reactive Protein (Hs-CPR) pada Penderita Diabetes Melitus Tipe 2. Seminar Ilmiah Perhimpunan Biokimia dan Bologi Molekuler Indonesia. Fakultas Kedokteran: Universitas Gadjah Mada. 2015; 51-55.

19.   Purwaningsih W. Pemberian Suspensi Bubuk Kedelai Dapat Menurunkan Kadar Malondialdehid (Mda) Serum pada Tikus Putih Diabetus Melitus yang Diinduksi Streptozotozin. 2012; 9(2):7.

20.   Sangeetha R. Jayaraj V. Stress Induced Type 2 Diabetes Mellitus among Industrial Workers – A Review. Research Journal of Pharmacy and Technology. 2019; 12(1):396–402.doi:10.5958/0974-360X.2019.00072.6.

21.   Maheshwari P. Shanmugarajan TS. Evaluation of Prescribing Practices of Metformin in Patients with Type-2 Diabetes Mellitus. Research Journal of Pharmacy and Technology. 2019; 12(2):531–534.doi:10.5958/0974-360X.2019.00093.3.

22.   Fayyadh AA. Ibrahim H. Zain HHM. Al-Qubaisi MS. The Effect of Agarwood Leaf Extracts on Blood Glucose Level of Type II Diabetes Mellitus in ICR Male Mice. Research Journal of Pharmacy and Technology. 2020; 13(1):237–242.doi:10.5958/0974-360X.2020.00048.7.

23.   Shita ADP. Perubahan Level TNF-α Dan IL-1 pada Kondisi Diabetes Mellitus. Pros Dent Sci Meet II, Fakultas Kedokteran Gigi, Universitas Jember. 2015; 1:1–7.doi:10.1017/CBO9781107415324.004.

24.   Aronson JK. Meyler’s Side Effects of Drugs. Elsevier Science. 2015.

25.   Senthilnathan B. Vivekanandan K. Bhavya E. Masilamani SPB. Impact of Nanoparticulate Drug Delivery System of Herbal Drug in Control of Diabetes Mellitus. Research Journal of Pharmacy and Technology. 2019; 12(4):1688–1694.doi:10.5958/0974-360X.2019.00282.8.

26.   Akash MSH. Rehman K. Liaqat A. Tumor Necrosis Factor-Alpha: Role in Development of Insulin Resistance and Pathogenesis of Type 2 Diabetes Mellitus. Journal of Cellular Biochemistry. 2018; 119(1):105–110.doi:10.1002/jcb.26174.

27.   Hassan MM. The Significance of Interleukin 4 (IL-4) (590-C/T) Gene Polymorphism in Iraqi Patients with Type 2 Diabetes Mellitus: A Case-Control Study. Research Journal of Pharmacy and Technology. 2019; 12(11):5133–5137.

28.   Hwang DH. Encyclopedia of Human Nutrition. Elsevier Inc., Philadelphia. 2013; 2–4.

29.   Maghzi AH. Minagar A. IL1-β Expression in Multiple Sclerosis. Journal of the Neurological Sciences. 2014; 343(1):1.doi:10.1016/j.jns.2014.05.009.

30.   Herder C. Donath MY. Interleukin-1 Receptor Antagonist: Friend or Foe to the Heart? The Lancet. Diabetes & Endocrinology. 2015; 3(4):228–229.doi:10.1016/S2213-8587(15)00035-2.

31.   Soelistijo SA. Novida H. Rudijanto A. Soewondo P. Suastika K. Manaf A. et al. Konsesus Pengelolaan dan Pencegahan Diabetes Melitus Tipe 2 di Indonesia 2015. Pengurus Besar Perkumpulan Endokrionologi Indonesia (PB PERKENI). 2015.

32.   Laeli H. Nazaruddin N. Werdiningsih W. Kajian Sifat Kimia Dan Organoleptik Yogurt Jagung Manis (Zea Mays Saccharata) dengan Menggunakan Beberapa Jenis Inokulum. Pro Food. 2017; 2(1):77–84.

33.   Gobetti M. Minervini F. Encyclopedia of Food Microbiology. Elsevier. 2013; 432-438.

34.   Kellow NJ. Coughlan MT. Savige GS. Reid CM. Effect of Dietary Prebiotic Supplementation on Advanced Glycation, Insulin Resistance and Inflammatory Biomarkers in Adults with Pre-Diabetes: A Study Protocol for a Double-Blind Placebo-Controlled Randomised Crossover Clinical Trial. BMC Endocrine Disorders. 2014; 14(1):55.doi:10.1186/1472-6823-14-55.

35.   Abbas AC. Al-Kraity WRH. Abbas EC. Effect Antioxidant and Serotonin Level in the Sera on Type II Diabetes Mellitus Males Patients and Compare with Control Group. Research Journal of Pharmacy and Technology. 2019; 12(5):2453–246010.5958/0974-360X.2019.00411.6.

36.   Ermawati T. Periodontitis dan Diabetes Melitus. Stomatognatic-Jurnal Kedokteran Gigi. 2015; 9(3):152–154.

37.   Yekti N. Rochmah YS. Mujayanto R. Analisa Profil Kadar C-Reactive Protein Pada Status Kesehatan Periodontal Pasien Diabetes Melitus Tipe 2 (Studi di Rumah Sakit Islam Sultan Agung Semarang). Odonto: Dental Journal 2014; 1(2):19–24.doi:10.30659/odj.1.2.19-24.

38.   Huang SC. Lei YP. Lin CP. Hsiao PW. Chi YT. The Suppressive Effect of Supplementation of Combined Probiotic on Helicobacter Pylori Infection. Current Developments in Nutrition. 2020; 4 (Suppl 2):1565–1565.doi:10.1093/cdn/nzaa062_022.

39.   Lesbros-Pantoflickova D. Corthésy-Theulaz I. Blum AL. Helicobacter Pylori and Probiotics. The Journal of Nutrition. 2007; 137 (3 Suppl 2):812S-8S.doi:10.1093/jn/137.3.812S.

40.   Azad MAK. Sarker M. Wan D. Immunomodulatory Effects of Probiotics on Cytokine Profiles. BioMed Research International. 2018; 2018:e8063647.doi:10.1155/2018/8063647.

41.   Miraghajani M. Dehsoukhteh SS. Hamedani SG. Sabihi S. Ghiasvand R. Potential Mechanisms Linking Probiotics to Diabetes: A Narrative Review of the Literature. Sao Paulo Med J. 2017; 135(2):169–178.doi: 10.1590/1516-3180.2016.0311271216.

42.   Wang G. Li X. Zhao J. Zhang H. Chen W. Lactobacillus casei CCFM419 Attenuates Type 2 Diabetes via a Gut Microbiota Dependent Mechanism. Food & Function. 2017; 8 (9):3155–3164.doi:10.1039/c7fo00593h.

43.   Li X. Wang E. Yin B. Fang D. Chen P. Wang G. et al. Effects of Lactobacillus casei CCFM419 on Insulin Resistance and Gut Microbiota in Type 2 Diabetic Mice. Beneficial Microbes. 2017; 8(3):421–432.doi:10.3920/BM2016.0167.

44.   Gurung M. Li Z. You H. Rodrigues R. Jump DB. Morgun A. Shulzhenko, N. Role of Gut Microbiota in Type 2 Diabetes Pathophysiology. EBioMedicine. 2020; 51:102590.doi:10.1016/j.ebiom.2019.11.051.

45.   Liu T. Zhang L. Joo D. Sun SC. NF-ΚB Signaling in Inflammation. Signal Transduction and Targeted Therapy. 2017; 2:17023.doi:10.1038/sigtrans.2017.23.

46.   Tripolt NJ. Leber B. Blattl D. Eder M. Wonisch W. Scharnagl H. et al. Short Communication: Effect of Supplementation with Lactobacillus casei Shirota on Insulin Sensitivity, β-Cell Function, and Markers of Endothelial Function and Inflammation in Subjects with Metabolic Syndrome--a Pilot Study. Journal of Dairy Science.2013; 96(1):89–95.doi:10.3168/jds.2012-5863.

 

 

 

Accepted on 06.05.2022         © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):107-110.