INTRODUCTION:

A naturally occurring derivative of amino acids, L-carnitine (LC) is necessary for the transport of long-chain fatty acids for β-oxidation within the mitochondria. It is a potent pharmacological substance that is mostly produced in the liver and kidneys from the amino acids lysine and methionine.¹ It is also taken as a supplement to help people lose weight.²

It may be involved in acetyl group transfer for acetylcholine production and moves acetyl-CoA into the cytoplasm during acetyl-l-carnitine export from mitochondria. Because of carnitine acetyltransferase, it is essential for the peroxisomal oxidation of very long-chain fatty acids.^3,4

L-carnitine L-tartrate (LCLT) is a nutritional compound that has potential health benefits, particularly in the recovery and fat metabolism.⁵ It is the most prevalent form of L-carnitine, comprising 68% of L-carnitine and 32% of L-tartaric acid, by weight. LCLT is a stable, white crystalline salt with a free-flowing consistency and a pleasant citric flavor.⁶

The immune system's intricate biochemical reaction to infections, wounds, or dangerous stimuli is inflammation. Chronic inflammation is essential to the onset of many diseases, such as cancer, diabetes, cardiovascular disorders, neurological diseases, and autoimmune ailments, but acute inflammation is protective and required for healing. Prolonged inflammation leads to tissue damage, altered cellular functions, and dysregulation of immune responses, contributing to disease progression.^7,8

The need for studying new anti-inflammatory agents arises due to the available anti-inflammatory medications, such as corticosteroids and non-steroidal anti-inflammatory medicines (NSAIDs), which have the potential to be beneficial but often come with significant side effects when used long-term, such as gastrointestinal issues, cardiovascular risks, and immune suppression.^9,10 Moreover, not all patients respond well to existing treatments, leading to a demand for alternative therapies.

Free radicals and reactive oxygen species (ROS) are extremely reactive substances that have two functions in cellular physiology. At low to moderate levels, they function as signaling molecules that regulate cell growth and immune response.¹¹ Free radicals like superoxide anion radicals (O₂·−) and hydroxyl radicals (OH·) as well as non-radical species like hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂) are examples of reactive oxygen species (ROS), which are byproducts of regular cellular metabolism, especially in the mitochondria. However, oxidative stress, which harms cellular constituents including DNA, proteins, and lipids, can result from an overabundance of ROS and free radicals.^11,12

Diseases such as diabetes, cancer, heart problems, and neurological illnesses are all significantly influenced by oxidative damage.¹¹ Chronic oxidative stress can worsen inflammation, contributing to disease progression. While ROS are crucial for cellular function, their imbalance is a major factor in disease etiology. The increasing recognition of ROS and free radicals in disease mechanisms has spurred research into antioxidant therapies, which aim to neutralize excess ROS and mitigate their harmful effects.¹³ However, creating effective remedies has been difficult due to the intricacy of ROS signaling and the dual function of these species in health and sickness.^12,14

Antioxidants can slow the progression of many chronic diseases and lipid peroxidation while shielding the body from the impacts of free radicals and ROS. Propyl gallate, ascorbic acid, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) are the most widely utilized antioxidants. However, liver damage and carcinogenesis are thought to be caused by BHA and BHT.¹¹

L-Carnitine L-Tartrate is known for its role in energy metabolism, but recent research suggests that it may have anti-inflammatory and antioxidant activities as well.¹⁵ By exploring these activities in vitro, we can assess its viability as a novel treatment for inflammation and oxidative stress-driven diseases and expand its use beyond its traditional role in metabolism and sports performance.

This investigation was carried out to assess the potential anti-inflammatory and antioxidant properties of LCLT using a variety of in vitro anti-inflammatory and antioxidant tests, such as hydrogen peroxide scavenging, proteinase inhibition, albumin denaturation, and free radical (DPPH) scavenging.

MATERIALS AND METHODS:

Drug and chemicals:

We purchased L-carnitine L-tartrate (LCLT) from Sigma Aldrich in the United States. We bought the following supplies from Merck Specialities Pvt. Ltd. in Mumbai, India: dimethyl sulfoxide (DMSO), bovine serum albumin, perchloric acid, tris-Hcl-buffer, diphenyl picrylhydrazyl (DPPH), hydrogen peroxide (H₂O₂), and phosphate buffer. A retail drugstore provided the reference standard ascorbic acid and diclofenac sodium. The analytical-grade chemicals used were all purchased from Merck Specialities Pvt. Ltd. in Mumbai, India. Analytical readings were taken using a Shimadzu UV-1800 UV-visible spectrophotometer.

In-vitro Anti-inflammatory Activity:

1. Albumin denaturation method:

By measuring its proteinase inhibitory activity using a method outlined by Dadoriya (2020), the proteinase inhibitory potential of LCLT was assessed.^16–18 Following the dissolution of the test sample (LCLT) at several concentrations (100–500µg/mL), a reaction mixture (2mL) was created by combining 0.06mg of trypsin with 1mL of 20mM Tris-HCl buffer (pH 7.4). Incubate the mixture at 37°C for 5minutes. To the mixture, add 1mL of a 0.8% (w/v) casein solution, and then incubate for another 20 minutes. Add 2 milliliters of 70% perchloric acid to halt the process. For ten minutes, the resulting mixture was centrifuged at 3000rpm. Measure the absorbance of the supernatant layer at 210 nm using a buffer solution as the blank.

The percentage inhibition of albumin denaturation was calculated using the formula below. With DMSO acting as the negative control and diclofenac sodium as the positive control, the results were displayed as IC₅₀ values.

% inhibition = [(A_c – A_t)/A_c] × 100

Where, A_crepresents the absorbance of negative control (i.e., DMSO) and A_trepresents the absorbance when LCLT is present.

2. Proteinase inhibitory activity:

The proteinase inhibitory potential of LCLT was evaluated by assessing its proteinase inhibitory activity following a procedure described by Dadoriya (2020).^19–22 After dissolving the test sample (LCLT) at different concentrations (100-500µg/mL), 0.06mg of trypsin and 1mL of 20mM Tris-HCl buffer (pH 7.4) were combined to form a reaction mixture (2mL). For 5minutes, incubate the mixture at 37°C. Add 1mL of 0.8% (w/v) casein solution to the mixture and incubate for an additional 20 minutes. To stop the reaction, add 2 mL of 70% perchloric acid. The resultant mixture was centrifuged at 3000rpm for 10minutes. Using a buffer solution as the blank, measure the absorbance of the supernatant layer at 210nm. Diclofenac sodium served as the reference standard at concentrations of 100-500 µg/mL.

The following formula was used to determine the percentage of proteinase inhibition. The results were shown as IC₅₀ values, with diclofenac sodium serving as the positive control and the reaction mixture devoid of the test sample as the negative control.

% inhibition = [(A_c – A_t)/A_c] × 100

Where, At denotes the absorbance in the presence of LCLT and Ac denotes the absorbance of the negative control, which is Tris-HCL buffer and trypsin.

In-vitro Antioxidant Activity:

1. Radical Scavenging Activity:

Gulcin (2006) described the radical scavenging assay, often known as the DPPH assay. The LCLT's ability to scavenge free radicals was measured using the assay.^22–24 When the sample is present, this assay calculates the rate at which the stable free radical DPPH bleaches at a specific wavelength. The highest absorption of DPPH in its radical form occurs at 517nm. However, when DPPH is lowered by a radical species or an antioxidant, its absorption is decreased. In conclusion, 1mL of 0.1mM DPPH solution in ethanol was mixed with 3mL of LCLT (15–30µg/mL) and standard antioxidant (15–30 µg/mL) and allowed to sit at room temperature for 30minutes. The absorbance was measured at 517nm using a UV spectrophotometer after 30minutes. Stronger radical scavenging activity is indicated by a lower absorbance in the reaction mixture.

The percentage of radical scavenging was calculated using the following formula. Ascorbic acid was used as the positive control and a reaction mixture devoid of the sample as the negative control. The results were displayed as IC₅₀ values.

% radical scavenging = [(A_c – A_t)/A_c] × 100

Where, At denotes the absorbance in the presence of LCLT and Ac denotes the absorbance of the negative control, or DPPH.

2. H₂O₂ Scavenging Activity:

Using a method described by Pandey (2017), the H₂O₂ scavenging activity of LCLT was measured spectrophotometrically to determine its potential for scavenging H₂O₂.^25–27 The phosphate-buffered solution having pH 7.4 was used to make a 40 mM hydrogen peroxide (H₂O₂) solution. The H₂O₂ solution and either LCLT (200-1000 µg/mL) or standard antioxidant (200-1000 µg/mL) were thoroughly mixed in equal proportions (1.5 mL). The absorbance of the mixture was measured at 230 nm after 10 minutes. The blank solution lacked both the phosphate buffer and H₂O₂.

The percentage of H₂O₂ scavenging was calculated using the formula below. Ascorbic acid was used as the positive control and a reaction mixture devoid of the sample as the negative control. The results were displayed as IC₅₀ values.

% H₂O₂ scavanged = [(A_c – A_t)/A_c] × 100

Where, At denotes the absorbance in the presence of LCLT and Ac denotes the absorbance of the negative control, or H₂O₂.

RESULTS:

In-vitro Anti-inflammatory Activity:

1. Albumin denaturation method:

The dose-dependent decrease in absorbance indicates that bovine serum albumin denaturation was decreased by LCLT between 100 and 500µg/mL. Within the same concentration range, conventional diclofenac sodium showed similar effects. The linear regression coefficients for diclofenac sodium and LCLT were r² = 0.9395 and r² = 0.9714, respectively. Based on linear regression analysis, diclofenac sodium and LCLT were shown to have IC₅₀ values of 194.44 and 282.03µg/mL. Figure No. 1 presents the detailed results.

Figure No. 1 Albumin denaturation method

2. Proteinase Inhibitory Activity:

The dose-dependent decrease in absorbance of LCLT suggested that it exhibited proteinase inhibitory activity within the range of 100-500µg/mL. Within the same concentration range, conventional diclofenac sodium showed similar effects. The linear regression coefficients for diclofenac sodium and LCLT were r² = 0.9674 and r² = 0.9883, respectively. Based on linear regression analysis, diclofenac sodium and LCLT were shown to have IC₅₀ values of 179.25 and 270.51µg/mL. Figure No. 2 presents the detailed results.

Figure No. 2 Proteinase inhibitory activity

In-vitro Antioxidant Activity:

1. Radical Scavenging Activity (DPPH Assay):

The dose-dependent decrease in absorbance of the purple color solution of LCLT indicates that LCLT suppressed DPPH radicals within the range of 20–100µg/mL. Within the same concentration range, conventional ascorbic acid showed similar effects. The linear regression coefficients for ascorbic acid and LCLT were r² = 0.9842 and r² = 0.9969, respectively. Based on linear regression analysis, ascorbic acid and LCLT were shown to have IC₅₀ values of 45.29 and 61.11µg/mL. Figure No. 3 presents the detailed results.

Figure No. 3 Radical Scavenging Activity (DPPH Assay)

2. H₂O₂ Scavenging Activity:

The dose-dependent reduction in absorbance of LCLT revealed H₂O₂ scavenging activity of within the range of 15–30 µg/mL. Within the same concentration range, conventional ascorbic acid showed similar effects. The linear regression coefficients for ascorbic acid and LCLT were r² = 0.9978 and r²= 0.9975, respectively. Based on linear regression analysis, ascorbic acid and LCLT were shown to have IC₅₀ values of 24.24 and 32.84µg/mL, respectively. Figure No. 4 presents the detailed results.

Figure No. 4 H₂O₂ Scavenging Activity

DISCUSSION:

The results of this study demonstrate that L-carnitine-L-tartrate (LCLT) exhibits significant in-vitro anti-inflammatory and antioxidant activities, with effects comparable to those of common reference substances like ascorbic acid and diclofenac sodium.

In-vitro Anti-Inflammatory Activity:

The albumin denaturation method demonstrated that, within the concentration range of 100–500µg/mL, LCLT efficiently decreased the denaturation of bovine serum albumin in a dose-dependent manner. The well-known anti-inflammatory medication diclofenac sodium and LCLT both have IC₅₀ values of 282.03 and 194.44 µg/mL, respectively, which demonstrate their similar performance. Similarly, in the proteinase inhibitory activity assay, LCLT demonstrated a dose-dependent inhibition within the same concentration range. The IC₅₀ value for LCLT (270.51µg/mL) was slightly higher than that of diclofenac sodium (179.25µg/mL), indicating that while LCLT is somewhat less potent, it still shows significant proteinase inhibitory activity.

These findings collectively suggest that LCLT has a promising anti-inflammatory potential, which could be harnessed in therapeutic applications, especially considering its relatively high correlation coefficients in both assays.

In-vitro Antioxidant Activity:

The DPPH assay and H₂O₂ scavenging activity were used to assess the antioxidant capacity of LCLT. Within the dosage range of 20–100µg/mL, LCLT demonstrated a dose-dependent decrease in absorbance in the DPPH experiment. The IC₅₀ value of LCLT (61.11µg/mL) was close to that of the standard ascorbic acid (45.29µg/mL). This suggests that LCLT has a robust capacity to neutralize DPPH radicals, which is a common indicator of antioxidant activity. In the H₂O₂ scavenging assay, LCLT exhibited effective scavenging activity in the 15–30µg/mL range, with an IC₅₀ value of 32.84 µg/mL, compared to 24.24µg/mL for ascorbic acid indicating that LCLT is almost as effective as ascorbic acid in scavenging hydrogen peroxide, a reactive oxygen species.

CONCLUSION:

The results of the study show that LCLT has antioxidant and anti-inflammatory properties in vitro, with a performance comparable to conventional standards like diclofenac sodium and ascorbic acid. While LCLT shows slightly lower potency in some assays, its high linear regression coefficients across all tests suggest a reliable and consistent efficacy. These results underscore the potential of LCLT as a therapeutic agent in managing inflammation and oxidative stress. According to this study, LCLT may serve as a lead molecule for the development of a strong anti-inflammatory medication that can be used to treat inflammation.

ACKNOWLEDGMENTS:

We sincerely thank each and every one of you for helping us to complete this research article. We also express our gratitude to Dr. D. Y. Patil College of Pharmacy in Akurdi, Pune, for giving us the chance and assistance to take part in and contribute to this research project.

REFERENCES:

1. Alhasaniah AH. L-carnitine: Nutrition, Pathology, and Health Benefits. Saudi J Biol Sci. 2023; 30(2):103555.

2. Sawicka AK, Renzi G, Olek RA. The bright and the dark sides of L-carnitine supplementation: a systematic review. J Int Soc Sports Nutr. 2020; 17(1).

3. Gülçin I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006; 78(8): 803–11.

4. Gupta A, Rawat S, Gupta P. Clinical research and therapeutic importance of dietary supplement L-carnitine: Review. Asian J Pharm Res 2018; 8(1): 47–58.

5. Adeva-Andany MM, Calvo-Castro I, Fernández-Fernández C, Donapetry-García C, Pedre-Piñeiro AM. Significance of L-carnitine for human health. IUBMB Life. 2017; 69(8): 578–94. Lovells H, FedEx B. GRAS Notice (GRN) No. 993 [Internet]. Hogan Lovells; 2021 [cited 2024 Jun 22]. Available from: https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory

6. Singh N, Baby D, Rajguru J, Patil P, Thakkannavar S, Pujari V. Inflammation and cancer. Ann Afr Med. 2019; 18(3): 121–6.

7. Parnham MJ. Inflammation research: modern design, more quality science. Inflamm Res. 2011; 60(1): 1.

8. Patil KR, Mahajan UB, Unger BS, Goyal SN, Belemkar S, Surana SJ, et al. Animal models of inflammation for screening of anti-inflammatory drugs: Implications for the discovery and development of phytopharmaceuticals. Int J Mol Sci. 2019; 20(18). Available from: https://pubmed.ncbi.nlm.nih.gov/31491986/

9. Shinde MG, Sayyad KD, Swami GV, Savant PB, Yalmar PR, Mane ST. A review on inflammation and its pharmacotherapy. Asian J Pharm Technol. 2023; 13(3): 201–6.

10. Darenskaya MA, Kolesnikova LI, Kolesnikov SI. Oxidative stress: pathogenetic role in diabetes mellitus and its complications and therapeutic approaches to correction. Bull Exp Biol Med. 2021; 171(2): 179–89. Available from: https://pubmed.ncbi.nlm.nih.gov/34173093/

11. Nimbalkar V, Vyawahare N. L-carnitine protects cisplatin-induced liver fibrosis in experimental animals via reducing oxidative and nitrosative stress. Int J Pharm Investig 2022;12(3):370–4.

12. Arya B, Krishnaveni K, Sambathkumar R. Review on antioxidant supplements use in cancer chemotherapy. Res J Pharmacol Pharmacodyn. 2020; 12(1): 21–4.

13. Nimbalkar V, Vyawahare N. L-carnitine ameliorates bile duct ligation-induced liver fibrosis via reducing the nitrosative stress in experimental animals: preclinical evidences. Heliyon. 2021; 7(11): e08488. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8642613/

14. Bremer J. Carnitine: Metabolism and functions. Physiol Rev. 1983; 63(4): 1420–80.

15. Goli V, Bhaskar KV, Macharla SP, Bhaskar J, Devi PS, Ramchander T. Effects of anti-inflammatory activity of Mimosa pudica. Asian J Pharm Res. 2011; 1(3): 69–71.

16. Narapusetty N, Sivaiah O, Balanasaraiah B, Haranadhbabu M, Prasad B, Crown BH, et al. Anti-inflammatory activity of ethanolic extract of Basella alba in acute and sub-acute model. Asian J Pharm Res. 2017;7(2):88.

17. Mahapatra DK, Shivhare RS, Kumar P. Murrayanine-chalcone transformed into novel pyrimidine compounds demonstrated promising anti-inflammatory activity. Asian J Pharm Res. 2018; 8(1): 6.

18. Dadoriya P, Dey YN, Sharma D, Yadav M, Wanjari MM, Gaidhani SN, et al. In-vitro anti-inflammatory and antioxidant activities of an Ayurvedic formulation – Trayodashang guggulu. J Herb Med 2020; 23: 100366.

19. G P, Pandiar D, Shanmugam R, Poothakulath Krishnan R. An in vitro evaluation of anti-inflammatory and antioxidant activities of Cocos nucifera and Triticum aestivum formulation. Cureus. 2023; 15(11). Available from: https://pubmed.ncbi.nlm.nih.gov/38090429/

20. Venkatesan A, Samy JVRA, Balakrishnan K, Natesan V, Kim SJ. In vitro antioxidant, anti-inflammatory, antimicrobial, and antidiabetic activities of synthesized chitosan-loaded p-coumaric acid nanoparticles. Curr Pharm Biotechnol. 2023; 24(9): 1178–94.

21. Khalequeuzzaman M, Al Hasan S, Haque M, Parul R, Alam MR. Evaluation of antioxidant and anti-inflammatory activity of ethanolic extract of Glochidion acuminatum leaves. Asian J Pharm Res. 2023; 13(2): 87–91.

22. Babu SN, Govindarajan S, Vijayalakshmi MA, Noor A. Evaluation of in vitro anti-diabetic and antioxidant activities and preliminary phytochemical screening of gel, epidermis and flower extract of Aloe vera. Res J Pharm Technol. 2019; 12(4):1761–8.

23. Krishna KV, Karuppuraj V, Perumal K. Antioxidant activity and folic acid content in indigenous isolates of Ganoderma lucidum. Asian J Pharm Anal. 2016; 6(4): 213.

24. Pandey A, Kaushik A, Wanjari M, Dey YN, Jaiswal BS, Dhodi A. Antioxidant and anti-inflammatory activities of Aerva pseudotomentosa leaves. Pharm Biol. 2017; 55(1): 1688–97.

25. Iqbal P, Ahmed D, Asghar M-N. A comparative in vitro antioxidant potential profile of extracts from different parts of Fagonia cretica. Asian Pac J Trop Med. 2014; 7: S473-S480. doi:10.1016/S1995-7645(14)60277-7.

26. Khobragade CN, Bodade R, Manwar AV. Synthesis and antioxidant activity of some flavone derivatives. Asian J Res Chem. 2010; 2(3): 97–105.

Received on 11.07.2025 Revised on 06.10.2025

Accepted on 04.12.2025 Published on 06.07.2026

Available online from July 20, 2026

Asian J. Pharm. Res. 2026; 16(3):237-241.

DOI: 10.52711/2231-5691.2026.00035

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.