Liquid Chromatography–Mass Spectrometry Study Focusing on Neuroprotective...

[Full title: Liquid Chromatography–Mass Spectrometry Study Focusing on Neuroprotective Effect of Compounds Obtained from Bacopa monnieri Juice]

Asian Journal of Pharmaceutic s • Jul-Sep 2024 • 18 (3) | 905 Liquid Chromatography–Mass Spectrometry Study Focusing on Neuroprotective Effect of Compounds Obtained from Bacopa monnieri Juice Prem Shankar Pandey, K. Ramachandra Reddy, Priya Kumari Department of Rasa Shastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Abstract Introduction: According to Ayurvedic literature, Brahmi is highly recommended for the management of psychiatric problems since it acts on the brain. In traditional Ayurvedic medicine, Brahmi , a herb is recommended for the management of various psychological disorders such as Alakshmi (inauspicious condition), Unmada (insanity), and Apasmara (epilepsy). Drug quality is a basic necessity in this day and age, and analytical testing can help combat impurities or subpar pharmaceuticals. Materials and Methods: Compound analysis of Brahmi Juice ( Swarasa ) using liquid chromatographic mass spectrometry (LC-MS). Results: LC-MS analysis shows major compound such as L-Phenylalanine, Phenylacetylene, DL-Arginine, Azelaic acid, Erucamide, D-(+)- Pyroglutamic acid, L-Glutamic acid, Valine, Choline, L-Isoleucine, DL-Tryptophan, L-(-)-Methionine, and Beta-Ionone which play a significant role in neuroprotection. Conclusion: The study showed the presence of compounds rich in neurotransmitters which also act as neurotonics having their specific neuroprotective effect in conditions such as Alzheimer’s Disease, Parkinsonism, and Dementia. By employing a wide array of testing procedures, manufacturers are able to ensure compliance with regulatory requirements and supply consumers with a better product that effectively delivers Brahmi’s therapeutic benefits Keywords: Analytical, Ayurveda, Brahmi, liquid chromatographic mass spectrometry, Swarasa Address for correspondence: Prof. K. Ramachandra Reddy, Department of Rasa Shastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India E-mail: krcreddy@bhu.ac.in Received: 29-05-2024 Revised: 05-07-2024 Accepted: 13-07-2024 INTRODUCTION H erb “ Brahmi ( Bacopa monnieri )” used for treating psychiatric problems is a well-known Ayurvedic traditional medicine. Based on research, most psychiatric disorders can benefit from the widespread internal and external use of Brahmi , [1] the word “ Brahmi ” comes from the name of the mythological creator of the Hindu pantheon, “ Brahma .” Since the brain is the center of creative activity in the human body, Brahmi refers to substances that promote brain health. The Charak Samhita has the earliest direct mention of Brahmi’s ability to improve memory. Brahmi is recommended as a treatment for mental disorders (retardation) that result in insanity [2] Nowadays, a drug’s traits are more crucial in the treatment of illness. A highquality medicine is more valuable in today’s world. To produce pharmaceuticals of high quality, we must choose raw materials of high quality and follow good manufacturing, storage, and other procedures. Sometimes, even after doing all of these procedures, the properties of that specific medication are unknown until analysis. Many analytical tests with standard values are mentioned to overcome this. A prepared medication can be regarded as high grade if it passes certain tests [3] These tests encompass both qualitative and quantitative analyses of its constituents, contaminants, and physical characteristics. To find and measure any compounds in the formulation, pesticide residue analysis uses techniques such as liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry [4] These qualities ORIGINAL AR TICLE

Pandey, et al .: Neuroprotective Effect of Bacopa monnieri Juice By LC-MS Asian Journal of Pharmaceutic s • Jul-Sep 2024 • 18 (3) | 906 shed light on the stability, uniformity, and acceptance of the formulation by the consumer [5] To accomplish the goal, for the first time, we carried out an liquid chromatography-mass spectrometry (LC-MS) analysis of Brahmi juice ( Swarasa ) MATERIALS AND METHODS Methodology of Brahmi juice extraction Juice was prepared in Ayurvedic Pharmacy of IMS, BHU with the help of End Runner. First, Brahmi plant material was cut in to small pieces, then crushed in End Runner till formation Kalka (Bolus), after that Kalka was pressed in clean cloth and juice is obtained [6] Chromatographic conditions Application Mode: CAMAG Linomat 5 – Applicator Filtering System: Whatman filter paper No. 1 Stationary Phase: MERCK - TLC/HPTLC Silica gel 60 F 254 on aluminum sheets Application (Y axis) Start Position: 10 mm development End Position: 80 mm from plate base Sample Application Volume: 10 µ L Standard Application Volume: 7.5 µ L Distance Between Tracks: 20 mm Development Mode: CAMAG TLC Twin Trough Chamber 2 of 15 Chamber Saturation Time: 30 min Mobile Phase: Chloroform: acetone:water (14:2:0.03 v/v) Pre-chromatographic washing: Pre-chromatographic derivatization was done by diethyl ether visualization: at 366 nm (after derivatization) Spray reagent: 1% LLP in hexane Derivatization mode: CAMAG - Dip tank for about 1 min Drying Mode, Temp, and Time: TLC Plate Heater Preheated at 100 ± 50 C for 3 min Methodology of LC-MS for compound analysis of Brahmi Ghrita Instrument: High-resolution Accurate Mass Spectrometry System Model: Orbitrap Eclipse Tribrid Mass Spectrometer Make: Thermo Fisher Scientific For Small Molecule UHPLC: DionexUltiMate 3000 RS UHPLC System Solvent Composition for Small Molecule Solvent A: 100% Water + 0.1% Formic Acid Solvent B: 80% Acetonitrile + 0.1% Formic Acid Solvent C: 100% Methanol + 0.1% Formic Acid Column Detail: Hypersil GOLD™ C 18 Selectivity HPLC Column Particle size 1.9 µ m, Diameter 2.1 mm, Length 100 mm Analysis All analyses were conducted using the online database “Compound Discoverer 3.3.2.31” with its default settings. An untargeted metabolomics workflow was employed, utilizing molecular networks, online databases, and mzLogic. Chemical identification was based on fragment patterns from ChemSpider (using formula or precise mass) and mzCloud (ddMS 2) RESULTS Compound analysis by LC-MS By performing LC-MS analysis, we found 2749 compounds [Table 1], out of which the compounds that play role in neuroprotection are presented in Table 1 DISCUSSION Similar to other Ayurvedic herbs, Brahmi derives its medicinal properties from particular botanical constituents [7] In herbal formulations, contaminants such as pesticides, heavy metals, and microbiological infections can present major health hazards. To determine and measure these impurities and guarantee that Brahmi is safe to consume, analytical testing is used [8] Global regulatory agencies set strict standards for the potency and security of herbal remedies. To prove compliance with these restrictions and enable the lawful distribution and sale of Brahmi in both domestic and international markets, analytical data are necessary [5] Analytical testing provides important information about the chemical composition and properties of Brahmi . Regular testing produces analytical data that can also be used to guide ongoing research and development projects that try to improve the method or develop new uses for Brahmi in the medical field. Ayurvedic medicine is constantly improving and innovating owing to this iterative approach Compounds from LC-MS having neuroprotective property Identifying compounds responsible for the treatment of neurological diseases or associated mechanisms requires a thorough understanding of their pharmacological properties and mechanisms of action. While several compounds listed

Pandey, et al .: Neuroprotective Effect of Bacopa monnieri Juice By LC-MS Asian Journal of Pharmaceutic s • Jul-Sep 2024 • 18 (3) | 907 may have potential therapeutic effects, some may be more directly relevant to neurological conditions The total ions chromatogram and extracted ion chromatograms for some of the significantly identified drugs together with the base peak chromatogram of the sample acquired by negative and positive ion mode along their molecular structure and ion chromatogram are supporting this study [9] Here are some compounds from the list that have been studied or implicated in neurological health [ Figure 1] L-phenylalanine Neurotransmitters including dopamine, norepinephrine, and epinephrine, which are essential for brain function and mood control, are derived from L-phenylalanine [10] It is also a part of the treatment for phenylketonuria, a hereditary condition that affects the metabolism of neurotransmitters DL-arginine Arginine is a precursor to nitric oxide, a signaling molecule involved in vasodilation and neurotransmission. It has been studied for its potential neuroprotective effects in conditions such as ischemic stroke and Alzheimer’s disease [11] L-glutamic acid The central nervous system’s main excitatory neurotransmitter is glutamate. While glutamate modulators are being researched for their potential as treatments for a variety of neurological conditions, excessive glutamate signaling can also lead to neurotoxicity [12] DL-tryptophan Serotonin, a neurotransmitter important in mood regulation, sleep-wake cycles, and cognitive function, is derived from tryptophan. Its possible antidepressant effects and significance in treating neurological disorders like anxiety and depression have been researched [13] L-(-)-methionine An important amino acid, methionine is involved in methylation processes and the creation of neurotransmitters. Its neuroprotective qualities and possible advantages in ailments including Parkinson’s disease and age-related cognitive decline have been studied in various researches [14] D-(+)-pyroglutamic acid D-(+)- Pyroglutamic acid, sometimes referred to as 5-oxoproline, is a metabolite that plays a role in the brain’s antioxidant defence systems and glutathione metabolism. Research indicates that pyroglutamic acid may have neuroprotective effects by promoting the synthesis of glutathione, scavenging free radicals, and reducing the damage that oxidative stress causes to neurons [15] Azelaic Acid Azelaic acid is a dicarboxylic acid that possesses antibacterial and anti-inflammatory characteristics. Although its direct contribution to neurological disorders is not well understood, its anti-inflammatory properties may have consequences for neuroinflammatory disorders such as multiple sclerosis or neurodegenerative diseases [16] Table 1: Results of isolated compounds in Brahmi juice Name Calc. MW m/z RT (min) Area (Max.) L-Phenylalanine 165.07883 166.08611 1.578 10561069992 Phenylacetylene 102.04691 120.08073 1.579 4565038628 DL-Arginine 174.11139 175.1187 0.406 3115604901 Azelaic acid 188.10394 187.09666 12.978 2584071035 Erucamide 337.334 338.34128 27.303 557172648 D-(+)-Pyroglutamic Acid 129.04245 130.04976 0.396 529943221.7 L-Glutamic acid 147.05247 146.0452 0.846 516581231.8 Valine 117.07887 118.08616 0.433 414187851 Choline 103.09979 104.10706 0.39 337116272.1 L-Isoleucine 131.09457 132.10184 0.574 310377312.1 DL-Tryptophan 204.08972 188.07047 3.163 283504547.7 L-(-)-Methionine 149.05091 150.05817 0.464 278705141.3 Beta-Ionone 192.15122 175.14795 13.828 257690188.2

Pandey, et al .: Neuroprotective Effect of Bacopa monnieri Juice By LC-MS Asian Journal of Pharmaceutic s • Jul-Sep 2024 • 18 (3) | 908 Valine Essential for the production of proteins, valine is also a building block for neurotransmitters. It contributes to the equilibrium of neurotransmitters and may have an indirect effect on neurological function by influencing protein metabolism [17] L-Isoleucine Another necessary amino acid involved in energy metabolism and protein synthesis is leucine. By promoting neurotransmitter synthesis and preserving cellular energy levels in the brain, it might benefit neurological health [18] Choline One essential vitamin that is used to make the neurotransmitter acetylcholine is choline. For healthy brain growth, cognitive function, and memory formation, an adequate choline intake is necessary [19] Phenylacetylene It is unclear whether phenylacetylene, a chemical molecule, directly contributes to neurological disorders. Nonetheless, studies on related substances point to possible neuroprotective benefits through processes including antioxidant activity or neurotransmitter system modification [20] Further studies are needed to elucidate its specific effects on neurological health. Nonetheless, pre-clinical research has indicated that certain fatty acid compounds have neuroprotective qualities, indicating possible directions for further investigation [21] Beta-ionone Some research indicates that it may have neuroprotective qualities through antioxidant processes. However, further research is needed to validate these findings CONCLUSIONS The present study of LC-MS belongs to Bramhi ( B. monnieri ) juice showed the presence of L-Phenylalanine, Phenylacetylene, DL-Arginine, Azelaic acid, Erucamide, D-(+)-Pyroglutamic Acid, L-Glutamic acid, Azelaic acid, and Valine L-Isoleucine which are rich in neurotransmitters which also act as neurotonics having their specific neuroprotective effect in conditions such as Alzheimer’s Disease, Parkinsonism, and Dementia. Ensuring the efficacy, safety, and purity of Brahmi are largely dependent on analytical testing. Utilizing an extensive range of tests, producers can guarantee adherence to legal specifications and offer customers a superior product that efficiently provides Brahmi’s medicinal advantages. To summarize, LC-MS is an effective analytical instrument that may be used for research and development, contaminant detection, chemical identification, quantification, and quality control when analyzing. Numerous processes, such as neurotransmitter modulation, neuroprotection, and metabolic regulation, may be employed by these substances to achieve their neurological effects. Nevertheless, more investigation is required to clarify their precise therapeutic uses and modes of action in neurological conditions ACKNOWLEDGMENTS The authors would like to thank the Department of Science and Technology (DST), India, and Sophisticated Analytical and Technical Help Institute - Banaras Hindu University (SATHI-BHU) for providing a “High-Resolution Mass Spectrometry” facility Figure 1: Chemical structure of compounds belongs to Brahmi juice, (a) L-Phenylalanine; (b) Phenylacetylene; (c) DL-Arginine; (d) Azelaic acid; (e) Erucamide; (f and g) D-(+)- Pyroglutamic Acid; (h) Valine; (i) Choline; (j) L-Isoleucine; (k) DL-Tryptophan; (l) L-(-)-Methionine; (m) beta-ionone a b c d e f g h i j k l m

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