High-Resolution Mass Spectroscopy Study of Smriti Panchak Yoga with An Emphasis on Antioxidant and Neuroprotective Effect

In Ayurveda, many neurodegenerative disorders have been mentioned with their pathophysiology and treatment. In accordance, with that we have designed a new formulation named Smriti Panchak Yoga having 5 herbal ingredients i.e. Ashwagandha Brahmi, Shankhpushpi, Vacha, Tagar, Smriti Panchak comprised of two words – Smriti & Panchak. The word Smriti means - Retention and recalling power. Panchak denotes the formulation comprised of five drugs. Smriti Panchak is an herbal Formulation containing a combination of drugs that are known for potential multiple benefits including mental illness and psychological disorders & boost memory power. The combination gives the properties such as brain tonic, memory enhancer, antioxidant and neuroprotective effect. Since its inception in 6000 B.C., Withania somnifera (L.) Dunal (Solanaceae), popularly known as Ashwagandha in Sanskrit, has been extensively implemented as herbal medicine. The species name has been ascribed as ‘somnifera’, which means “sleep-inducer” in Latin, owing to its prodigious anti-stress characteristics; Withania somnifera include the ability to improve concentration, memory, mood, anxiety, insomnia and neurological disorder. Bacopa monnieri (L.)– (BM) (Common names: Brahmi, Bacopa, Waterhyssop), renowned as a nootropic plant; belonging to the family Scrophulariaceae, is a well-known ayurvedic medicinal plant used in India, as a neural tonic to improve intelligence and memory, increase brain function and promote longevity. Convolvulus pluricaulis (also called Shankhpushpi in Hindi) is a herb that has been extensively investigated for its pharmacological and therapeutic effects. Convolvulus pluricaulis (Convolvulaceae) has long been used as a traditional herbal medicine in India as a nerve tonic. We investigated the neuroprotective effects of aqueous extract from Convolvulus pluricaulis (CP) against aluminium chloride-induced neurotoxicity in rat cerebral cortex. Vacha botanical name Acorus calamus (Family Araceae) is an important medicinal plant of the Himalayan region largely used in the preparation of cosmetics and traditional medicines. Total phenolic content and antioxidant activity varied among the populations and solvent systems. Solvents with medium polarity such as acetone and methanol were found best for extraction of total phenolic content and antioxidant activity. Tagara (Valeriana wallichii of family Valerianaceae), an ayurvedic drug that possesses protective effects on several aspects of brain and nervous conditions is chosen for the present study. It is well used in various pharmaceutical preparations for the treatment of migraine Insomnia and anxiety. Tagara is also reported to have an antioxidant effect.

MATERIALS METHOD:

Authentication And Preparation Of SPY: The Raw material of SPY for the analysis by liquid chromatography-mass spectrometry was collected from the local market Gola-Deena Nath Varanasi, Uttar Pradesh. The same Raw materials of SPY is later authenticated in the department of Botany, Institute of science Banaras Hindu university and accession No of Withania somnifera Solena. 2023/02, Acorus calamus Acora. 2023/01, Bacopa monnieri Plantagina 2023/01, Valeriana wallichi caprifolia 2023/01, Convolvulus pluricauulis convolvuli. 2023/01. After procurement and authentication of raw drugs Preparation of Churna of Raw material and preparation of hydroalcoholic extract of each drug separately. To Prepare Smriti Panchak yoga, equal quantity of each dried and solvent-free extract i.e., Withania somnifera (Ashwagandha), Bacopa monnieri (bramhi), Convolvulus pluricaulis (Shakhpushpi), Acorus calamus (Vacha), and Valeriana wallichii (Tagar) were combined and thoroughly mixed using a ceramic mortar and pestle. The homogenous mixture was formed within 20 min of trituration. The colour of SPY was dark brown with a characteristic odor.

Method Employed for HRMS Analysis: The sample preparation for HRMS analysis started with the addition of an individually optimized sample of Smriti Panchak Yoga (100 mg) with 1.5 ml of solvent (methanol:water; 80:20) and homogenized using an Eppendorf  Thermomixer (Eppendorf SE) Cat 750 rpm for 30 min at 25 °C. Then, the sample was centrifuged (3500 rpm/10 min/25 °C). The supernatant was filtered with a 0.22 µ polytetrafluoroethylene (PTFE) syringe filter, and 4 µl of the filtrate was used as injection volume on the C18 reverse-phase high-performance liquid chromatography (RP-HPLC) column (Hypersil GOLD™, Thermo Fischer Scientific: particle size 1.9 µ, 2.1 mm × 100 mm).

The reversed-phase chromatographic separation starts with a high aqueous phase (+0.1% formic acid) and ends on a highly organic phase (MeOH+ 0.1% formic acid), typically 100% aqueous to 100% organic. The liquid chromatography gradient parameters were as follows: 0 to six minutes 5% MeOH, six to 10 minutes 30% MeOH, 10-20 minutes 50% MeOH, 20-25 minutes 90% MeOH, 25-27 minutes 90% MeOH, and 27-30 minutes 5% with a flow rate of 300 L/Min and column oven temperature 40 °C. The optimized sample of Smriti Panchak Yoga was tested for metabolomics analysis. Thermo Fisher Scientific high resolution accurate mass spectrometry system of the model Orbitrap Eclipse Tribrid mass spectrometer coupled with nano liquid chromatography and ultra high-pressure liquid chromatography (Dionex Ultimate 3000 rapid separation liquid chromatography (RSLC)) system, a heated electrospray ionization (HESI) source was used to feed the sample to the mass spectrometer post chromatographic separation. The Orbitrap analyzer was utilized at 60,000 resolutions separately for positive/negative polarity with a mass range (m/z) of 100-1,000, a 35% RF lens, and a 25% normalized automatic gain control (AGC) target, keeping 2.0e5 as the intensity threshold to perform MS-OT (master scan). To obtain ddMS2 OT higher collisional dissociation (HCD), the selection parameters were quadrupole isolation mode with 1.5 isolation window (m/z) HCD activation type, 30, 45, and 60 HCD collision energy (%), 15,000 Orbitrap resolution, and a 20% normalized AGC target.  The raw data obtained from the mass analyzer were performed through the default parameters of Compound Discoverer 3.3.2.31 (Thermo Fischer Scientific) using online databases. The chosen workflow was the Natural Product Unknown ID with both online and local database searches, focused on untargeted food research without statistical analysis. This approach detects and identifies unknown compounds by performing retention time alignment, detecting unknown compounds, and grouping them across all samples. It predicts the elemental compositions for all compounds and filters out chemical backgrounds using blank samples. Compound identification is carried out through mzCloud (HighChem LLC, Bratislava, Slovakia) (ddMS2 and/or DIA), ChemSpider (Royal Society of Chemistry, Cambridge, UK) (using exact mass or formula), and local database searches against mass lists (with or without retention time). Spectral similarity searches are conducted on mzCloud for ddMS2 compounds and spectral distance scoring is applied for ChemSpider and mass list matches¹.

RESULTS: The total ion chromatogram (TIC) depicts the total intensity of all ions detected as a function of time. It is a vital tool for interpreting the composition of a sample and understanding the various compounds present in it. It is a visual representation of all the ions detected during a chromatographic separation, allowing us to gain insights into the composition of a sample. The TIC of the components present in Smriti Panchak Yoga is depicted in Figure 1.

Figure 1: Total ion chromatogram of Smriti Panchak Yoga obtained by UHPLC HRAMS analysis of the Smriti Panchak Yoga sample in positive and negative ion modes.

Neuroprotective and Antioxidant activities of the phytochemical constituents found in Smriti Panchak Yoga

By performing HRMS analysis, a total of 3213 phytochemical constituents were identified in the Smriti Panchak yoga. Among those phytochemical components, the following components, namely withanolide, Coumarin, Caffeic acid, Apigenin, Eugenol, P-Cymene, and beta-caryophyllene oxide exhibit neuroprotective and antioxidant activity as per the following references.

Withanolide – A: Withanolide-A (WA), a major bioactive component of Withania somnifera, is a potential medicinal natural compound showing improvement in some neurological diseases, such as cerebral ischemia. In the present study, we examined whether the administration of WA can exert the beneficial effects involved in neuroprotection and anti-inflammatory effects in a mouse model of pilocarpine-induced SE. Our results showed that WA treatment ameliorated SE-induced apoptotic neuronal cell death in the hippocampus. Moreover, WA treatment reduced immunoreactivity of both ionized calcium-binding adapter molecule 1-positive microglia/macrophage and glial fibrillary acidic protein-positive reactive astrocytes, and the SE-induced increase in both interleukin-1 β and tumour necrosis factor in the hippocampus, suggesting that inhibiting pro-inflammatory factors by WA treatment might induce neuroprotection after SE. These results suggest that WA may be useful in improving the treatment efficacy for pathophysiology following SE².

Coumarin: Coumarin derivates LM-031 and LMDS-1 to -4 were examined (Figure 1A). Anti-aggregation and anti-oxidative stress are important treatment approaches for neurodegenerative diseases. The inhibition of ?K280 tauRD aggregation was measured by thioflavin T assay. Congo red, known to reduce misfolded aggregation, was included for comparison. The free radical-scavenging activity was examined using DPPH as a substrate and kaempferol as a positive control. Kaempferol, LM-031 and LMDS-1 to -4 had EC50 values of 28, 93, 122, 132, 132 and 126 µM, respectively. In addition, the oxygen radical absorbance capacity of the LM-031 and LMDS-1 to -4 was examined based on a Trolox standard curve. LM-031 and LMDS-1 to -4 at 100 µM had activity equivalent to 51, 10, 15, 12 and 16 µM Trolox, respectively. The cytotoxicity of LM-031 and LMDS-1 to -4 was examined by MTT assay. All compounds had cell viability up to 75–92% in 100 µM compound-treated ?K280 tauRD-DsRed SH-SY5Y cells³.

Caffeic acid: Inhibition of lipid peroxidation assay confirmed antioxidant activities of caffeic acid isolated from butanol fraction of E. annuus on both ferric ion and vitamin-C induced lipid peroxidation on mouse brain homogenates. Caffeic acid suppressed lipid peroxidation on mouse brain homogenates (Figure 4A). Caffeic acid showed less effectiveness than (+)-catechin at all concentrations; more than 50% of inhibitory activity of lipid peroxidation was observed at the concentration of 50 μg/ml. However, caffeic acid had an EC50 value of 38.43 μg/ml, compared to (+)-catechin (31.17 μg/ml). Previous studies indicated that caffeic acid had excellent antioxidant and neuroprotective effects [26]. These results suggested a potential use of the crude extract of E. annuus as well as the isolated compounds for treating neurodegenerative diseases such as AD⁴.

Apigenin: Apigenin (4,5,7–trihydroxyflavone) is a flavonoid found in abundance in many fruits and vegetables, that has shown important effects in controlling the inflammatory response. This study evaluated the neuroprotective and neuroimmunomodulator potential of apigenin using in vitro models of neuro-inflammation associated with Alzheimer’s Disease (AD). Co-cultures of neurons and glial cells were obtained from the cortex of newborn and embryonic Wistar rats. After 26 days in vitro, cultures were exposed to lipopolysaccharide (LPS; 1 μg/ml), or IL-1β (10 ng/ml) for 24 h, or to Aβ oligomers (500 nM) for 4 h, and then treated with apigenin (1 μM) for further 24 h. It was observed that the treatment with apigenin preserved neurons and astrocytes integrity, determined by Rosenfeld’s staining and immunocytochemistry for β-tubulin III and GFAP, respectively. Moreover, it was observed by Fluoro-Jade-B and caspase-3 immunostaining that apigenin was not neurotoxic and has a neuroprotective effect against inflammatory damage. Additionally, apigenin reduced microglial activation, characterized by inhibition of proliferation (BrdU+ cells) and modulation of microglia morphology (Iba-1 + cells), and decreased the expression of the M1 inflammatory marker CD68. Together these data demonstrate that apigenin presents neuroprotective and anti-inflammatory effects in vitro and might represent an important neuro-immunomodulatory agent for the treatment of neurodegenerative conditions. Neurodegenerative disorders (ND) are characterized by the progressive and irreversible loss of neurons. Alzheimer’s Disease (AD) is the most incident age-related ND, in which the presence of a chronic inflammatory compound seems to be related to its pathogenesis. Different stimuli in the central nervous system (CNS) can induce activation, proliferation, and changes in phenotype and glial function, which can be modulated by anti-inflammatory agents⁵.

Stearic acid: Stearic acid can protect brain slices (cortical or hippocampal) against injury induced by OGD or glutamate. Its neuroprotective effect may be mainly mediated by the activation of PPAR-γ. Experiments using brain slices have the advantages of in vivo and in vitro studies: they not only maintain anatomic relations and natural synaptic connectivity in vitro, but also eliminate such in vivo variables as blood flow, temperature and ionic environment, and closely match in vivo conditions. Therefore, increasing numbers of brain slice models have been used to study brain function and brain protection. In the present study, we used 3 different damage models to reflect the pathological characteristics of different phases of I/R injury (metabolism disorder, toxic amino acid and oxidative stress) [11]. The present study is the first to demonstrate that stearic acid can dose-dependently protect rat brain slices against OGD and glutamate toxicity, but not against NaN3 toxicity⁶.

P-cymene: P-cymene, monoterpenes of aromatic plants. This study investigated these compounds’ possible anti-cholinesterase, anti-α-amylase, and neuroprotective effects. We evaluated the anti-acetylcholinesterase and anti-α-amylase activities at different concentrations of the compounds. The maximum non-toxic dose of carvacrol and p-cymene against SH-SY5Y neuroblastoma cells was determined using an MTT assay. The neuroprotective effects of the compounds were evaluated on H₂O₂-induced stress in SH-SY5Y cells, studying the expression of caspase-3 using Western blotting assays. Carvacrol showed inhibitory activities against acetylcholinesterase (IC50 = 3.8 µg/mL) and butyrylcholinesterase (IC₅₀ = 32.7 µg/mL). Instead, the anti-α-amylase activity of carvacrol resulted in an IC₅₀ value of 171.2 μg/mL After a pre-treatment with the maximum non-toxic dose of carvacrol and p-cymene, the expression of caspase-3 was reduced compared to cells treated with H₂O₂ alone. Carvacrol and p-cymene showed in vitro anti-enzymatic properties, and may act as neuroprotective agents against oxidative stress⁷.

Betain: In addition, betaine has a neuroprotective role, preserves myocardial function, and prevents pancreatic steatosis. Betaine also attenuates oxidant stress, endoplasmic reticulum stress, inflammation, and cancer development. To conclude, betaine exerts significant therapeutic and biological effects that are potentially beneficial for alleviating a diverse number of human diseases and conditions. A large number of studies report that medicinal herbs and many food ingredients protect against the development of liver disease because they possess antioxidant, anti-inflammatory, or anti-necrotic activities. This review focuses on the biological and beneficial effects of dietary betaine (trimethylglycine), a naturally occurring and crucial methyl donor, that restores methionine homeostasis in cells⁸.

Gallic acid: Gallic is also able to reverse the scopolamine-induced amnesia in mice, probably through inhibiting oxidative stress and decreasing acetylcholinesterase (AChE) enzyme activity in the brain. Several beneficial effects are reported for gallic acid, including antioxidant, anti-inflammatory, and antineoplastic properties. This compound has been reported to have therapeutic activities in gastrointestinal, neuropsychological, metabolic, and cardiovascular disorders⁹.

DISCUSSION: Ayurveda includes various medicinal herbs which are responsible for the development of the brain. In accordance, with that we have designed a new formulation named Smriti Panchak Yog (SPY) having 5 herbal extracts i.e. Brahmi, Shankhpushpi, Vacha, Tagar, and Ashwagandha. Ensuring the quality, safety and efficacy of herbal drugs through standardization is crucial in advancing scientific understanding. Liquid chromatography equipped with high-resolution mass spectrometry (LC-HRMS) is deployed for the identification of secondary metabolites in various crude extracts. The plant extract showed antioxidant activity through free radical scavenging activity. The analysis of the phytoconstituent revealed that SPY have antioxidant capacity refers to the ability of a substance to neutralize or prevent the harmful effect of reactive oxygen species. DPPH and the total phenolic content study revealed that SPY has a high antioxidant capacity and neuroprotective effect reflecting the better neuropharmacological activity of SPY such as Withanolide-A (WA), a major bioactive component of Withania somnifera, is a potential medicinal natural compound showing improvement of some neurological diseases, such as cerebral ischemia². Citrus coumarin, effectively inhibited microglia activation, cyclooxygenase-2 expression by astrocytes, and neuronal cell death in the hippocampus following ischemic insults. These results suggest that auraptene (AUR) acts as a neuroprotective agent in the ischemic brain¹⁰, Caffeic acid, one of the most common phenolic acids, is widely distributed in several species of plants and regularly consumed in the human diet. Caffeic acid and derivatives show that these chemicals have a variety of functions including acting as antioxidants, suppressing cerebral lipid peroxidation, and reducing brain infarct after cerebral ischemia in rats. It has been also suggested that caffeic acid and its derivatives have therapeutic potential in the treatment of neurodegenerative diseases¹¹. Using the MPTP-induced parkinsonian mouse model, Patil and colleagues found that the neuroprotective role of Apigenin is mediated in part by its potent antioxidant activity (increased SOD, CAT, GSH and decreased LP), prevention of TH and BDNF decrease and Glial fibrillary acidic protein (GFAP) and TNF- increase¹². Stearic acid dose-dependently protected cortical neurons against glutamate or H2O2 injury and increased glutamate uptake in cultured neurons. This protection was concomitant to the inhibition of lipid peroxidation and the promotion activity of Cu/Zn SOD and CAT in cultured cortical neurons¹³. p-cymene showed in vitro anti-enzymatic properties and may act as a neuroprotective agent against oxidative stress. These compounds have possible anti-cholinesterase, anti-α-amylase, and neuroprotective effects¹⁴. Level of the biomarkers induced by brain injury in the groups fed with betaine, omega-3 and betaine+omea-3 combination before the traumatic damage approximated to that of control group values, suggesting that these products may have a neuroprotective role¹⁵. It can be inferred that the neuroprotective and antioxidant activity of the specified components is likely associated with their molecular structure, as it profoundly influences their chemical and neuropharmacological activities. Factors such as functional groups, shape, stereochemistry, charge distribution, molecular weight, conformational flexibility, and hydrophobic/ hydrophilic characteristics all play integral roles in determining how a molecule interacts with biological systems.  Hence, the molecular structures of the active components found in Smriti Panchak Yoga are depicted in Figure.

FIGURE 2: Molecular structure of isolated components from Smriti Panchak Yoga possessing neuroprotective and antioxidant action as identified through high-resolution mass spectrometry analysis. These images have been created by the authors.