A Novel Densitometric HPTLC Method Integrated with FTIR for The Detection of Rutin in Senna auriculata Flower Extract

Introduction

Senna auriculata (L.) Roxb.belonging to the family Fabaceae (Previously known as Caesalpiniaceae), is recognized as one of the medicinal flora utilized traditionally in Ayurveda practices since the 15^th century (Fig. 1.)¹. Senna auriculata commonly referred to as Tanner’s Cassia in English and Avaram poo in Tamil represents a notable traditional plant this evergreen species is native to India².The shrub is a small, perennial plant that can grow as tall as 2 meters³. The flowers are large, asymmetrical and hermaphroditic vibrant yellow, measuring almost 5cm in diameter. The pedicels are sleek and have a length of 2.5cm. The racemes are brief, sparsely flowered and upright, clustered in the axils of the upper leaves to form a distinct terminal inflorescence. The stamens are non-functional and the ovary is superior, single-chambered, with ovules situated along the edges⁴.The word ‘Flavonoid’ originates from the Latin term ‘flavus’, which translates to yellow. The examination of flavonoids has a longstanding history that extends over several decades⁵. Flavonoids are secondary metabolites found in plants that constitute a category of naturally occurring polyphenols found widely in the plant kingdom. These extraordinary substances are chemically characterized by a structure comprising fifteen carbon atoms. These molecules possess a carbon backbone organized as C6-C3-C6, featuring a benzo-γ-pyrone structure features a phenyl ring. In this configuration the benzene and phenyl rings are referred to as the A ring and B ring respectively while the oxygen containing γ-pyrone ring is classified as the C ring⁶.

The main subcategories consist of flavones, flavonols, flavanones, flavanonols, flavan-3-ols, anthocyanins, isoflavones and chalcones. Each category of bioflavonoids fulfills a unique biochemical role and demonstrates a specific distribution among plant species⁷. The word Quercetin comes from the Latin term “Quercetum”, it falls under the category of flavonols, which are not synthesized by the human organism⁸.Quercetin is a yellow compound that exhibits low solubility it is soluble in alcohol and lipids but remains insoluble in cold water. It is recognized as one of the most commonly utilized bioflavonoids for addressing metabolic and inflammatory conditions ⁹. Rutin is a type of flavonols often referred to by several names including rutoside, sophorin, vitamin P, or quercetin-3-O-rutinoside (Fig. 2.)¹⁰. Rutin being a lipophilic compound is soluble in organic solvents additionally it exhibits poor stability and bioavailability primarily attributable to its low solubility in water¹¹. Phytochemical screening analysis of Senna auriculata (L.) Roxb. shade dried flowers extract identified the existence of different categories of secondary plant metabolites including flavonoids (Shinoda test), alkaloids (Dragendroff’s test), phenolic compounds (Gelatin test), proteins and amino acids (Millon’s test), tannins (Braymer’s test), saponins (Foam test), glycosides (Legal’s test)¹². HPTLC is a widely utilized technology for the identification and analysis of the stability of herbal raw materials and formulations. Its minimal solvent usage, cost-effectiveness and shorter analysis time are making the HPTLC method a promising analytical tool¹³. As per the reviewed literature the identification, quantification and estimation of flavonoids in HPTLC analytical technique by using different crude extract. This study develops a High Performance Thin Layer Chromatography profile for flavonoids, identifying quercetin in Senna auriculata flowers revealing its antioxidant potential and suggesting further exploration of its chemical composition and medicinal use¹⁴. Cassia auriculata shows significant antioxidant activity in leaves and twigs with quercetin presence confirmed through HPTLC analysis¹⁵.  This study analyzed flavonoids, phenolic acids and xanthones in six herbal materials using HPTLC. Quercetin was found in varying concentrations except Cassia lanceolate establishing a reliable methodology for routine phytochemical analysis¹⁶. HPTLC method validates emodin and quercetin quantification in Cassia fistula and Cassia tora identifying optimal solvent systems with high linearity and low detection limits¹⁷. Study presents a HPTLC method to quantify quercetin in Michelia champaca flowers revealing its antioxidant potential and benefits for herbal pharmaceutical quality control and standardization¹⁸. Research developed an HPTLC method to quantify flavonoids in Cassia occidentalis demonstrating significant larvicidal activity against malaria vectors¹⁹. Study developed a phenolic fingerprint for differentiating genus Cassia L.sensu lato using HPTLC identifying compound concentrations notably higher Quercetin and Curcumin aiding taxonomic classification and chemotaxonomic insights^20. Study identifies 19 flavonoids in Aerva lanata using HPTLC emphasizing its medicinal potential especially against cancer and heart issues while supporting quality assurance in herbal products²¹. Study presents a validated HPTLC method for quantifying rutin, quercetin and gallic acid in Pterospermum acerifolium extracts showing excellent linearity and high reliability for quality control²². Study analyzes the phenolic content, rutin levels and bioactive properties of methanolic extracts from Verbesina Sphaerocephala leaves and flowers. HPTLC confirmed rutin presence with total phenolic content varying and significant antioxidant and antibacterial activities observed²³. Study investigates Dodonaea angustifolia using HPTLC and ultrasound assisted methanol extraction to quantify flavonoids and phenolic acids in leaves and flowers. Strong antibacterial activity was observed²⁴. Research examines HPTLC to analyze flavonoid content in safflower varieties identifying APRR3 as the safest and most bioactive emphasizing the need for analytical assessment in herbal applications²⁵. Assessment considers analyzes flavonoids in Cyperus rotundus using TLC and HPTLC identifying quercetin, establishing essential metrics for assessing its medicinal quality²⁶. Inquiry delves into  uses HPTLC to quantify quercetin and rutin in Melia azedarach extracts validating HPTLC for phytochemical assessment²⁷. This study develops a validated HPTLC method for rutin quantification in Morus alba, Morus nigra and Morus indica leaves achieving for quality assurance²⁸. This study establishes HPTLC technique for assessing rutin, quercetin and liquiritin in Cocculus hirsutus leaves achieving high accuracy and precision for quality assurance in herbal products²⁹.Study developed HPTLC method for quantifying rutin and quercetin in Ocimum basilicum seeds achieving effective separation and good precision with low detection limits³⁰. Research developed HPTLC method to quantify gallic acid rutin and quercetin in Terminalia chebula revealing significant phytochemical properties³¹. A validated HPTLC technique measures flavonoids rutin and quercetin in Anogeissus latifolia bark achieving ideal separation with high linear regression coefficients and specific concentrations³². Quercetin and rutin content in Benincasa hispida seeds and Carissa congesta roots using HPTLC and HPLC, revealing significant levels and highlighting their ethnopharmacological importance³³. Analyzes six Senna species using HPTLC revealing variations in chrysophanol levels. Morphological and anatomical assessments aid species identification ensuring accuracy in herbal medicine³⁴. HPTLC to analyze flavonoids in Syzygium cumini leaves finding quercetin, rutin and others. Ethanol extract showed high Quercetin content and significant antimicrobial effects against various pathogens³⁵. HPTLC and HPLC to analyze flavonoid content in Capparis moonii extracts revealing notable immune modulating effects but limited anticancer activity³⁶. Introduces a validated HPTLC method for quantifying berberine and rutin in Tinospora cordifolia extracts and formulations confirming specificity, linearity and accuracy with notable antioxidant activity³⁷. HPTLC method for rutin quantification in Hibiscus micranthus, achieving accurate results rutin content with excellent precision and reliability³⁸. An advanced HPTLC method was developed for simultaneous quantification of quercetin and rutin in Punica granatum, Tamarindus indica and Prunus domestica. Technique is efficient, accurate, cost effective and meets ICH validation standards ensuring reliable assessment of bioactive flavonoids³⁹. HPTLC to quantify flavonoid rutin in Verbesina sphaerocephala revealing high phenolic content, strong antioxidant potential and antibacterial activity, supporting its ethnopharmacological value⁴⁰. Compares HPTLC and spectrophotometry for rutin measurement in Amaryllis belladonna L. confirming HPTLC’s superior accuracy, efficiency and lower solvent use for bioactive compound analysis⁴¹. Developed a cost effective HPTLC method for analyzing flavonoids in eight Indocalamus species revealing six key markers and specific totals⁴². Flavonoids and phenolic acids in seven Croatian Stachys taxa using HPTLC. Notable include chlorogenic acid in six taxa and the highest flavonoid content in S. recta subsp. recta⁴³. Flavonoids in bamboo leaf extract offer health benefits. A precise HPTLC method measures vitexin and related compounds in Phyllostachys and Pleioblastus species differing significantly in flavonoid concentrations⁴⁴. Examines HPTLC techniques to evaluate flavonoids in Cyclanthera pedata extracts revealing significant antioxidant activity particularly in leaf extracts compared to fruits⁴⁵. HPTLC method for flavonoid analysis, detecting apigenin, quercetin,rutin and luteolin in medicinal plants like Bauhinia variegate and Ginkgo biloba with high accuracy and efficiency⁴⁶. Quantitatively evaluated flavonoids from Satureja hortensis L. using HPTLC with Reflux extraction with ethanol yielded highest levels of rosmarinic acid and luteolin⁴⁷. Research combines HPTLC with MALDI-TOF MS to effectively identify flavonoids like rutin and luteolin, enhancing resolution and mass identification for complex phytochemical analysis⁴⁸. Developed a novel method using HPTLC and multivariate image analysis to differentiate Apiaceae species analyzing samples and enhancing quality control in herbal drugs through flavonoid measurement⁴⁹. Enhances methods for separating phenolic compounds notably flavonoids, using RP-HPLC and NP-HPTLC, improving identification in herbal extracts like Polygonum species⁵⁰. Employs HPTLC and ATR-FTIR to analyze flavonoids and polyphenolics in Olive leaf extracts revealing enhanced extraction through fermentation, improving antioxidant activity⁵¹. Clinacanthus nutans highlights antiviral and anti-inflammatory properties. An analytical technique using HPTLC and HPLC-UV/DAD identified key flavones. Prominent indicator for quality control in herbal products⁵². A rapid UPLC method analyzes five indole alkaloids and four flavonoids from Passiflora species in eight minutes providing high sensitivity and improved authentication for supplements with HPTLC supporting quality assurance⁵³. Research employed HPTLC and HPLC-DAD-MS to identify flavonoids and phenolic compounds in extracts, caffeoyl glucaric acids and consistent results for Galinsoga parviflora and Galinsoga⁵⁴. Study employs HPTLC, HPLC and GC-MS to analyze flavonoids in Morinda tinctoria leaves identifying rutin and scopoletin. Results confirm significant flavonoid content supporting its pharmacological potential⁵⁵. HPTLC, HPLC-MS and NMR to analyze flavonoids in Cymbopogon giganteus identifying epicatechin, luteolin 8-C-glucosideand luteolin 6-C-glucoside. Results confirm high flavonoid content⁵⁶. Develops a method combining HPLC, ESI-MS and HPTLC to distinguish Dendrobium officinale from D.devonianum identifying flavonoids for accurate differentiation essential for consumer safety⁵⁷. Study uses HPTLC-DPPH to isolate a novel flavonoid from Abrus precatorius leaves showing strong antioxidant activity⁵⁸. Antioxidant properties of Asparagus racemosus aerial segments through HPTLC and ATR-FTIR high phenolic and flavonoid content in methanolic extracts, significant DPPH scavenging activity and identification of beneficial compounds⁵⁹. This analytical study investigates the Bioactive compound profile of Senna auriculata methanolic flower extract, with focus on quantification of the flavonoids especially rutin and quercetin using densitometry analysis employing High Performance Thin Layer Chromatography (HPTLC)⁶⁰. The concentrations of these flavonoids in the extract will be measured and compared with standard references to confirm their presence and assess their potential for future non rinse radiation-induced skin protection herbal formulation sheet mask in the International Space Station (ISS). Additionally, the study aims to identify and characterize the chemical constituents of the extract through Fourier Transform Infrared Spectroscopy (FTIR), enabling detailed elucidation of its phytochemical profile⁶¹. 

Materials and Methods

Plant Collection and Authentication

Fresh flowers of Senna auriculata were systematically collected from Chengalpattu district in December month. The botanical identity of the flower was confirmed through Identification of crude drug –Macroscopic study of the coded Drug examination authenticated and officially certified by the Captain Srinivasa murthy Central Ayurveda Research Institute, Anna Nagar, Chennai, Tamil Nadu, India. A voucher specimen (No.2502005) has been archived for future reference.

Chemicals and Solvents

Rutin reference standard (90-101%) was obtained from HIMedia Laboratories Pvt. Ltd., Maharashtra, India. Quercetin reference standard (95%) was obtained from LOBA CHEMIE Pvt. Ltd., Mumbai, Maharashtra. Solvent for mobile phase used in the developed were toluene, methanol, ethyl acetate, formic acid all of HPLC grade quality. HPTLC plate used; E.Merck KGaA silica gel 60 F254.

Instruments

HPTLC instruments of Aetron AE260321 include an automatic syringe sample applicator with a 10ml capacity in Glass spraylin HPTLC – Thin Layer Chromatography software. Photo documentation was made using Aetron IDS, quantification using just TLC software.

Preparation of Senna auriculata Flower Extract

Senna auriculata flowers are thoroughly rinsed with tap water and then exposed to drying in a shaded environment at ambient temperature for a duration 15 days. The dried flowers are grind using an electrical blender equipped with a 40-mesh sieve to obtain a fine powder. From this powder,10g are taken and dissolved in 100ml of HPLC-grade methanol. The maceration extractive technique is employed in this process. On the third day the sample undergoes purification using Whatman Grade 1 filter paper. The filtered extract is then transferred into a Petri dish to undergo open evaporation for one day. After this period, a solid residue is obtained for further use.

Preliminary Phytochemical Test for Senna auriculata Flower Extract

Initial phytochemical analysis performed according to Indian pharmacopoeia procedure the liquid extract from the flowers of Senna auriculata demonstrated the presence of flavonoids, alkaloids, phenolic compounds, proteins and amino acids, saponins, tannins and glycosides (Table 4.).

Preparation of Standard Stock Solutions

Standard stock solutions were constituted by dissolving 10.2mg of Rutin and 10.8mg of Quercetin in 50mL of methanol. The final concentrations obtained were 0.204mg/ mL for Rutin and 0.216mg/ mL for Quercetin.    

Preparation of Sample Stock Solution

1.003g of Senna auriculata flower solid extract was dissolved in 10mL HPLC grade methanol. Then 1.5mL of this was diluted to 10mL, yielding a working concentration of 15.046mg/mL for HPTLC Densitometry analysis. 

Method Development

The employed HPTLC plates were HPTLC silica gel 60 F254 (E. Merck KGaA) having 0.2mm layer thickness and 10x 10cm in plates size; The standard and sample solutions of Rutin and Quercetin were applied to the HPTLC plates; Each at 10 μl. The compositure of the overall mobile phase that was applied in gas chromatography was toluene, ethyl acetate and formic acid, 25% each for the formic and 20% respectively for the two and 1% in volumes for both detection types. The analysis was done in a twin trough glass chamber 10×10 cm pre saturated with the mobile phase and maintained for 20min with the moving front up to 85 mm.  They were then left in the air to dry for a period of forty – five minutes after going through the development stage.  At 254 nm detection, eight bands were distinguished with the following values: 3-4 = 82px, 5-6 = 84px and for the flower extract of the Senna auriculata 5-6 = 80px, across the total length of 190mm (Fig. 3, Fig. 4, table 1.). The standard Rf values recorded for Rutin at 254 nm wavelength was 0.014 and for Quercetin 0.385 and the sample Rf value for the Senna auriculata extract was 0.032.  Under 366 nm detection, six bands were recorded, with band widths of 81 px for Rutin, 75 px for Quercetin, and 65 px for the Senna auriculata extract, maintaining the same band length of 190 mm. The corresponding standard Rf values at 366 nm were 0.017 for Rutin and 0.388 for Quercetin, whereas the sample Rf value was 0.026. These optimized chromatographic conditions enabled efficient separation and detection of the bioactive constituents present in the Senna auriculata flower extract at both detection wavelengths. 

Figure 1: Senna auriculata (L.) Roxb.syn. flower Click here to View Figure

Figure 2: Chemical structure of Rutin Click here to View Figure

Figure 3: HPTLC plate at 254nm showing visible bands for Rutin (Lane 1), Quercetin (Lane 2) and the Senna auriculata flower extract (Lane 3), Click here to View Figure

Figure 4: The HPTLC Densitometric Chromatogram recorded at 254nm showing the lane profiles of Standard Rutin (Lane 1), Standard Quercetin (Lane 2), and the Senna auriculata flower extract (Lane3). Click here to View Figure

Lanes

 Bands

Table 1: Densitometric analysis at 254nm revealed bands corresponding to Rutin in the herbal extract,confirmed by matching Rf values and peak profiles with those of standard compounds

Figure 5: HPTLC plate at 366nm showing fluorescent bands for Rutin (Lane 1), Quercetin (Lane 2), and the Senna auriculata flower extract (Lane 3). Click here to View Figure

Figure 6: HPTLC Densitometric Chromatogram captured at 366nm illustrating the lane profiles of standard Rutin (Lane 1), Standard Quercetin (Lane 2) and the Senna auriculata flower extract (Lane 3). Click here to View Figure

Lanes

Bands

Table 2: Densitometric analysis at 366nm the flower extract demonstrated fluorescent bands corresponding to those of the Rutin standard, suggesting its presence based on closely aligned Rf values and fluorescence intensities

FTIR Spectrum for Identification and Characterization of Phytoconstituents in Senna auriculata

Preparation of Senna auriculata Flower Extract

Senna auriculata flowers are thoroughly rinsed with tap water and then exposed to drying in a shaded environment at ambient temperature for a duration 15 days. The dried flowers are grind using an electrical blender equipped with a 40-mesh sieve to obtain a fine powder. From this powder,10g are taken and dissolved in 100ml of HPLC-grade methanol. The maceration extractive technique is employed in this process. On the third day the sample undergoes purification using Whatman Grade 1 filter paper. The filtered extract is then transferred into a Petri dish to undergo open evaporation for one day. After this period, a solid residue is obtained for further use.

Instrument

Spectrum 2 Type: ATR-FTIR Spectrometer Service Spectrometer Make: PerkinElmer Location: USA.

FTIR Analysis working Procedure

The solid extract of Senna auriculata flowers was analyzed using an ATR-FTIR spectrometer. The powder sample was placed and pressed on the ATR crystal and spectra was recorded from 4000 to 450cm^-1with a resolution of 4cm^-1 and 32 scans. Absorption bands appeared at 3270.78 cm^-1 (O-H), 2924.00 and 2853.37 cm^-1 (C-H) ,1611.62 and 1505.51cm^-1 (C=C) ,1448.44 cm^-1 (CH₂),1226.57-1068.10cm^-1 (C-O/C-O-C),967.30cm^-1 (=C-H),834.53 cm^-1 (C-H bending), and 499.00cm^-1 (C-Br). These peaks indicate the presence of phenolic, aromatic, ether these functional groups (Fig. 7. and Table 3.).

Figure 7: FTIR spectrum of Senna auriculata shows prominent peaks confirmed by Phenols or Alcohols, Alkanes, Aromatic compounds, and Ethers functional group vibrations. Click here to View Figure

Table 3: FTIR peak table of Senna auriculata identifies functional groups indicating the presence of phenols, alcohols, alkanes, aromatic compounds, ethers, alkenes and halides reflecting a rich phytochemical composition

 Qualitative Phytochemical Profiling of Senna auriculata Flower Extract

Qualitative Phytochemical analysis was conducted following the established standard protocols of Trease and Evans (1989) (Table 4.).

Table 4: Preliminary Phytochemical Analysis of Senna auriculata Flower extract Click here to View Table

Results and Discussion

This work describes the optimized HPTLC-Densitometry method for the qualitative analysis of the extract of Senna auriculata flower and consists Toluene: Ethyl acetate: Formic acid (25: 20: 1) is used on the plates silica gel 60 F 254 and the detection was done at 254 nm and also at 366 nm. The Rf values for the standard Rutin and Quercetin were determined as 14 and 385 at 254nm and 17 and 388 at 366nm respectively.  Therefore, the Senna auriculata flower extract has provided the Rf values of 32 at 254nm and 26 at 366nm almost close to the standard Rutin is indicating the presence of Rutin in the extract.  The selected mobile phase toluene, ethyl acetate and formic acid were found to be effective as far as resolution and separation of phytoconstituents is concerned. Total Rutin concentration of the extract was analysed using densitometry quantification based on the ration of the sample ‘peak area’ with the standard ‘peak area’ (Formulas 3 & 4) The functional groups of hydroxyl -OH, aromatic C=C, and ether C-O-C were detected in the extract based on the characteristic IR peaks of Rutin. 

Conclusion

The study successfully developed a dual- analytical approach combining HPTLC densitometry with FTIR spectroscopy for the quantification and characterization of Rutin in Senna auriculata flower extract. The HPTLC method exhibited high specificity, precision and reproducibility under dual wavelength detection enabling accurate quantification of flavonoid. FTIR analysis confirmed the presence of functional groups associated with flavonoid structures, supporting compound integrity and identity. This integrated cost effective and time efficient method provides a reliable phytochemical standardization and quality control indicates strong potential for future non rinse radiation-induced skin protection herbal formulation sheet mask during space missions particularly on the International space station (ISS). 

Acknowledgement

The authors would like to deeply thank writers VISTAS – Vels Institute of Science, Technology & Advanced Studies (VISTAS) old pallavaram Chennai-600117, Tamilnadu, India for their unending support for all the facilities and ideas provided, which have had a direct effect in developing the current work.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval. 

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