Rapid Screening of Adulterants of Mesua ferrea L. by FTIR and GC-MS Techniques

Medicinal plants have long been utilized for their unique phytoconstituents, offering therapeutic benefits and promoting health[1]. Mesua, a substantial genus encompassing approximately 48 species, with Mesua ferrea L. being the most extensively studied, stands out as an epitome. This species is endangered in India, native to tropical Sri Lanka, and holds the official status of being the tree of Tripura. Commonly known as Cobra's saffron in English and Nagakeshara in Hindi, it continues to be a subject of significant exploration and interest [2]. In Ayurveda, 'Nagkesar' holds a prominent position as a crucial medicinal remedy, with Mesua ferrea L.'s stamen being its authentic source. The therapeutic effects of M. ferrea L. stamens are evident by the presence of steroids, terpenoids, and volatile oil components. These stamens demonstrate notable bactericidal and antioxidant activities [3]. Despite these benefits, market samples often suffer from adulteration, with unripe fruits of Cinnamomum tamala T.Nees & Eberm. and fruits of Dillenia pentagyna   Roxb. being falsely presented as 'Kala nagkesar' and 'Malabar nagkesar' respectively. 'Kala nagkesar' are sold in the markets of Gujrat and Bombay whereas 'Malabar nagkesar’ in South India. The substituted products lack any connection to the genuine drug and may or may not contain essential phytoconstituents  [4], [5]Thus, the development of biomarkers becomes crucial for distinguishing between authentic and adulterated plant samples. The objective of this present study was to develop FTIR and GC-MS fingerprints of Mesua. ferrea L., Cinnamomum tamala T. Nees & Eberm.  and Dillenia pentagyna Roxb. This approach seeks to distinguish authentic Mesua ferrea L. from its adulterants, ensuring the integrity and quality of this valuable medicinal plant in the market.

MATERIALS AND METHODS

Collection and authentication of plant materials

The blooms of Mesua ferrea L. were gathered in February from Veermata Jijabai Bhosale Udyan in Mumbai. Additionally, the immature fruits of Cinnamomum tamala T. Nees & Eberm.  and the fruits of Dillenia pentagyna Roxb. were acquired in June from a botanical garden in Dahanu (Palghar, Maharashtra), and Dapoli Urban Bank Senior Science College (Jalgaon, Maharashtra) respectively. The procured plant sample Mesua ferrea L. was identified and authenticated at Blatter Herbarium, St. Xavier's College, Fort, Mumbai. The voucher specimen number given was NDG-2259.

Preparation of sample powder

The flowers of Mesua ferrea L., unripe fruits of Cinnamomum tamala T. Nees & Eberm., and fruits of Dillenia pentagyna Roxb. were air-dried in the shade for one week. Later, the stamens from Mesua ferrea L. flowers were separated and ground into powder using a mixer blender. The shade-dried unripe fruits of Cinnamomum tamala T. Nees & Eberm.  and fruits of Dillenia pentagyna Roxb. were also mechanically ground.

FTIR Spectrum Analysis

To identify the characteristic functional groups present, the powdered plant samples underwent Fourier transform infrared (FTIR) spectrum analysis. This analysis was conducted at the Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Mumbai. The FTIR spectra were generated using the Bruker, Germany Vertex 80 FTIR System with a 3000 Hyperion Microscope. The samples were scanned across the range of 4000 to 440 cm-1, and the recorded peak values of the plant materials were documented.

GC-MS analysis

The GC-MS analysis of the soxhleted methanolic extract derived from the stamens of Mesua ferrea L., unripe fruits of Cinnamomum tamala T. Nees & Eberm., and fruits of Dillenia pentagyna Roxb. was conducted at the Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Powai, Mumbai. This analytical process aims to examine the presence of active constituents and the chemical composition of the samples. The equipment utilized for this analysis included the Agilent 7890 GC instrument and the Joel Accu TOF GCV MS instrument. The solvent mixture employed consisted of toluene, chloroform, ethanol, and ethyl acetate. Helium gas (99.999%) served as the carrier gas with a flow rate of 1 ml/min. The analytical setup featured an HP5 column with specifications including a length of 30 mm, internal diameter of 0.32 mm, film thickness of 0.25 mm, and a temperature range from -60 °C to 325 °C (350 °C). The GC runtime was set at 35 minutes, during which the oven temperature increased from 70 °C to 280 °C at a rate of 8 °C per minute. A sample size of 4 µl was injected through the injector. The MS was performed at 70eV. Compound identification was achieved by comparing the spectrum of unknown compounds with the spectrum of known compounds in the library, enabling the determination of their names, molecular weights, and structures.

RESULTS

FTIR spectrum analysis

The powdered stamens of Mesua ferrea L. exhibited 11 distinct functional groups, as indicated by the FTIR spectrum. Table 1 and Figure 1 illustrate the FTIR spectrum specific to M. ferrea L. stamens. The absorption peak at 3416.73 cm^-1 corresponds to the stretching vibration of the H-bonded and O-H groups. Additionally, the peaks at 2926.42 and 2854.43 cm^-1 signify the asymmetric and symmetric stretching of saturated (sp3) carbon, respectively. The band at 1729.48 cm^-1 is attributed to C=O stretching related to the carbonyl skeletal mode in the plant sample. Various other groups, including aromatic compounds, phenols or tertiary alcohols, acids, alkenes, alkyl halides, phosphate ions, and halogen compounds, exhibit absorption at 15252.27, 1445.81, 1376.38, 1341.51, 1284.42, 1244.89, 1656.27, 823.48, 779.32, 702.18, 1158.29, 1101.79, 614.29, 559.31, and 518.47 cm^-1, respectively. The identification of the functional group in unripe fruits of Cinnamomum tamala T. Nees & Eberm.  relied on the analysis of the FTIR spectrum, focusing on specific peak values within the infrared radiation range. Figure 2 and Table 2 present the FTIR spectrum for unripe fruits of Cinnamomum tamala T. Nees & Eberm... A prominent absorption band at 3433.07 cm^-1 indicated the presence of bonded H/O-H stretch. The C-H asymmetrical stretching methylene group was observed at 2922.86 cm^-1, while the band at 2852.33 cm^-1 represented the C-H symmetric stretching of methylene groups in aliphatic compounds. The absorbance at 1639.64 cm^-1 signified the existence of carboxyl compounds (C=O stretch). Other observed bands included 1459.37 cm^-1 (C=C stretch), 1381.85 cm^-1 (O-H bend), and 1048.78 cm^-1 (PO³ stretch). FTIR spectroscopy was employed to analyze the functional groups present in the powdered fruits of Dillenia pentagyna Roxb. (Figure 3 and Table 3), with the separation of these groups based on peak values. An expansive absorption band at 3734.82 and 3430.78 cm^-1 signifies the presence of non-bonded, O-H stretch and H-bonded, O-H stretch respectively. A narrower band at 2924.6 cm^-1 indicates asymmetric stretch and at 2853.26 cm^-1 shows symmetric stretch in saturated aliphatic compounds. A strong intensity band at 1640.88 cm^-1 reveals a C=O stretch, indicating the presence of a ketone compound. Peaks at 1373.99, 1321.99, and 1230.32 cm^-1 are assigned to the O-H bend and C-O stretch, respectively. The absorption band at 1108.28 cm^-1 is attributed to amine and alkyl halide. An intense band at 1051.08 cm^-1 corresponds to PO³ stretch vibration, indicating the presence of phosphate ions. Multiple bands at 884.31, 829.35, 783.35 cm^-1 confirm the presence of alkenes. Peaks at 607.03 and 522.90 cm^-1 are associated with halogen compounds.

Table 1: Structural features of the M. ferrea L. stamens by FTIR spectrum

Figure 1: FTIR Spectrum analysis of stamens of Mesua ferrea L.

Table 2: Structural features of the C. tamala T. Nees & Eberm. unripe fruits by FTIR spectrum

Figure 1: FTIR Spectrum analysis of unripe fruits of Cinnamomum tamala T. Nees & Eberm.

Table 3:  Structural features of the D. pentagyna Roxb. fruits by FTIR spectrum

Figure 2: FTIR Spectrum analysis of fruits of Dillenia pentagyna Roxb.

GC-MS analysis

The bioactive constituents found in the soxhleted methanolic extract of Mesua ferrea L. stamens, unripe fruits of Cinnamomum tamala T. Nees & Eberm., and fruits of Dillenia pentagyna Roxb. are detailed in Tables 5-7, listing their names, molecular formulas, retention times, and quantities. Mesua ferrea L. stamens, upon GC-MS analysis, revealed the presence of 12 compounds (table 4 and figure 4), with 3-Furanacetic acid, 4-hexyl-2,5-dihydro-2,5-dioxo (28.192%), Spathulenol (18.958%), and 2- [4-methyl-6- [2,6,6-trimethylcyclohex-1-enylhexa-1,3,5-trienylcyclohex-1-en-1-carboxaldehyde (12.670%) identified as the top three major compounds. The GC-MS chromatogram of C. tamala T. Nees & Eberm.  unripe fruits (figure 5) exhibited 8 peaks, representing 8 compounds (table 5), with Palmitin,1-mono (57.468%), Stearin,2-mono (34.529%), and Anodendroside E 2 (4.618%) as the top three major compounds. Methanolic extract of D. pentagyna Roxb. fruits revealed 4 compounds (table 6 and figure 6), highlighting (E)-13-Docosenoic acid (86.530%) followed by 15-Tetracosenoic acid, methyl ester, (Z)- (12.614%) as major components. Five specific biologically active compounds, namely 2-Propanone, 1-hydroxy (C₃H₆O₂), Hexanoicacid,2-methyl(C₇H₁₄O₂), 1b,4a-Epoxy-2H-cyclopenta[3,4]cyclopropa[8,9] cycloundec[1,2b]oxiren5(1aH0one,2,7,9,19tetrakis(acetyloxy)decahydro-3,6,8,8,10a pentamethyl (C₂₈H₃₈O₁₁), Furanacetic acid, 4-hexyl-2,5-dihydro-2,5-dioxo (C₁₂H₁₆O₅), Formic acid, 3,7,11-trimethyl-1,6,10-dodecatrien-3-yl ester (C₁₆H₂₆O₂), are identified as potential biomarkers. These compounds are present in the authentic drug (Mesua ferrea L. stamens) but absent in the adulterants (Cinnamomum tamala T. Nees & Eberm.  unripe fruits and Dillenia pentagyna Roxb. fruits), suggesting their utility in detecting adulteration. Notably, the adulterants contain numerous other biologically active compounds like flavonoids and phenolic acids, indicating their potential use as distinct drugs.

Table 4: Biologically active chemical compounds of methanol extract from M. ferrea L. stamens.

Figure 3:  GC-MS chromatogram of stamens of Mesua ferrea Linn.

Table 5: Biologically active chemical compounds of methanol extract from C. tamala T. Nees & Eberm. unripe fruits

Figure 4: GC-MS chromatogram of unripe fruits of Cinnamomum tamala T. Nees & Eberm.

Table 6: Biologically active chemical compounds of methanol extract from D. pentagyna Roxb. fruits

Figure 5: GC-MS chromatogram of fruits of Dillenia pentagyna Roxb.