Analytical Profiling of Saffron (Crocus sativus) using 1H-NMR and FTIR based Metabolomics approach and UV-Vis, HPTLC and TLC Chromatography Fingerprinting

Manas Ranjan Sahoo¹*, Ramesh R. varrier¹, Anithakumari R², Guruvaurappan Palanichamy³, Bala Tirupura Sundari H¹, Bala Guru¹

¹AVN Ayurveda Formulation Pvt Ltd, Quality Control, Madurai, Tamil Nadu, India, 625004.

²AVN Ayurveda Formulation Pvt Ltd, Research and Department, Madurai, Tamil Nadu, India, 625004.

³AVN Ayurveda Formulation Pvt Ltd, Microbiology Department, Madurai, Tamil Nadu, India, 625004.

*Corresponding Author E-mail: manasranjansahoo09@yahoo.com, manas@avnayurveda.in

ABSTRACT:

Crocus sativus L. commonly known as saffron or Kesar in India, is an important medicinal herb in Ayurveda and has been traditionally used for treatment of neurological disorders, for depression, anxiety and sleep disorders. It is uses as a coloring and flavouring agent in the preparation of various foods. Modern research has high lighted its beneficial effects in treatment of cardiovascular action, diabetic cataract, and as a potent antiinflammatory herb. Due to its high cost its quality control is of utmost importance to ensure its authenticity, purity and its medicinal properties. In the preset study the we have used Thin Layer Chromatography (TLC) analysis, High performance thin layer chromatography (HPTLC) fingerprint, UV-Vis spectrophotometric analysis, ¹H nuclear magnetic resonance (¹H NMR) and Fourier transform infrared spectroscopy (FTIR) based metabolomic study for quality control and characterization of saffron. The antibacterial and antifungal activity were evaluated using agar well-diffusion method in two pathogenic bacterial strains, Escherichia coli, Staphylococcus aureus and two pathogenic fungal strains, Candida albicans, and Aspergillus brasiliensis. The ¹H NMR spectroscopy couples with FTIR analysis leads to identifcation of the secondary metabolites of saffron like crocetin, picrocrocin and safranal on basis of reported diagonostic signals and peaks. The antimicrobial activity showed moderate antibacterial and antifungal activity. The TLC and HPTLC profile reveals the characteristic fingerprint. Overall the present study showed that the ¹H-NMR, FTIR based metabolomics approach and TLC and HPTLC metabolite profiling can be powerful strategy for maintaining the holistic quality of the saffron.

KEYWORDS: Crocus sativus, Quality Control, FTIR, ¹H-NMR, HPTLC, TLC, UV-Vis spectroscopy.

INTRODUCTION:

Saffron (Crocus sativus L.) commonly known as kesar in India is the most expensive and a very profitable spice in the world wide. The herb belongs to the family of Iridaceae and propagated through the vegetative method by the underground part corm of the plant. Saffron is obtained from the long and dried red colored stigma of the flower having its characteristic bitter taste and aromatic odour. It is mainly used as a flavouring and coloring ingredient in various food preparations. It is used as a nutraceutical, herbal-medicine and cosmetic applications for their various biological activities ^1-2. It is used in various traditional medicines like Ayurveda, Unani, Persian and Chinese medicine system. It is cultivated in India, Iran, Afghanistan, Italy, Morocco, Turkey, Spain and Greece and the Iran is the largest producer and supplier of saffron of approx 90% of world supply³. Various traditional uses of saffron are sedative, treatment of asthma, aphrodisiac, in treatment of anxiety, and immunity enhancer¹. It is reported for various pharmacological properties like neuroprotective, for treatment of alzheimer’s disease, anti-microbial, anti-inflammatory, antihypertensive, relaxant, antitussive, hypolipidemic, anticonvulsant, analgesic, nervous disorders like depressant, anxiolytic and sleep disturbance, cardio protective, for improvement of cognition, anticancer, and antioxidant activity, for prevent of eye cataract^4-8. Various phytochemicals have been reported from the saffron such as carotenoids, terpenoids, flavonoids, terpenoids, anthocyanins, vitamins such as riboflavin, thiamine, proteins, starch, amino acids. The flavonoids like rutin, kaempferol and quercetin, luteolin, and hesperidin. The major phytochemicals like crocins (a groups of glycosides of C₂₀ carotenoid called crocetin [C₂₀H₂₄O₄], with sugar substitutions like glucose and gentiobiose), picrocrocin (C₁₆H₂₆O₇-glucopyranosyl glycoside of monoterpenoid safranal) and safranal (C₁₀H₁₄O) a monoterpenoid aldehyde are responsible for characteristic color, bitter taste and aromatic odour of saffron respectively. The crocin is a water soluble glycoside that imparts the orange-yellow color to saffron when added in water^9-10.

Quality control of herbal drugs, raw materials are essential part of the herbal medicine development process for ensuring the therapeutic efficacy and consistency of the products. Various analytical techniques that are used in the quality control and pharmaceutical analysis of the products are preliminary phytochemical analysis, TLC, HPTLC, ¹H-NMR, FTIR, and UV-Vis spectroscopy methods^11-16. Chromatography techniques like TLC and HPTLC analysis has been found to be immensely useful for development of characteristic fingerprint of various herbal drugs and analysis of the corresponding finished drugs. These methods can be used for qualitative analysis as well as the quantification of characteristic phytochemical marker or bio-active marker compounds in the herbal materials or finished formulations^17-25. Due to enormous application of saffron in pharmaceutical products, Natural Health Products, Dietary supplements, food, nutraceutical and various cosmetic application quality control is indispensable to ensure its desired properties.

Various metabolomics approach has been used for authentication of quality control of saffron like DNA barcoding, transcriptomics and metaboomics. ¹H NMR is an exclusive technique used in structure elucidation of organic molecules in pharmaceutical chemistry and the phytochemicals. From the ¹H NMR metabolomics the structural information of components from a complex mixture can be obtained without prior-isolation and chromatographic purification²⁶. In the present study chemical fingerprints of saffron extract was evaluated using FTIR and ¹H NMR and to identify the major compounds.

MATERIALS AND METHODS:

Saffron samples were procured from Suppliers in India and the samples was varified by the Botanist and a specimen samples was kept in the department harbarium for future reference. The samples were dried at ambient temperature and carefully stored in dark protected from light until analyzed. All the chemicals used in the experiment are of analytical grade. All the solvents used in the experiment were purchased from Qualigens. Napthol-Yellow was purchased from Loba-Chemie, Sudan Red-G was purchased from Tokyo chemical industries, 4-Methoxy Benzaldehyde was purchased from Avra synthesis pvt ltd.

Sample preparation:

Dried and powdered sample (500mg) of saffron (Crocos sativus L.) stigma was extracted with 10mL water and water: methanol (1:1) separately by maceration for 24 h at a room temperature. The soluble extract was filtered with whatman paper and dried in rotary evaporator at temperature of 40℃ under vacuum. The sample was used for NMR analysis and Thin layer chromatography (TLC) and High performance Thin layer chromatography (HPTLC).

Physicochemical analysis of saffron:

The dried saffron were used for quantitative determination of water soluble extractive value, alcohol soluble extractive value, total ash values as per the pharmacopoeia standard method mentioned in Ayurvedic Pharmacopoeia²⁷.

Spectrophotometric Analysis:

The UV-Vis analysis for saffron was done as per the reported method of ISO 3632 [13]. Aqueous extract of saffron is prepared using BIS method. Accurately weighed One gram of sample of saffron powder was transferred to 50ml of distilled water in a volumetric flask. It was extracted under sonication for 30 minutes and kept in dark for 24 hours. Then the clear supernatant of the saffron solution was diluted to 0.004% with with distilled water. UV-Vis Spectrum was recorded using with Shimadzu (UB-1700) UV-Vis spectrophotometer.

FTIR Spectroscopy:

The FTIR analysis was carried out using a fine powder of saffron. The FTIR spectra for powdered saffron was recorded with Shimadzu-FTIR-8400S using KBr pellets. FTIR measurements were performed in transmission mode. All spectra were recorded in the range between 4000 and 500cm^-1frequency region.

NMR Analysis:

The ¹H NMR spectra was taken for aqueous extracts extracts of saffron. The NMR analysis were carried out on a Bruker Avance III-600 spectrometer. The spectra was recorded using D2O as a deuterated solvents.

Evaluation of antifungal and antibacterial activity of saffron extract:

The standard cultures of gram-negative Escherichia coli-ATCC-8739^TM, gram-positive Staphylococcus aureus-ATCC® 6538™, and fungus Candida albicans-ATCCC 10231 and Aspergillus brasiliensis-ATCC 16404 used in antimicrobial assay were purchased as from HiMedia. The bacteria and fungus culture was rejuvinated in Trypton Soya Broth by incubating at temperature of 37 ℃ for 18h and than stocked in soyabein caseine digest agar and sabouraud dextrose emmons agar respectively. The subculture was prepared from the stock for anti-microbial assay. The agar well-diffusion method was employed to determine antibacterial and antifungal activity of saffron extract. For antimicrobial activity 500 mg of saffron was extracted with aqueous medium under reflux. The concentration of saffron extract used in the assay was 100mg/mL in DMSO. The bacterial and fungus inoculum was spread over the nutrient-agar plate using a sterile cotton swab to provide an uniform microbial growth. The holes of 6mm diameter were made on the agar plates and 75ml of each of the saffron extract were added to wells in triplicate to evaluate antibacterial and anti-fungal activity. The plates were incubated for 48 hour at incubation temperature of 37℃. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone in millimeters using zone-scale.

RESULTS AND DISCUSSION:

UV-Vis Spectrophotometric analysis:

The UV-Vis spectra (Fig-1) of aqueous extract saffron showed showed three peaks at around 440nm that represents polyne-conjugation chain of crocin and crocetin and lesser intense peak at around 257 and 330 nm are attributed to picrocrocin and safranal respectively. The absorptions were as per the reported values in the literature^10,28-31.

Figure 1. UV–Vis spectra of aqueous extract of Saffron (Crocus sativus L.)

Physicochemical parameters and TLC analysis of Saffron:

Water soluble extractive (%w/w) value of saffron was found to be 69.99% and Alcohol soluble extractive value (%w/w) was found to be 51.85% for saffron. The TLC analysis was carried out to to detect presence of any synthetic orange and red color was mixed with the saffron. TLC (Fig-2) of saffron extract was run with napthol yellow (monosodium salt 2,4-dinitrophenol) and sudan red-G (2-hydroxy-[1-(2-methoxy) phenylazo]napthalene) as per the BIS test method mentioned [17-18]. The TLC was developed with the methanolic extract of saffron with the above reagent using mobile phase ethyl acetate: propanol: water (6.5:2.5:0.1). The TLC (Fig 2-I) showed absence of both napthol yellow and sudan red-G in the saffron. The TLC (Fig 2-I) showed three yellow color spots at bottom portion with lowest spot is the most deep yellow color attributed to crocin as per the reported literature. Both the coloring agents were having different Rf and was not matching with any of the bands from saffron extract. The TLC was derivatized with chromogenic reagent prepared by mixing 2.0 ml of 4-methoxybenzaldehyde with 18 ml of ethyl alcohol followed by mixing with 2.0 ml of sulphuric acid. After spraying with the reagent the TLC plate was heated at temperature of 110℃ for 5 to 10 minutes that showed greyish green color spots (Fig 2-II) attributed to crocin and violet color spots attributed to picrocrocin. Spraying with vanillin and anisaldehyde sulphuric acid showed grey color spots on the TLC as show in Fig-2 III and IV. The methanolic extract of saffron on reverse phase TLC (RP₁₈ TLC) showed characteristic yellow bands in white light. The chromatogram in show in the figure-2-V.

HPTLC Fingerprinting:

The high performance HPTLC analysis showed characteristic fingerprint of saffron. The chromatogram revealed three yellow bands at R_f0.11, 0.31 and 0.72 at 520nm, under 254nm it showed dark bands at Rf of 0.09, 0.20, 0.36, 0.52, and fluorescent spots under 366nm at Rf of 0.15, 0.23, 0.32, 0.35, 0.46, 0.56. The chromplate and the densitogram of the TLC is showed in below figure.

Figure 3: HPTLC fingerprint of saffron extracts

FT-IR analysis:

FTIR spectroscopy is non-invasive and can rapidly provide metabolite fingerprints that reveals reliable information on molecular structure and composition of herbal extracts. The FTIR analysis showed various stretching frequencies of distinct functional groups that represent presence of phytochemical profile of saffron. The FTIR spectra showed a broad band at frequency 3443.66 cm^-1due to stretching vibration of O-H bond from alcoholic functional group from crocin, at 2925.81 cm^-1, 2853.49 cm^-1is due to C-H stretching vibrations, 1658.67cm^-1 due to conjugated carbonyl (C=O) groups of picrococin, vibration at 1747.39 is due to carbonyl (C=O) groups of crocetin esters. Vibration frequency between 1500 to 1200 cm^-1is due to the presence of OH, C-C, C-O and C-C-O functional group for carbohydrate moiety of glycosides crocetin and picrocrocin. The frequency at 1227.61 cm^-1is due to C-O stretching of ester group (O=C-O) present in crocetin and its glycosides. Absorption at 1069.45 is due to C-O-C stretching of sugar rings and glycosides and the peaks between 900 to 700cm-1 (748.33cm^-1, 689.50 cm^-1) is due to C-H bending vibrations. All these peaks were supported by earlier literature evidence of FTIR spectral data of saffron³².

Figure 4: FT-IR Spectra of Saffron powder

¹H NMR analysis:

The NMR spectra of saffron water extract in D₂O is shown in figure-5. The spectra showed characteristic peaks. The characteristic NMR data is presented in the table-1. The NMR values were in accordance with that of the reported literature^33-35. The NMR shift value is mentioned below.

¹H-NMR (d) 9.85, 7.68 (d), 7.57, 7.26, 7.04, 5.10, 4.69, 4.52, 4.51, 4.282, 3.884, 3.867, 3.810, 3.780, 3.704, 3.606, 3.578, 3.364, 3.341, 3.292, 3.271, 3.231, 3.079, 2.298, 2.276, 2.249, 2.106, 2.022, 1.239, 1.105, 1.078, 0.840, 0.812, 0.805

Figure 5: ¹H NMR spectrum of aqueous extract of saffron (Crocus sativus L.) in D₂O

Antibacterial and antifungal activity of saffron extracts:

Both the antibacterial and antifungal activity was evaluated for aqueous extracts of saffron using agar well-diffusion method. In the 6mm well 75ml of saffron aqueous extract dissolved in DMSO was added. All the experiments were done in triplicate. After loading of the extracts the agar plates were incubated for 48 hours at incubation temperature of 37℃. The aqueous extract of saffron showed moderate antibacterial and antifungal activity. The zone of inhibition was calculated as mean of three zone measurement of agar plate for each of the organisms. The saffron extract showed clear zones on the agar plate indicating its antimicrobial activity against the tested pathogenic organism. The zone of inhibition was found to 16mm in E.coli, 12 mm in S.aureus, 10mm in A.brasilensis and 12 mm in C.albicans. The antibiogram of the antimicrobial assay is presented below. The saffron is a well known ingredients in many food preparation for their attractive color and flavours. So the moderate antibacterial and antifungal activity displayed above can act as a natural preservative for food preparations that extends the self life of the product up to some extent.

Figure 6: Antibiogram of saffron antimicrobial activity

CONCLUSION:

Our study established a multi-prong approach including TLC analysis, HPTLC analysis, ¹H-NMR, FTIR spectroscopy techniques for quality assessment of saffron. Characteristic HPTLC fingerprint, FTIR spectra and NMR spectra were gained. TLC was found to be a rapid and simple approach method for detection of synthetic color adulteration in saffron. The saffron extracts showed moderate anti-bacterial and anti-fungal activity, that supports the use of saffron in various traditional skin care application. ¹H-NMR and FTIR spectroscopic analysis of saffron samples led to rapid identifcation of secondary metabolites in the crude extract of saffron without going for time consuming chromatography purification that involves many steps. As the herbal extracts are rich of several secondary metabolites NMR and FTIR based maetabolomics can be used as a powerful tool for quality assessment of herbal samples. The strategy proposed in this study provides a useful, effective, rapid and practical method to evaluate the quality consistency of sample.

ACKNOWLEDGMENTS:

The authors are thankful to Ayya Nadar Janaki Ammal College-deparment of chemistry (Tamil Nadu-India) for FTIR analysis and The Gandhigram Rural Institute-Department of Chemistry (Tamil Nadu-India) for NMR analysis.

CONFLICTS OF INTEREST:

There is no conflict of interest.

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Received on 04.01.2023 Modified on 09.02.2023

Res. J. Pharmacognosy and Phytochem. 2023; 15(3):191-197.

DOI: 10.52711/0975-4385.2023.00029

¹H-NMR Characteristics Signals	Description	Metabolites identified
7.68, 7.57, 7.26, 7.04, 5.10, 4.282, 3.884, 3.867, 3.810, 3.780, 3.704, 3.606, 3.578, 3.364, 3.341, 3.292, 3.271, 3.231, 2.106, 2.022,	Proton at 2.10 and 2.02 is the characteristic methyl group of crocin attached to the double bond carbons. 7.68-7.04 for the conjugated double bond CH=CH group of crocetin moiety of crocins	Crocin ³³
5.54, 4.28, 3.86, 3.60, 3.56, 3.36,3.23, 3.85,	5.54 for anomeric proton of the gentiobiosyl moiety	β-gentiobiosyl moiety ³⁴
2.29, 2.27, 2.10, 1.23, 3.86, 9.85 Glucose units: 3.34, 3.29, 3.70, 3.60, 3.78,3.27,3.12,	The characetrstic peak at 9.85 is due to aldehyde proton of picrocrocin	Picrocin ³⁴
4.52,3.86, 3.07,3.34, 3.27, 3.60	The shift at 4.52 is due to the anomeric protons	Beta-Glucose ³³
5.10,3.81, 3.78, 3.70, 3.34, 3.29	The shift at 5.10 is due to the anomeric protons	Alfa-glucose ³⁴
1.239, 1.105, 1.078, 0.840, 0.812, 0.805	for -CH3 and -CH2- groups of fatty acids	linoleic and linolenic fatty acids ³⁵