FTIR

IDENTIFICATION AND COMPARISON OF BIOMOLECULES IN MEDICINAL PLANTS OF TEPHROSIA TINCTORIA AND ATYLOSIA ALBICANS BY USING FTIR J. KOMAL KUMAR, A.G. DEVI PRASAD Department of Environmental Science, University of Mysore, Mysore – 570006, Karnataka, India, e mail: kompaddy@yahoo.com Abstract. The aim of this study is to adopt the approach of metabolic fingerprinting through the use of Fourier Transform Infrared technique to understand the composition, chemical structure and discrimination of biomolecules in medicinal plants of Tephrosia tinctoria and Atylosia albicans. IR spectrum in mid infrared region (4000–400cm–1) was used for discriminating and indentifying various functional groups present in two different species of medicinal plants belonging to the family Leguminosae. Presence of C=O, C–H, C=C and C–O, C–C, C–O were identified. These bonding structures are responsible for the presence of alkyl groups, methyl groups, alcohols, ethers, esters, carboxylic acid, anhydrides and deoxyribose. In the present study FTIR Spectroscopy was used as a sensitive and effective assay for the detection and comparison of biomolecules between the two species of medicinal plants. The results showed that Tephrosia tinctoria and Atylosia albicans are rich in phenolic compounds. Key words: FTIR, biomolecules, Atylosia albicans, Tephrosia tinctoria, leguminosae. INTRODUCTION Medicinal plant research includes much more than the discovery of new drugs. This field has been expanding to also include diverse subjects as negotiation of power based on medicinal plant knowledge [11]. A wide range of our recently used medicines had their roots directly or indirectly from plants. Some of these medicines are no longer synthesized in large quantities by competitors because they have shown toxicity to humans and other animals. This has made possible for more investigations to be carried out on plants so as to enable us to know the therapeutic status of newly discovered drugs of plant origin. In this respect, plant based research has made promising results in the fields of anticancer and antimalarial therapies [5]. _________________________ Received: November 2010; in final form January 2011. ROMANIAN J. BIOPHYS., Vol. 21, No. 1, P. 63–71, BUCHAREST, 2011 64 J. Komal Kumar, A.G. Devi Prasad 2 Attempts to apply IR technology to biology began as early as the 1910s, when the use of IR spectroscopy for the analysis of biological samples was first suggested. By the late 1940s, the technique was being successfully explored for the study of biological materials and, in fact, IR spectroscopy has become an accepted tool for the characterization of biomolecules [16]. FTIR has been proven to be useful in studying compositional changes in plant cell walls during development. Therefore, it can possibly be used to determine changes in cell wall architecture upon exposure to organic contaminants [18]. Initially, the use of infrared spectroscopical method is restricted only for structural elucidation of isolated compounds from the herbal matrices. It was also found to be useful in phytochemical studies as a fingerprinting device, for comparing natural with synthetic sample [14]. Drug discovery from the medicinal plants continues to provide new and important leads against various pharmacological targets including cancer, HIV/AIDS, Alzheimer’s, malaria, infections and pain [1]. A number of events preceding the interaction of a drug with biological target and/or pharmacological effects have to be considered, and these involve absorption, distribution, metabolism and elimination. In order to assess the importance of each of these factors on drug action, both structural and physicochemical properties of the drug should be taken into account [12]. In biological systems, properties such as electrostatic bonds, hydrogen bonds, van der Waals bonds, as well as effects related to electron-transfer and hydrophobic effects are of major importance. Although the hydrogen bond is fairly weak compared to other interactions, it is of paramount importance in biological systems. Investigations of drug metabolism, its biotransformation pathways and structure of formed metabolites are of toxicological, pharmacological and biomedical interest [25]. In the present study FTIR spectroscopy was used for identification and comparison of biomolecules in two different species of medicinal plants. MATERIALS AND METHODS COLLECTION AND IDENTIFICATION OF PLANT MATERIAL Two medicinal plants Atylosia albicans and Tephrosia tinctoria were collected from the Western Ghats region of Hassan District, Karnataka, India. The leaf, flower, fruit and stem were carefully excised from the plant. These cleaned plant parts were shade dried and placed in polythene bags. The Herbaria of these plants are kept in the Department of Environmental Science, University of Mysore. 3 Identification and comparison of biomolecules in medicinal plants 65 PREPARATION OF THE PLANT MATERIAL The plant parts were shade dried at room temperature in a clean environment to avoid contamination for 14 days and powdered in a domestic grinder. The powdered samples were stored in air tight glass bottles at room temperature for further analysis. SAMPLE PREPARATION The plant powders were kept in a Lyophilizer to remove water. The samples were again ground in an agate mortar and pestle in order to obtain fine powder. Each powdered plant material was mixed with completely dried potassium bromide (at a ration of 1/100), and the mixture was subjected to a pressure of 5 × 106 pa in an evacuated die to produce a Kbr pellet for use in a FTIR spectrometer. TEST CHEMICALS The AnalaR grade Alcohol and Kbr was obtained from Sigma Aldrich Company, Bangalore, India, and were used without further purification for the experiment. SPECTROSCOPIC ANALYSIS FTIR spectra were recorded with a FTIR 460 plus Jasco. The powdered samples of both Atylosia albicans and Tephrosia tinctoria were mixed with dried potassium bromide and prepared as pellets, scanned at room temperature (25±2 ºC) at 4000–400cm–1 spectral range. To improve the signal to noise ratio for each spectrum, 100 interferograms with a spectral resolution of ±4cm–1 were averaged. Background spectra, which were collected under identical conditions, were subtracted from the sample spectra. Each sample was scanned under the same conditions with six different pellets. Special care was taken to prepare the pellets at the same thickness by taking the same amount of sample and applying the same pressure. Therefore, in the present study it was possible to directly relate the intensities of the absorption bands to the concentration of the corresponding functional groups [3]. RESULTS AND DISCUSSION The results of functional group analysis using FTIR revealed the existence of various characteristic functional groups in leaves, stem, flower and fruit of A. albicans and T. tinctoria (Fig. 1 to Fig. 5). The absorption bands, the wave number (cm–1) of dominant peak obtained from absorption spectra were defined in Table 1. 66 J. Komal Kumar, A.G. Devi Prasad 4 The dominant bands at 1655cm–1 and 1546 cm–1 were attributed to protein amide І and ІІ bands [8]. The shoulder at about 1750 cm–1 was attributed to lipid C=O stretching vibration [8]. The band at 1465 cm–1 was assigned to the CH2 bending mode of the cell lipids. The band at 1460 cm–1 represents asymmetric CH3 bending modes of end ethyl group proteins [26]. The band at 1402 cm–1 represents C=O symmetric stretching of COO– and assigned to lipids [26 and 17]. And band at 1377 cm–1 represents C–H bending mode of CH2 [2]. From information obtained from previous studies we assigned the remaining IR bands as follows: the peaks at 1237 cm–1 and 1082 cm–1 were attributed to PO–2 asymmetric and symmetric stretching vibrations and phospholipids [8]. The peak at 1064 cm–1 resulted from the overlap of several bands, including absorption due to the vibration modes of CH2OH and the C–O stretching vibration coupled to the C–O bending mode of cell carbohydrates [27]. The very strong absorption band observed around 3373–3422 cm–1 may be due to the presence of bonded N–H/C–H/O–H stretching of amines and amides [19]. The very strong absorption at 3400 cm–1 shows the presence of amino acids and the very strong absorption band appearing in the region 2933– 2922 cm–1 is due to N–H stretching. The lone C=O stretching vibration band corresponding to saturated aliphatic ester 1743 cm–1 is present in all parts of the plants. The bands at 900–1350cm–1, 1020 cm–1, 1024 cm–1 and 1050–100 cm–1 are attributed to phosphodiester stretching bands region (for absorbances due to collagen and glycogen), DNA, glycogen (C–O stretch associated with glycogen, phosphate and oligosaccharides PO–2 stretching modes), P–O–C antisymmetric stretching mode of phosphate ester, and C–OH stretching of oligosaccharides respectively. A band at 1051 cm–1 is attributed to C–O–C stretching of DNA and RNA [10]. The more intense bands occurring at 3419 cm–1, 2927 cm–1, 2853 cm–1, 1633 cm–1,1421 cm–1, 1260 cm–1, 1073 cm–1, 816 cm–1, and 635 cm–1 corresponding to O–H/N–H, C–H, C–O and C–CI/C–S stretching/bending vibrations respectively indicate the presence of amino acids, alkenes, nitrates, ethers, organic halogen compounds and carbohydrates in A. albicans and T. tinctoria [21]. 80 60 3754.72 cm-1 %T 2131.92 cm-1 40 1064.51 cm-1 20 3343 cm-1 0 4000.6 633.501 cm-1 1586.16 cm-1 2000 1000 Wavenumber [cm-1] 399.193 Fig. 1. FTIR spectra of Tephrosia tinctoria leaves. 5 Identification and comparison of biomolecules in medicinal plants 150 100 3807.76 cm-1 2144.45 cm-1 %T 50 1051.98 cm-1 2814.6 cm-1 1507.1 cm-1 1348.96 cm-1 1589.06 cm-1 3400.85 cm-1 0 4000.6 2000 762.709 cm-1 1000 Wavenumber [cm-1] 399.193 Fig. 2. FTIR spectra of Atylosia albicans leaves. 100 3848.25 cm-1 80 2138.67 cm-1 60 %T 1057.76 cm-1 2814.6 cm-1 40 1348 cm-1 1589.06 cm-1 20 3323.71 cm-1 0 4000.6 2000 657.607 cm-1 1000 Wavenumber [cm-1] 399.193 Fig. 3. FTIR spectra of Tephrosia tinctoria flower. 150 100 3758.58 cm-1 2146.38 cm-1 %T 50 2815.56 cm-1 3403.74 cm-1 0 4000.6 1054.87 cm-1 1348.96 cm-1 506.223 cm-1 1588.09 cm-1 2000 1000 Wavenumber [cm-1] 399.193 Fig. 4. FTIR spectra of Atylosia albicans fruit. 67 68 J. Komal Kumar, A.G. Devi Prasad 6 100 80 3882 cm -1 60 %T 40 2136.74 cm -1 2814.6 cm -1 20 3361.32 cm -1 0 4000.6 1349.93 cm -1 1585.2 cm -1 2000 676.892 cm -1 435.834 cm -1 1000 W a venumbe r [cm-1] 399.193 Fig. 5. FTIR spectra of Atylosia albicans stem. Table 1 General band assignments of the FTIR spectra of biological tissue based on literature [20] S. No. 1 2 3 4 5 6 PEAK 521 cm–1 600–900 cm–1 892 cm–1 940 cm–1 1000–140 cm–1 1000–200 cm–1 7 1000–350 cm–1 8 9 10 11 12 13 14 15 16 17 19 1020–50 cm–1 1030 cm–1 1105 cm–1 1145 cm–1 1180–300 cm–1 1206 cm–1 1244/5 cm–1 1255 cm–1 1312–1317 cm–1 1456 cm–1 1482 cm–1 1504 cm–1 20 21 22 2800–3000 cm–1 3500–600 cm–1 3000–700 cm–1 ASSIGNMENT torsion and ring torsion of phenyl CH out-of-plane bending vibrations C–C, C–O deoxyribose Carotenoid Protein amide I absorption C–OH bonds in oligosaccharides such as mannose & galactose Region of the phosphate vibration carbohydrate residues attached to collagen and amide III vibration (in collagen) Glycogen Collagen Carbohydrates Phosphate & oligosaccharides Amide III band region Amide III Collagen PO–2 asymmetric (phosphate I) Amide III Amide III band components of proteins collagen CH3 bending vibration (lipids and proteins) Benzene In-plane CH bending vibration from the phenyl rings C–H Lipid region OH bonds O–H stretching (water) A symmetrical stretching of NO2 group results in strong absorption in the region 1660–1625 cm–1. The observed absorption band at 1630 cm–1 indicates the presence of amines (protein) [15]. This gives the evidence that the plants A. albicans and T. tinctoria are rich in proteins. The weak absorption band 7 Identification and comparison of biomolecules in medicinal plants 69 observed between 1421 and 1415 cm–1 in the plant parts of A. albicans and T. tinctoria may be due to the presence of bonded C–O/O–H bending. The medium absorption band of 620 cm–1 indicates the presence of sulphate. A strong absorption band occurs at 597 and 580 cm–1 in the stem, leaves, fruits and flower of two species which is possibly due to aliphatic C–CI absorption and brominate compounds. The brominate compounds show an infrared band region at 600–500 cm–1 [24]. The weak absorption band at 539 cm–1 indicates the presence of phosphates in the leaves, fruits, flowers and stem in the examined plants. The very weak band occurring at 780 cm–1 in the flowers, leaves, stem and fruits of these plants can be attributed to out-of-plane N–H wagging, primary and secondary amide and nitrite group. Five major peaks 1590 cm–1, 1348 cm–1, 1051, 3385 cm–1, 1063 cm–1 and 456 cm–1 were observed in the FTIR spectra. A weak absorption peak at 453 cm–1 has appeared which is attributed to the absorption of Y–O bond [28]. This is a significant observation made in A. albicans as it is not reported in any legumes so far. A weak absorption band at 940 cm–1 is attributed to carotenoid being present only in the fruit of A. albicans [13]. A weak absorption band at 965 cm–1 is attributed to C–O stretching of the phosphodiester and the ribose and is present only in A. albicans [4]. A very weak peak at 892 cm–1 is attributed to C–C, C–O deoxyribose and seen only in A. albicans [7]. A medium peak at 1340 cm–1 is due to CH2 wagging collagen present in A. albicans [9, 27]. A very strong peak at 1581 cm–1, 1358 cm–1, 520 cm–1 is attributed to ring C–C stretch of phenyl, stretching C–O, deformation C–H, deformation N–H and phenyl group respectively, are present in fruits, leaves and the stem of A. albicans and leaves and flowers of T. tinctoria [6, 23]. A medium peak at 3300 cm–1 and 1020–1050 cm–1 is attributed to amide І bands stemming from N–H stretching modes in proteins, nucleic acids and glycogen [12]. CONCLUSIONS By the above investigations, it can be concluded that A. albicans and T. tinctoria are rich in phenolic compounds and also show the presence of oligosaccharides, phosphates, proteins, carbohydrates and carotenoid. The present work also indicates the presence of biomolecule concentration is different in different parts of the plants. This work offers scope for further research in phytochemical analysis and biological activity of medicinal plants. 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