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Biochemical Analysis of Indigofera L. Species with Special Emphasis on Protein Content and Phylogenetic Analysis

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Biochemical Analysis of Indigofera L. Species with Special Emphasis on Protein Content and Phylogenetic Analysis International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109 Special Issue, ICRAFHN 2018 pp 119-121 Original Research Article Biochemical analysis of indigofera L. species with special emphasis on protein content and phylogenetic analysis Smita P Gudadhe1, Prashant J Gadge2*, Varsha S Dhoran3 1Department of Botany, Arvindbabu Deshmukh Mahavidyalay Barshingi Katol, Dist. Nagpur, Maharashtra, INDIA. 2 Department of Botany, A.S.C. College, Badnapur, Dist Jalna, Maharashtra, INDIA. 3 P.G. Department of Botany Sant Gadge Baba Amravati University, Amravati, Maharashtra, INDIA. Email: [email protected] Abstract Indigofera L. is a dicotyledonous plant and is a member of Leguminoceae-Papillionaeae family of largely herbs, shrubs and trees with a great variety of habitat. Indigofera has important medicinal uses. All the parts are useful. For the present study three species were selected Viz. Indigofera linifolia (Linn.f.) Retz, I. cordifolia Heyne ex Roth.. I. trita Linn. The sample proteins (seed proteins) were compared with a range of molecular weight marker. In Indigofera trita Linn. 15 bands were observed. The highest number of bands were observed in the range 75-50 i.e.. Lowest number of bands in the range 225-150, 100-75, 25-15 was 1 band in each range. Above 225 bands were absent likewise in the range 15-10 also bands absent. In Indigofera cordifolia Heyne ex Roth. 16 bands were observed. In Indigofera linifolia (Linn.f.) Retz 15 bands were observed. The highest number of bands in the range 25-15 is 4 bands and the lowest number of bands in the range above 225, 225-150, and 100-75 is 1 band in each range. It was found that the seeds have low molecular weight proteins. On the basis of banding pattern, the data was collected and analyzed for the phylogenetic analysis with the help of NTSYS software, showed close relation in I. trita and I. linifolia on the other hand I. cordifolia showed distantly related species. Key Words: Indigofera, Protein, SDS PAGE. *Address for Correspondence: Dr. Prashant J. Gadge, Quarter No. A1 Agri. Research Centre, Badnapur, Dist. Jalna 431202 Email: [email protected] of a biomolecules in a biological processes or systems the Access this article online biochemist typically needs to design a strategy to detect that biomolecule, isolate in pure form among thousands Quick Response Code: Website: of molecules that are been found in an extracts from a www.statperson.com biological sample, characterize it, and analyze its function. An assay, the biochemical test that characterizes a molecule, whether quantitative or semi-quantitative or qualitative, is important to determine the presence and the Accessed Date: quality of biomolecule at each step of the study. In 10 March 2018 Indigofera there are many morphological variations within the species in different regions of the world. In India about 60 species and 10 varieties of Indigofera are INTRODUCTION found (Hajra et al., 1995). There are thirty-five species of Biochemical analysis techniques refer to a set of methods, Indigofera that are reported from Maharashtra (Almeida, assays and procedures that enable scientist to analyze the 1998) at different places. They are Indigofera angulosa substances found in living organisms and the chemical Edge worth from khandesh; I. aspalathoides Val ex DC. reactions underlying life processes. The most from Deccan; I. astragalina DC. from Ambali, sophisticated of these techniques are reserved for Sawantwadi, Malvan, Marathwada and Vidarbha; I. specially research and diagnostic laboratories, also barberi Gamble from (Osmanabad) Marathwada; I. simplified sets of these techniques are used in various cassioides Rottle. DC. from Koina nagar, Purandhar, fields. To perform a comprehensive biochemical analysis Mahabaleshwar, Pune, Marathwada, Vidarbha and Copyright © 2018, Statperson Publishing Corporation, International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ICRAFHN 2018 Smita P Gudadhe, Prashant J Gadge, Varsha S Dhoran Khandesh; I. colutea (Burm. f.) Merrill. from Khandesh Twigs are used as tooth brushes and preferred over more and Nagpur. I. coerulea Roxb. from Ahamednagar and commonly used Acacia nilotica (Linn.) or Azadirachta Pune and Marathwada; I. coerulea Roxb., var. indica Linn. twig. Siddhuraju and his colleagues (1995) occidentalis Gillet and Ali from Ahamednagar and analyzed the nutrients composition and antinutritional Marathwada; I. constricta (Thw) trimen from Kumbharli factors of I. linifolia (Linn.f.)Retz and their weight were ghat and Ratnagiri; I. cordifolia Heyne ex Roth. from determined. The mature seeds contained 296.6g Kg-1 Thane, Nasik, Pune, Malvan, Marathwada, Vidarbha and crude proteins, 47.2g Kg-1 crude lipid, 56.7g Kg-1 Khandesh; I. dalzelli Cooke from Panchgani, Carbohydrates. The seeds of I. linifolia (Linn.f.) Retz Sawantwadi, Mahabaleshwar, Uran and Ramghat; I. were rich in K, Ca, Mn, and Cu, while albumins and deccanensis Sanjappa reported from Sautada (Beed); I. globulins constituted the major portion of seed proteins. trifoliata Linn. from Ramling forest, (Osmanabad); I. glabra Linn. from Vidarbha and Melghat; I. glandulosa MATERIALS AND METHODS Wendl. from Thane, Khopli, Sion, Purandar, Matunga For the present study three different species of Indigofera and Marathwada; I. heteranta Wall. ex Brandis from viz. I. linifolia (Linn.f.)Retz, I. cordifolia Heyne ex Roth, Vashi (New Bombay); I. karuppiana Pallithanum from I. trita Linn. were collected from Sant Gadge Baba dry hill slopes of Osmanabad, Marathwada, Vidarbha; I. Amravati University Campus. Variability of total seed karnatakana Sanjappa from Konkan; I. linifolia storage proteins was investigated by using SDS-PAGE (Linn.f.)Retz from Juhu, Sawantwadi, Matheran, (Laemmli, 1970). Protein was extracted from dry seed by Marathwada, Vidarbha and Khandesh;. I. linifolia Retz. grinding it to a fine powder. An appropriate quantity var. campbellii Wight ex Baker in Hook. from Malad, (0.01 g) of this powder was taken in a 1.5 ml Eppendorf Pune, Ahamednagar, Marathwada Osmanabad and tube. Protein extraction buffer (400 ul) containing 0.05M Vidarbha; I. echinata Willd. from Konkan, Malvan, Tris-HCl (pH 8.0), 0.4% SDS, 5M Urea and 1.5% 2- Vidarbha, Wardha, Tadoba, Ballarpur; I. oblongifolia Mercaptoethanol then was added to the tube. The sample Forsk. from Khandesh Amalnar, Vidarbha, Wamanpalli; was vortexed for 10 min followed by centrifugation at I. parviflora Heyne from Sawantwadi, Konkan, Junnar 12000 rpm for 10 min at room temperature. The proteins fort, Marathawada and Osmanabad; I. santapau Sanjappa extracted in the supernatant were size fractionated using from Purandar, Vazirgad fort; I. semitrijuga Forsk. from SDS-PAGE (9.5% Acrylamide) and stained with Bombay, Pune, Marathwada, khandesh and Vidarbha; I. Coomassie Brilliant Blue (CBB) dye. After destaining, spicata Forsk from Purandar, Sinhagad, Panchgani, Pune, gels were photographed by placing them on white light. Marathwada, Vidarbha and Melghat; I. tinctoria Linn. Data Analysis: A matrix (for SDS-PAGE) was generated from Mumbai, Pune, Marathwada, Vidarbha and by scoring reproducible bands as 1 for their presence and Melghat; I. suffruticosa Miller. from Powai and Victoria as 0 for their absence across the lines. Genetic similarity Garden-Bombay; I. trifoliate Linn. from Sawantwadi, coefficients were computed following Nei and Li (1979). Igatpuri, Purandar and Vidarbha; I. trita Linn. from Pune, The data were subsequently used to construct a Pashan, Solapur, Khandesh, Marathwada and Vidarbha. dendrogram using the unweighted pair group method of Three varieties of Indigofera trita i.e. I. maffei (Chiv) arithmetic averages (UPGMA) (Sneath and Sokal, 1973) Gillet, I. purandharensis Sanjappa and I. flccida (Koen.ex employing sequential, agglomerative hierarchic and non- Roxb) are reported by Almeida from the same regions overlapping clustering (SAHN). All the computations and some from different regions of Maharashtra; I. were carried out using the software NTSYSpc (Numerical aspalathoides Vahl ex DC. I. wightii Graham from Taxonomy and Multivariate Analysis System), version Konkan and Marathwada; I. hochststeri Baker from 2.1 (Rohlf, 2000). Correlations coefficients were Kolhapur, Pune and Chakan. These species are commonly calculated using similarity coefficients obtained from found within Maharashtra State and they show great SDS-PAGE analyses. morphological variations. Out of these 35 species, 11 Species are present in Amravati district (Dhore, 2002). RESULT AND DISCUSSION They are having edible and medicinal value. Indigofera The genetic information in a cell which is present in has important medicinal uses. As there are large number DNA, converts into proteins. These proteins are of species each found its use in different manner remarkable molecules and largely responsible for the depending upon its secondary constituents. I. cordifolia structure and function of the cells of plants. The protein Heyne ex Roth.. used as a forage crops for animals due to content can also determine some medicinal properties of the high crude protein and calcium contents and palability the plants. Electrophorograms showing the protein was good (Nath et al., 1971). The seeds of I. trita Linn. banding patterns of Indigofera lines. A total of forty two are used as nutritive tonic, collected for goat fodder. bands were scored among the 3 species studied. Of these International Journal of
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