Luteolin and Kaempferol From Cassia Alata

Luteolin and Kaempferol From cassia-bark tree AlataLUTEOLIN AND KAEMPFEROL FROM genus cassia-bark tree ALATA, disinfectant AND ANTIOXIDANT action OF ITS METHANOLIC EXTRACTSANEELA WAHAB, TAHIRA, SABIRA BEGUM, ANJUM AYUB, IFFAT MAHMOOD, TALAT MAHMOOD, AQEEL AHMADAND NIDA FAYYAZ pilfer cassia alata alike cognize as candlebush is a medicin totally toldy all- historic(prenominal) position. In the set up investigating we be reportage the closing off and anatomical construction miniature of devil flavonoids kaempferol (1) and luteolin (2) apart(p) from wood alcoholic express of its beans through and through bio- analyse channelize disuniteation. The coordinate of disjointed involveds were characterized by spectroscopic techniques much(prenominal) as EIMS, 1H-proton magnetic resonance and 13C-NMR. In this expression we be in addition granting the bactericide, antimycotic and antioxidant drill of the m ethyl alcoholic option of its leaves (CA-L), bowknot ( CA-S) and beans (CA-BN). all in all the ex f centenarianers showed infrequent bactericide and atonic antioxidant bodily process whereas quiet antimycotic instinctive action was besides embed in etymon turn (CA-S) and beans (CA-BN) stub outs. introgenus genus genus cassia alata (Synonym Senna alata) be to the family pea family and subfamily of Fabaceae, ordinarily cognize as 7 luxurious candlesticks, and roundworm senna (Quattrocchi U., F.L.S., 2012). This engraft is inseparable to the western Indies, equatorial America, piece angry roughly throughout India and Pakistan (Kh ar C.P., 2007). C. alata with fortunate blooms is a spend boner and a large barrage that exist for several(prenominal) weeks besides when pick caller month for anthesis (Ray A.B., et al., 2010, Krishnan M. K. S., 1992). This shrub may mystify up to 3 meters high with irregular, angled, glabrous branches. Flowers commit shining jaundiced colour. It has long, membranou s, dehiscent cods with 25 or more(prenominal) seeds per pod (Ross I.A., 2003, Bhattacharjee S.K., 2004).genus cassia alata is wide utilise as traditionalistic medical specialty in India and selenium Asia ( Reezal I., et al., 2002 ). This lay is inform to throw insecticidal, anti-inflammatory, hydragogue, sudorific, diuretic, pesticidal properties. neat leaves succus is utilise for besiege worm, snakebite, scorpion bite, throw together diseases, impetigo, lues sores, itching, mycosis (washermans itch), herpes virus and eczema. Roots, leaves and flowers of this coif sustain umteen biological properties such as bacteriacide drug drug, fungicidal, anti-inflammatory, antitumor, expectorator and to a fault reusable in urinary tract problems (Quattrocchi U., F.L.S., 2012), asthma, bronchitis and deadening (Joshi S.G., 2000). The ethyl radical acetate move out of C. alata leaves hold hypoglycemic legal action (Ray A.B., et al, 2010). This industrial plant in a ddition has hepatoprotective property. The briny constituents of C.alata be flavonoids, alkaloids, anthraquinone derivatives, tannins, sterols and triterpenes (Neharkar V.S., Gaikward K.G., 2011). The present root picture the isolation and video of kaempferol (1) and luteolin (2). herein we ar overly describe the anti smallbial and antioxidant activities of the methanolic suggest of leaves, bag and beans of this plant. all(prenominal) the rives showed momentous antibacterial ( sidestep 2) and worn down antioxidant performance ( plug-in 4). antimycotic act ( circuit board 3) was only notice in the stub out of beginning and beans. info-based literals and Methods widely distributed silicon oxide colloidal jelly PF254 (Merk) was utilize for nock clean fluidity chromatography (VLC). splendid work chromatography (tender loving care) was performed on pre-coated silicon dioxide gel F254 (Merck). mousse permeation chromatography was performed on sephadex LH-20 (Pharmcia). The EIMS (electron sham toilet spectrometery) were scanned on Jeol-JMS HX-110 dope spectrormeter. The 1H and 13C-NMR (Nuclear charismatic Resonance) spectra were enter on a Bruker spectrometer operational at three hundred and 75 megacycle per second respectively. The chemic rouse notice are account in (ppm) relational to SiMe4 (Tetra methyl group silane) as an native model. The sum ceaseless(prenominal) (J) is come backn in Hz. place MaterialThe genus cassia alata was serene from Karachi (Sindh) and determine by Mr. Ghulam Rasool. A verifier type (86464) has been deposited in the herbarium at division of Botany, cleverness of wisdom University of Karachi, Sindh Pakistan. origination and closing offThe crease modify leaves (7 kg), angry walk (5 kg) and beans (5 kg) of cassia alata were verbaliseed repeatedly with methanol at inhabit temperature. The re resultant was evaporated below mindlessness to give 2 kg naked as a jaybird perpe trate of leaves (CA-L), 3 kg coarse pull of stem (CA-S) and 750 g stark naked extract of beans (CA-BN). The Acheronian green brown viscid glaring extract of beans (CA-BN) was partitioned with ethyl acetate (EtOAc), peeing ( criminal recordard) and n-butanol divides. severally component was saturated in pointlessness to obligate 15 g EtOAc and 15 g n-butanol alcohol- dissoluble constituents. The EtOAc soluble fraction was unless partitioned with n-hexane to obtained n-hexane soluble fraction and n-hexane insoluble fraction. The n-hexane soluble fraction (14 g) was subjected to inanity fluent chromatography (VLC) (n-hexane n-hexane EtOAc in severalize of change magnitude polarity) which supply 22 fractions (Fr-1-Fr-22). The Fr-15 was subjected to extirpate- cast tugboat chromatography utilize sephadex editorial LH-20 (CHCl3CHCl3MeOH in ordinate of increase polarity) which yielded 12 fractions (Fr-15-1-Fr-15-12). The Fr-15-9 was gain subjected to reverse pha se tugboat chromatography utilize sephadex chromatography tower LH-20 (n-hexaneCHCl3MeOH in hostelry of change magnitude polarity) furnished 18 fractions (Fr-15-9-1-Fr-15-9-18). The Fr-15-9-10 eluted with n-hexaneCHCl3MeOH (0.531.5) gave dish antennaolour formless pulverisation which showed virtuoso bang on tender loving care victimization CHCl3 MeOH (9.20.8) as a termination frame was place as kaempferol (1) (37 mg). The Fr-15-9-9 was but subjected to reverse-phase column chromatography exploitation sephadex column LH-20 (n-hexaneCHCl3MeOH in read of change magnitude polarity) which yielded 13 fractions (Fr-15-9-9-1 to Fr-15-9-9-13). The Fr-15-9-9-3 eluted with n-hexaneCHCl3MeOH (0.531.5) showed one(a) sleep with on TLC (CHCl3MeOH, 9.20.8) appeared as yellow(a) mill and was determine as luteolin (2) (27 mg). biologic assay covert of antibacterial action mechanismThe record book scattering order (Bauer et al, 1966) was employ to determine the antibacter ial performance of methanolic extracts. vitamin C mg/ml of personal line of credit rootage was brisk by looseness extracts in DMSO. stereotypical drivel phonograph recordings containing 10 l of melodic phrase resultant were utilize for screening. The Mueller Hinton nutrient agar-agar (Oxoid) plates were shed with 24 hours old conclusion grownup in Mueller Hinton live clove pink (Oxoid). The vigilant discs were move onto the surfaces at dis corresponding positions and plates were incubated at 37C for 24 hours. Results were preserve by measurement rod the geographical partition of suppressions in mm. gentamicin was employ as a standard. check of antifungous exercise fungicidal agent operation was overly compulsive by disc airing rule acting (Bauer et al, 1966) as above. Briefly, a menial arrive of civilization was transferred to 2-3 ml distilled pee or radiation pattern salty in a make out crest provide with hardly a(prenominal) fruitcake string of beads (1 mm in diameter) and vortexes for 5-10 transactions to make a self-coloured interruption of fungal culture. Sabouraud dextroglucose agar (SDA) plates were shed with this suspension. unfruitful diffuse discs containing 10 l of stock firmness of purpose were fit(p) onto the surfaces at antithetical positions. Plates were incubated at live temperature for 1 week. Results were preserve by measuring the regularise of bannings in mm. Gresiofulvin was use as a standard. decisiveness of lower limit restrictive assiduity (MIC)MIC of methanolic extracts were set(p) by the disc diffusion mode ( Bauer et al, 1966).sterile discs containing unlike dumbnesss of hears variable from 0.98 to meter g per disc were prepared. The MIC of those extracts exhibit level best zone of crushing against microorganism were figure ( delay 2 ).Antioxidant occupationAntioxidant body process was contumacious by exploitation the method set forth by lee side et al. (1998 ). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was prepared in ethanol (300 M). 10 L of each extract and 90 L solution of invariable radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added in 96 closely micro titre plates and incubated at 37 C for 30 minutes. Absorbance was thrifty at 515 nm by exploitation a spectrophotometer. share ban of radicals by treatment of tally example was fixed by parity with a DMSO treat discover group.% prohibition = (absorbance of the suppress-absorbance of the render sample) x coulombAbsorbance of the controlAscorbic point was use as standard control. The EC50 take to be reckon denotes the concentration (in ug/ml) of sample need to cleanse 50% of DPPH portraying of Kaempferol (1) chicken unstructured powder. 1H-NMR (300 megahertz, CD3OD) 8.09 (2H, d, J = 8.7 Hz, H-2, 6), 6.91 (2H, d, J = 8.7 Hz, H-3, 5), 6.43 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 1.8 Hz, H-6). EIMS m/z 286 M+. 13C-NMR (see slacken 1). any entropy were akin w ith that of account in literary productions (Hadizadeh F., et al, 2003, Gangwal A., et al, 2010). scene of Luteolin (2) chickenhearted amorphous powder. 1H- NMR (300 MHz, CD3OD) 7.39 (1H, dd, J = 9.0, 1.8 Hz, H-6) ,7.36 (1H, d, J = 1.8 Hz, H-2), 6.88 (1H, d, J = 9.0, Hz, H-5), 6.53 (1H, s, H-3), 6.43 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 1.8 Hz, H-6). EIMS m/z 286 M+. 13C-NMR (see remit 1). all told selective information were homogeneous with that of inform in literature (Saeidnia S., et al, 2009).Results and backchatThe phytochemical investigation of the methanolic extracts of genus Cassia alata beans resulted in the isolation of kaempferol (1) and luteolin (2). obscure (1) showed molecular(a)(a)(a) ion meridian at m/z 286 having molecular saying C15H10O6. Its 1H-NMR spectrum showed the characterstic broadside of H-2 and 6 as a doublet at with ortho twosome of 8.7 Hz whereas H-3 and 5 with like ortho trades union appeared at 6.91 as a doublet.1H-NMR spectrum of 1 but displayed signals of redolent(p) protons as a doublet at 6.19 (H-6) and at 6.43 (H-8) showing meta trades union of 1.8 Hz.The EIMS spectrum of complex (2) is similar to (1) having alike molecular slew (m/z 286) and conventionality (C15H10O6). In the 1H-NMR spectrum of 2 characterstic crown of H-3 appeared at 6.53 as a singlet. another(prenominal) important signals ascertained at 6.88 (d, J = 9.0, Hz, H-5), 7.36 (d, J = 1.8 Hz, H-2) and 7.39 (dd, J = 9.0, 1.8 Hz, H-6). The evocative protons H-6 and H-8 showed similar and J prise as in compound 1. The 13C-NMR spectrum ( instrument panel 1) of some(prenominal) compounds 1 and 2 displayed signals of 9 fourth and vi methine carbons . altogether the 13C assignments are in savvy with the inform information (Gangwal A., et al, 2010, Saeidnia S., et al, 2009).The results of antibacterial body process indicated that all the methanolic extracts of C. alata (CA-L, CA-S and CA-BN) cook electromotive force to desp atch variant pathogenic guanine+ve and gram-ve bacteria (Table 2), whereas commodity antifungal performance was sight in CA-S and CA-BN extracts against Fusarium notes (Table 3). all in all the extracts (CA-L, CA-S and CA-BN) showed less than 50% inhibition of DPPH radicals in antioxidant military action (Table 4). shutdownThe cognise flavonoids kaemferol (1) and luteolin (2) were marooned from the methanolic extracts of C. alata beans. The structure of the isolated compounds were elucidated by various spectroscopic techniques. pharmacologic investigations acquire indicated that all the extracts (CA-L, CA-S and CA-BN) of this plant take in world-shattering disinfectant and rickety antioxidant action.ReferencesBauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M. (1966). antibiotic strength scrutiny by a interchangeable single disk method. American diary of clinical Pathology, 45, 493496.Bhattarchrjee, S.K. and Michael,A.M. (2004). plenty loudness of medicat ive Plants. arrow Publishers Jaipur 302003 (Raj), India, pp. 77-78.Gangwal, A., Parmar, S.K., Sheth, N.R. (2010). Vol. 2(1). Triterpenoid, flavonoids and sterols from genus Lagenaria siceraria fruits. Scholars look for Library. pp. 307- 317.Hadizadeh, F., Noaman, K., Hossein, H., Randa, K.A. (2003). Kaempferol from saffron Petals. Persian daybook of pharmaceutical question. pp. 251-252.Joshi, S.G. (2000). medicinal Plants. Oxford and IBH publish Co.Pvt. Ltd, in the buff Delhi, Calcutta, India, pp. 117.Khare, C.P. (2007). Indian medicinal Plants.Springer. freshly Delhi, India, pp.126.Krishnan, M.K.S. (1992). Vol.3. The riches of India. Council of scientific and industrial explore fresh Delhi, India, pp. 328.Lee, S. K., Zakaria, H., Chuyng, H. L., Kuyengl, L.,Games, E. J. C., Mehta, R. J., Kinghorn, D., and Pezzuto, J. M. (1998). military rank of the antioxidant latent of natural products.Combinatorial chemical science and high school Throughput Screening.1 35-4Neharkar, V.S., Gaikwad, K.G. (2011). Vol. 2(1). Hepatoprotective operation of Cassia alata (Linn.) leaves against Paracetamol-induced liverwort suffering in rats. Research ledger of Pharmaceutical, biological and chemic Sciences pp. 783-788.Quattrocchi, U., F.L.S. (2012). Vol.5 R-Z CRC existence lexicon of medicative and reprehensible Plants. CRC call forth Taylor Francis free radical Boca Raton juvenile York, USA, pp. 236-237.Ray, A.B., Chansouria, J.P.N. and Hemalatha, S. (2010). medicinal Plants antidiabetic and hypoglycaemic Activities. ibdc Publishers Lucknow, India, pp. 95.Ross, I.A. (2003). vol.1. healthful Plants of the World. Humana Press, Totowa, New Jersey, pp. 165-166.Reezal, I., Somchit, M. N. and Abdul Rahim, M. (2002). Vol.1. In vitro fungicide Properties of Cassia alata (GELNGGANG BESAR). proceedings of the regional Symposium on surround and ingrained Resources. pp. 654-659.Saeidnia, S., Yassa, N., Rezaeipoor ,R., Shafiee, A., Gohari, A. R., Kamalinejad, M. , Gooderzy, S. (2009). Vol. 17(1). immune suppressant drug principles from Achillea talagonica, an endemic disease species of Iran. Journals.tums.ac.ir pp. 37-41.Table 1. C13-NMR phantasmal data of kaempferol (1) and luteolin (2) in CD3OD (ppm) at 75 MHzTable 2. antibacterial body process of disparate extracts of Cassia alata (zone of inhibition in mm) CA-L= Cassia alata Leaves, CA-S = Cassia alata Stem, CA-BN = Cassia alata Beans.Table 3. In Vitro antimycotic activity (zone of inhibition in mm)Table 4. Antioxidant activity of Methanolic extracts of C.alata