Phytochemistry (v.62, #2)
Rosmarinic acid by Maike Petersen; Monique S.J Simmonds (121-125).
Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. It is commonly found in species of the Boraginaceae and the subfamily Nepetoideae of the Lamiaceae. However, it is also found in species of other higher plant families and in some fern and hornwort species. Rosmarinic acid has a number of interesting biological activities, e.g. antiviral, antibacterial, antiinflammatory and antioxidant. The presence of rosmarinic acid in medicinal plants, herbs and spices has beneficial and health promoting effects. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. The biosynthesis of rosmarinic acid starts with the amino acids l-phenylalanine and l-tyrosine. All eight enzymes involved in the biosynthesis are known and characterised and cDNAs of several of the involved genes have been isolated. Plant cell cultures, e.g. from Coleus blumei or Salvia officinalis, accumulate rosmarinic acid in amounts much higher than in the plant itself (up to 36% of the cell dry weight). For this reason a biotechnological production of rosmarinic acid with plant cell cultures has been proposed.Rosmarinic acid is a widely occurring natural product in the plant kingdom with interesting biological activities. The compound is synthesised in Lamiaceae from the amino acids l-phenylalanine and l-tyrosine. All the enzymes and several genes of the biosynthetic pathway are known.
Keywords: Antioxidant; Boraginaceae; Caffeic acid esters; Lamiaceae; Plant cell cultures; Rosmarinic acid;
A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations by Sabrina Cacace; Gudrun Schröder; Elke Wehinger; Dieter Strack; Jürgen Schmidt; Joachim Schröder (127-137).
Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase (OMT) activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis. This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose. SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38–43 kDa). Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs. Homology-based RT-PCR identified cDNAs for four new OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT. The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases. The enzyme functions were investigated with purified recombinant proteins after cDNA expression in Escherichia coli. Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT. It represented a new type of OMT that performs two sequential methylations at the 3′- and 5′-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for C. roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.Purification and molecular characterization of an unusual flavonoid O-dimethyltransferase that explains the 3′,5′-methylation in flavonols and anthocyanins of Madagascar periwinkle.
Keywords: Catharanthus roseus; Apocynaceae; Madagascar periwinkle; Homology based cDNA cloning; Recombinant protein expression; O-Methyltransferase; Flavonoids;
Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde by David Fournand; Bernard Cathala; Catherine Lapierre (139-146).
Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)–H2O2 system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (β-5, β-β, and β-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The β-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the β,β′-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. “shuttle oxidant”) for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.The effect of pH on the dehydrogenative dimerization of coniferyl alcohol and/or sinapyl aldehyde, using capillary zone electrophoresis as an analytical tool, is reported.
Keywords: Horseradish peroxidase; Coniferyl alcohol; Sinapyl aldehyde; Dehydrogenative polymerization; Dehydrodimers; Hydrogen peroxide; Lignin; Lignification; Radical coupling reaction;
Sterol composition of mycelia of the plant pathogenic ascomycete Leptosphaeria maculans by K.M Griffiths; A Bacic; B.J Howlett (147-153).
Analysis of sterols in mycelia of the ascomycete, Leptosphaeria maculans by gas chromatography–mass spectrometry revealed that ergosterol comprised 95% of the total sterols, with eight other sterols comprising the remaining 5%. Six of these latter sterols were putative precursors of ergosterol and their presence suggested a pathway for ergosterol biosynthesis in this fungus. Ergosterol biosynthesis in fungi is inhibited by the triazole antifungal agent flutriafol. When L. maculans was grown in the presence of flutriafol, ergosterol content decreased while two 14α-methylated sterols, 24-methylene dihydrolanosterol and obtusifoliol, accumulated.The ergosterol biosynthetic pathway of Leptospheria maculans and effects of a triazole fungicide on sterol composition are described.
Keywords: Leptosphaeria maculans; Ascomycota; Plant pathogen; Gas chromatography–mass spectrometry; Sterols; Triazole fungicide; Flutriafol;
Transformations of testosterone and related steroids by Botrytis cinerea by Ewa Huszcza; Jadwiga Dmochowska-Gładysz (155-158).
Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7α-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14α-position.The unusual hydroxylation of 4-ene-3-oxo-steroids mainly at 7α-position by Botrytis cinerea is presented.
Keywords: Botrytis cinerea; Testosterone; Testosterone relatives; Biotransformation;
The biochemical origin of pentenol emissions from wounded leaves by A.J Fisher; H.D Grimes; R Fall (159-163).
Experiments are presented demonstrating that 1-penten-3-ol and 1-penten-3-one, C5 volatile wound compounds, are products of lipoxygenase-dependent reactions in soybean leaves.Large releases of 1-penten-3-ol (pentenol) and 1-penten-3-one (pentenone) were recently observed from a variety of leaves subjected to freeze–thaw damage in the presence of oxygen. In order to understand the biochemical origins of these volatiles, soybean leaf extracts were used to determine if the formation of pentenol and pentenone can be explained by known O2-dependent lipoxygenase (LOX) reactions. Enzymatic formation of these C5 volatiles was found to be dependent on α-linolenic acid or the 13(S)-hydroperoxide of α-linolenic acid [13(S)-HPOT] and blocked by LOX inhibitors. Five soybean leaf LOX isozyme genes (VLXA, VLXB, VLXC, VLXD, and VLXE) were then expressed in Escherichia coli and used in in vitro incubations with 13(S)-HPOT to test for volatile formation. Each of the LOX isozymes catalyzed the formation of low levels of pentenol, but not pentenone. It therefore seems likely that the C5,13-cleavage activity of LOX is the direct source of abundant pentenol and the indirect source of pentenone observed upon leaf wounding.
Keywords: Glycine max; Leguminosae; Soybean; Pentenol; Pentenone; Lipoxygenase; Volatile organic compound; Gas chromatography; Reduction gas detector;
3β,13-Dihydroxylated C20 gibberellins from inflorescences of Rumex acetosa L. by Tania S Stokes; Lewis N Mander; Stephen J Croker; Bruce Twitchin; David E Hanke (165-174).
Using full scan GC–MS a wide range of gibberellins (GAs) was identified in the young inflorescences of the dioecious species Rumex acetosa L., consistent with the ubiquitous early 13-hydroxylation pathway in both male and female plants. In addition, R. acetosa is the first species in which all three 3β,13-dihydroxylated C20-GAs—GA18, GA38 and GA23—have been identified in the same organism, suggesting an early 3β,13-dihydroxylation biosynthesis pathway in this species. Authentic GA18, GA38 and GA23 were synthesized and their effects and that of GA1, a GA common to both pathways, on the time to inflorescence emergence was investigated. GA1 accelerated the emergence of inflorescences in both male and female plants. In addition some evidence for biological activity per se of the C20-GA38 was obtained.Graphic
Keywords: Rumex acetosa; Polygonaceae; Sorrel; Synthesis; GC–MS; Gibberellin; Early 13-hydroxylation pathway; Early 3β,13-hydroxylation pathway; GA18; GA38; GA23;
Volatile emissions of eastern hemlock, Tsuga canadensis, and the influence of hemlock woolly adelgid by Corey D Broeckling; Scott M Salom (175-180).
The volatile emissions of eastern hemlock, Tsuga canadensis Carriere, were identified and quantified using standard and chiral gas chromatography and mass spectrometry. All of the identified compounds were monoterpenes, and included α-pinene, myrcene, tricyclene, camphene, α-phellandrene, β-pinene, limonene, β-phellandrene, terpinolene, and bornyl acetate. α-Pinene, myrcene, and camphene comprised greater than 75% by mass of the total release. Infestation by the exotic insect, hemlock woolly adelgid (HWA, Adelges tsugae Annand), resulted in an increased release rate of monoterpenes from branch tips. Release rate was negatively correlated to the amount of the branch tip sample that was new growth, suggesting that release rate is greater from previous-year foliage. Additionally the percent composition of the volatile profile is slightly altered by infestation, with α-pinene comprising 57% of volatiles from infested foliage and 66% from uninfested foliage.We report the identification and quantification of volatile emissions from Tsuga canadensis and determine the effect of hemlock woolly adelgid on the volatile profile.
Keywords: Tsuga canadensis; Pinaceae; Eastern hemlock; Adelges tsugae; Homoptera; Monoterpenes; Volatile emission;
Aluminum-induced oxidative stress in maize by Patricia R.S. Boscolo; Marcelo Menossi; Renato A. Jorge (181-189).
Oxidative stress induced by Al-toxicity was studied in two inbred lines of maize (Zea mays L.) by determination of the peroxidase, catalase superoxide dismutase activities, lipid peroxidation, protein oxidation and cell death, in root tip cells.The relation between Al-toxicity and oxidative stress was studied for two inbred lines of maize (Zea mays L.), Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive). Peroxidase (PX), catalase (CAT) and superoxide dismutase (SOD) activities were determined in root tips of both lines, exposed to different Al3+ concentrations and times of exposure. No increases were observed in CAT activities in either line, although SOD and PX were found to be 1.7 and 2.0 times greater than initial levels, respectively, in sensitive maize treated with 36 μM of Al3+ for 48 h. The results indicate that Al3+ induces the dose- and time dependent formation of reactive oxygen species (ROS) and subsequent protein oxidation in S1587-17, although not in Cat100-6. After exposure to 36 μM of Al3+ for 48 h, the formation of 20±2 nmol of carbonyls per mg of protein was observed in S1587-17. The onset of protein oxidation took place after the drop of the relative root growth observed in the sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition. The presence of Al3+ did not induce lipid peroxidation in either lines, contrasting with the observations in other species. These results, in conjunction with the data presented in the literature, indicate that oxidative stress caused by Al may harm several components of the cell, depending on the plant species. Moreover, Al3+ treatment and oxidative stress in the sensitive maize line induced cell death in root tip cells, an event revealed by the high chromatin fragmentation detected by TUNEL analysis.
Keywords: Aluminum toxicity; Oxidative stress; Protein oxidation; Lipid Peroxidation; Maize; ROS; Cell death;
Fatty acid variations in symbiotic dinoflagellates from Okinawan corals by Natalia V Zhukova; Eduard A Titlyanov (191-195).
Fatty acids of polar lipids and triacylglycerols of different morphophysiological types of symbiotic dinoflagellates (zooxanthellae) were investigated for determination of taxonomic differences among them.The fatty acid composition of polar lipids and triacylglycerols was determined in different morphophysiological types of symbiotic dinoflagellates (SD) isolated from the hydrocoral Millepora intricata and the scleractinian corals Pocillopora damicornis, Seriatopora caliendrum, Seriatopora hystrix and Stylophora pistillata from a fringing reef of Sesoko Island, Okinawa, Japan. The distribution of the fatty acids among the morphophysiologically distinct types of SD reported in these corals makes it possible to readily distinguish one type of SD from the other. Moreover, differences were found both in polar lipids and triacylglycerols. The polar lipids of SD from M. intricata showed a very distinctive fatty acid profile. A combination of large proportions of 18:4 (n-3), 18:5 (n-3), 22:5 (n-6), and 22:6 (n-3) and negligible amounts of 20:4 (n-6), and 20:5 (n-3) in SD from M. intricata was particularly noteworthy. The fatty acid profiles of SD from P. damicornis and SD isolated from S. caliendrum and S. hystrix differed in the proportion of 18:4 (n-3) and 22:6 (n-3). It is suggested that fatty acids might provide useful information on possible taxonomic differences among symbiotic dinoflagellates. It is assumed that biochemical differences can reflect the genetic diversity of the morphophysiological types of SD associated with several species of hermatypic corals from this region.
Keywords: Dinophyceae; Symbiotic dinoflagellates; Zooxanthellae; Hermatypic corals; Taxonomy; Fatty acids;
Cytotoxic triterpenes from the twigs of Celtis philippinensis by Bang Yeon Hwang; Hee-Byung Chai; Leonardus B.S Kardono; Soedarsono Riswan; Norman R Farnsworth; Geoffrey A Cordell; John M Pezzuto; A Douglas Kinghorn (197-201).
Two triterpene esters, 3β-trans-sinapoyloxylup-20(29)-en-28-ol (1) and 3β-trans-feruloyloxy-16β-hydroxylup-20(29)-ene (2), were isolated as cytotoxic constituents from the chloroform-soluble extract of the twigs of Celtis philippinensis, along with five known triterpenes, 3β-O-(E)-feruloylbetulin (3), 3β-O-(E)-coumaroylbetulin (4), betulin (5), 20-epibryonolic acid (6), and ursolic acid (7). The structures of 1 and 2 were assigned from their 1D and 2D NMR spectroscopic data. All isolates were evaluated for cytotoxicity against several human cancer cell lines.Two triterpene esters, 3β-trans-sinapoyloxylup-20(29)-en-28-ol (1) and 3β-trans-feruloyloxy-16β-hydroxylup-20(29)-ene (2), were isolated as cytotoxic constituents from the chloroform-soluble extract of the twigs of Celtis philippinensis.
Keywords: Celtis philippinensis; Ulmaceae; Twigs; Triterpenoids; 3β-trans-Sinapoyloxylup-20(29)-en-28-ol; 3β-trans-Feruloyloxy-16β-hydroxylup-20(29)-ene; Cytotoxicity evaluation;
Antimicrobial constituents from the rhizomes of Rheum emodi by K Suresh Babu; P.V Srinivas; B Praveen; K Hara Kishore; U Suryanarayana Murty; J Madhusudana Rao (203-207).
The bioassay-guided chemical examination of the rhizomes of R. emodi resulted in the isolation of two new oxanthrone esters, revandchinone-1, revandchinone-2, a new anthraquinone ether revandchinone-3 and a new oxanthrone ether, revandchinone-4. Their structures were established based on spectroscopic and degradative evidence. Occurrence of oxanthrone ether is reported for the first time. The anti bacterial and anti fungal activity of the isolates is studied.The bioassay-guided fractionation of the rhizomes of Rheum emodi afforded two oxanthrone esters, revandchinone-1 (1), revandchinone-2 (2), an anthraquinone ether, revandchinone-3 (3) and an oxanthrone ether, revandchinone-4 (4). The anti bacterial and anti fungal properties of the new compounds were established.
Keywords: Rheum emodi; Rhubarb; Polygonaceae; Revandchinone-1; Revandchinone-2; Revandchinone-3; Revandchinone-4; Chrysophanol; Physcion; β-Asarone;
Chemical composition and antibacterial activities of the essential oils of Lippia chevalieri and Lippia multiflora from Burkina Faso by I.H.N. Bassole; A.S. Ouattara; R. Nebie; C.A.T. Ouattara; Z.I. Kabore; S.A. Traore (209-212).
The chemical composition of the essential oils of Lippia chevalieri and Lippia multiflora obtained from the air-dried leaves by hydrodistillation were analysed using GC and GC–MS. L. chevalieri and L. multiflora belonged to thymol/p-cymene/2-phenyl ethyl propionate and thymol/p-cymene/thymyl acetate chemotypes, respectively. The essential oils were also tested against 09 strains using a broth microdilution method. The Gram-negative bacteria were the most sensitive. The essential oil of L. multiflora was the most active.The essential oils of Luppia chevalieri and Lippia multiflora air-dried leaves chemical composition were determined and tested against Gram-positive and Gram-negative bacteria.
Keywords: Lippia chevalieri; Lippia multiflora; Verbenaceae; Essential oils; Antibacterial;
Inhibition of LPS-induced NO production by the oleogum resin of Commiphora wightii and its constituents by Meselhy R. Meselhy (213-218).
Three new (1–3) and five known compounds (4–8) were isolated from the oleogum resin of Commiphora wightii (Arnott.) Bhanol. Their structures were elucidated by spectroscopic and chemical methods. The MeOH extract and the EtOAc-sol. fraction were found to demonstrate significant inhibition of NO formation in lipopolysaccharide (LPS)-activated murine macrophages J774.1 in vitro (IC50 values of 16.4 and 12.8 μg/ml, respectively). When compared with curcumin (IC50 value of 12.3 μM), Z- and E-Guggulsterones (4 and 5, respectively) were the most potent inhibitors of NO production (IC50 values of 1.1 and 3.3 μM, respectively), followed by myrrhanol A (7) and myrrhanone A (8) (IC50 values of 21.1 and 42.3 μM, respectively). Guggulsterone-M (1) and its didehydro derivative (2) were weak inhibitors, while guggulsterols I (6) and Y (3) were inactive (IC50 >500 μM).Three new and five known compounds were isolated from the oleogum resin of Commiphora wightti (Arnott.) Bhanol. Z- and E-Guggulsterones (4 and 5, respectively) were the most potent inhibitors of LPS-induced NO production (IC50 values of 1.1 and 3.3 μM, respectively).
Keywords: Commiphora wightii; Burseraceae; Oleogum resin; Steroids; LPS-induced nitric oxide; Murine macrophages J774.1;
Antioxidant flavonoids from leaves of Polygonum hydropiper L. by Zhao Feng Peng; Dieter Strack; Alfred Baumert; Ramanathan Subramaniam; Ngoh Khang Goh; Tet Fatt Chia; Swee Ngin Tan; Lian Sai Chia (219-228).
Ten flavonoid compounds were isolated from the dried leaves of Polygonum hydropiper L. (Laksa leaves), and identified as 3-O-α-l-rhamnopyranosyloxy-3′,4′,5,7-tetrahydroxyflavone; 3-O-β-d-glucopyranosyloxy-4′,5,7-trihydroxyflavone; 6-hydroxyapigenin; 6″-O-(3,4,5-trihydroxybenzoyl) 3-O-β-d-glucopyranosyloxy-3′, 4′, 5, 7-tetrahydroxyflavone; scutillarein; 6-hydroxyluteolin; 3′,4′,5,6,7-pentahydroxyflavone; 6-hydroxyluteolin-7-O-β-d-glucopyranoside; quercetin 3-O-β-d-glucuronide; 2″-O-(3,4,5-trihydroxybenzoyl) quercitrin; quercetin. Evaluation of the antioxidative activity, conducted in vitro, by using electron spin resonance (ESR) and ultraviolet visible (UV–vis) spectrophotometric assays, showed that these isolated flavonoids possess strong antioxidative capabilities. Measurement of the Trolox equivalent antioxidant capacity (TEAC) values, against ABTS (2,2′-azinobis(3-ethyl-benzo-thiazoline-6-sulphonic acid) radicals and phenyl-tert-butyl nitrone (PBN) azo initiator (AI) also showed strong anti-oxidative activity. The most powerful of the antioxidants was 2″-O-(3,4,5-trihydroxybenzoyl) quercitrin (galloyl quercitrin). A combination of two flavonoid compounds was tested for synergistic anti-oxidative capacity, but no significant improvement was observed.Ten flavonoid compounds were isolated and identified from the dried leaves of Polygonum hydropiper L. Among these, 2″-O-(3,4,5-trihydroxybenzoyl) quercitrin (galloyl quercitrin) showed high-yield occurrence and the strongest antioxidant activity. A combination of two flavonoid compounds was tested for synergistic antioxidative capacity, but no significant improvement was observed.
Keywords: Polygonum hydropiper L.; Flavonoid; Antioxidant; Trolox equivalent antioxidant capacity (TEAC); Galloyl quercitrin;
Malonylated flavonol glycosides from the petals of Clitoria ternatea by Kohei Kazuma; Naonobu Noda; Masahiko Suzuki (229-237).
Three flavonol glycosides, kaempferol 3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-glucoside, quercetin 3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-glucoside, and myricetin 3-O-(2″,6″-di-O-α-rhamnosyl)-β-glucoside were isolated from the petals of Clitoria ternatea cv. Double Blue, together with eleven known flavonol glycosides. Their structures were identified using UV, MS, and NMR spectroscopy. They were characterized as kaempferol and quercetin 3-(2 G - rhamnosylrutinoside)s, kaempferol, quercetin, and myricetin 3-neohesperidosides, 3-rutinosides, and 3-glucosides in the same tissue. In addition, the presence of myricetin 3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-glucoside was inferred from LC/MS/MS data for crude petal extracts. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.Two malonylated kaempferol and quercetin glycosides and one myricetin glycoside were isolated from the petals of Clitoria ternatea, together with eleven known flavonol glycosides. Their structures were identified by UV, MS, and NMR spectroscopic methods. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.
Keywords: Clitoria ternatea L.; Phaseoleae; Butterfly pea; Kaempferol 3-O-(2″-O-α- rhamnosyl-6″-O-malonyl)-β-glucoside; Quercetin 3-O-(2″-O-α-rhamnosyl-6″-O- malonyl)-β-glucoside; Myricetin 3-O-(2″,6″-di-O-α-rhamnosyl)-β-glucoside;
5H-Furan-2-ones from fungal cultures of Aporpium caryae by Laura M Levy; Gabriela M Cabrera; Jorge E Wright; Alicia M Seldes (239-243).
Four furanones 1–4 with an unusual skeleton containing an acetylene unit, named aporpinones, were isolated from the culture of the basidiomycete Aporpium caryae and their structures were elucidated by spectroscopic methods. Compounds 3 and 4 showed weak to moderate antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Four new furanones (1–4) with an unusual skeleton, named aporpinones, were isolated from the culture of the basidiomycete Aporpium caryae.
Keywords: Aporpium caryae; Basidiomycete; 5H-Furan-2-ones;
Corrigendum to “An immunosuppressive tryptophan-derived alkaloid from Lepidagathis cristata” [Phytochemistry 58 (2001) 1263–1266] by V Ravikanth; V.L.Niranjan Reddy; P Ramesh; T Prabhakar Rao; P.V Diwan; Ashok Khar; Y Venkateswarlu (243).