IJLSSR, VOLUME 3, ISSUE 4, JULY 2017:1137-1141

Research Article (Open access)

Identified Substances from the Leaves of Tephrosia cinerea (Leguminoseae)
Crude Extracts and their Phytotoxic Effects

Antonio Jose Cantanhede Filho1*, Lourivaldo Silva Santos2, Giselle Maria Skelding Pinheiro Guilhon2, Raissa
Costa Moraes2, Reinaldo Araujo Dos Santos2, Antonio Pedro Da Silva Souza Filho3, Juliana Feitosa Felizzola3

1Federal Institute of Education Science and Technology of Maranhao, Campus Sao Luis- Monte Castelo, Sao Luis- MA, Brazil
2Federal University of Para, Institute of Exact and Natural Sciences, Belem- PA, Brazil
3Embrapa Amazonia Oriental, Agroindústria Laboratory, Belem- PA, Brazil

*Address for Correspondence: Dr. Antonio Jose Cantanhede Filho, Teacher, Department of Natural Products, Federal
Institute of Education, Science and Technology of Maranhao, Sao Luis Campus- Monte Castelo, Getúlio Vargas
Avenue 04, CEP 65030-005, Sao Luis- MA, Brazil

Received: 07 March 2017/Revised: 05 May 2017/Accepted: 17 June 2017

ABSTRACT- This research evaluated the phytotoxic effect of the hexane (H.E), ethyl acetate (EtOAc.E) and methanolic (MeOH.E) crude extracts of the Tephrosia cinerea leaves on the seed germination of seeds using two weed species, Mimosa pudica (Malícia) and Senna obtusifolia (Mata-pasto), as test plants. The compounds were isolated using classic chromatography techniques and the structural elucidation of the compounds was performed by 1H and 13C NMR (1D and 2D) techniques. The ethyl acetate and methanolic extracts of T. cinerea were the most active, as they inhibited the germination of seeds in 92.0% and 81.0% respectively of malícia and mata-pasto, the ethyl acetate extract inhibited germination by 81.0% and the methanolic extract by 32.0%. The chemical study led to isolation of cinnamic acid and rotenone from the ethyl acetate extract, and mixture containing triacylglycerol and ß-sitosterol fatty acids from the hexane extract and the disaccharide trehalose from methanolic extract.
Key-words- Invasive species, Phytotoxicity, Crude extracts, Rotenone

Tephrosia cinerea, (Leguminoseae/Papilionoideae) is a Brazilian non-endemic native bush that presents two relevant synonyms, Tephrosia littoralis (Jacq). Benth. and Tephrosia villosior Benth [1]. Commonly known in Brazil as “anil bravo”, this plant is narcotic, vermifuge and poisonous, useful against swollen glands, ulcers and nerve disorders [2].
Four substances have been identified from the leaves of T. cinerea: Flemichapparin B, Isolonchocarpin, Anhydrolanceolatin-A and Rutin [2]. Methylapollinin 7-O-ß-glucopyranoside, and cineroside (A) have been isolated from the aerials parts of this species [3].
Weed or invasive species, which often spoil the crop are one of the most important factors to impose limitations to the world agricultural activity development [4]. The known toxic effects of commercial herbicides on human health range from nausea and vomiting to certain kinds of cancer. As for environmental damages, they can accumulate in the biota or contaminate waters and soil, which may lead to ecological imbalance [5].
Natural products based herbicides and agrochemicals are attractive for many reasons. These products are often considered to not cause environmental damages, causes many of them are easily biodegradable and due to the fact they are, at least partially, water-soluble, favoring their absorption by plants, it’s possible to use them in smaller amounts, therefore offering less risks to crop rotation [6-8]. In order to investigate extracts and/or substances that may be used as bioherbicides, Tephrosia cinerea leaves was subject to chemical investigation and phytotoxic effects evaluation, using germination inhibition assays against two weed species, Mimosa pudica (Malicia) and Senna obtusifolia (Mata-pasto).

Plant collection and extraction-
Tephrosia cinerea leaves were collected on June 2011 in an area belonging to the Atico Seabra Herbarium, located at Maranhao Federal University (UFMA). A voucher specimen (#1256) was deposited in the same Herbarium.
Part of the botanical material (780.2 g) was ground using a knife mill after drying for one week in a low humidity environment. The crude extracts were prepared by maceration with organic solvents for seven days with each solvent in the following sequence: hexane (Hex), ethyl acetate (EtOAc) and methanol (MeOH). The solutions were concentrated in a rotary evaporator, giving finally the hexane (9.3 g), ethyl acetate (5.7 g) and methanolic extracts (5.1 g).

Isolation and purification of compounds (1-5)- (1-5)- All three crude extracts were submitted to column chromatography (CC) on silica (70-230 mesh) using hexane/EtOAc and EtOAc/methanol mixtures as eluents in order of increasing polarity. Successive CC procedures were used to purify some fractions. These fractions were analyzed by thin layer chromatography (TLC) in aluminum backed silica gel 60 F254 plates and as revealed in UV light fluorescence chambers (254 and 366 nm) and by a ceric sulphate acid solution, followed by heating.
Fractioning of the ethyl acetate extract (5.7 g) resulted on 58 fractions. Needle-shaped crystals (34.0 mg) pre-cipitated from fractions 10-12, eluted from the column with a Hex- EtOAc 8:2 solutions. These crystals were identified as (E)-cinnamic acid (substance 1). After solvent evaporation, fractions 21-25 (eluted with a Hex-EtOAc 7:3 mixture) were purified through successive washings with a Hex- CH2Cl2 1:1solution, at low temperature, giving a syellowish solid, identified as rotenone, substance 2 (24 mg).
The hexane extract (9.3 g) CC fractioning led to 68 fractions. Fractions 4-11, eluted from the column with a Hex - EtOAc 9:1 solution was identified as triacylglycerols, substance 3 (38.8 mg). The major com-pounds of fractions 12-25 (eluted with Hex - EtOAc 8:2) were fatty esters of ß-sitosterol (substance 4.12 mg). Fractions 39 and 40, eluted from the column with a Hex-EtOAc 1:1 solution, produced, after precipitation and washing with a Hex-CH2Cl2 1:1 mixture, at low temperature, an additional amount of substance 1 (19.0 mg).
The methanolic extract originated 64 fractions after CC fractioning. Fractions 1-4 provided an additional amount of 1 and 2 in a mixture (20.0 mg). A C-18 cartridge filtration a MeOH solution of fractions 18-20 (eluted with a mixture of EtOAc-MeOH result on a mixture of the trehalose disaccharides isomers a,a-trehalose, a,ß-trehalose and ß,ß-trehalose, substance 5 (38 mg).

Nuclear Magnetic Resonance (NMR) Analysis- 1H and 13C (1D e 2D) NMR spectra were obtained from a Varian spectrometer, model MERCURY-300 (300 MHz for 1H and 75 MHz for 13C), using CDCl3 (1-4) and CD3OD (5) as solvents. The chemical shifts (d) were recorded in ppm based on the TMS signal. The experimental data were compared with those found in literature.

Bioassays methodology- Phytotoxic effects assays were performed according to methodologies described in literature [9-11]. Tephrosia cinerea crude extracts were evaluated (hexane, ethyl acetate, and methanolic extracts). Seeds of the test plants, M. pudica and S. obtusifolia were collected in the area of Castanhal country in the state of Pará. The seeds went through cleaning process and treated for dormancy break by immersion in sulfuric acid (H2SO4) solution for 15 minutes (M. pudica) and 20 minutes (S. obtusifolia) and then they were dried at room temperature [12].

Seeds germination- A BOD germination chamber was used at the constant temperature of 25ºC and 12 hours photoperiod. Each 9.0 centimeters diameter Petri dish lined with filter paper received 3 ml of each 1% concentrated (m/v) crude extract. After solvent evaporation, the filter paper was moistened with a fungicide aqueous solution (mycostatin– 1%), and then were inserted 20 seeds of the recipient plants. The control samples received only the fungicide aqueous solution and considered as germinated seeds, which presented root extension equal to or greater than 2.0 mm. Seeds germination was monitored for a period of 4 days, being the first germination count made 24 hours after assembly of the experiment. Count was realized daily with germinated seeds elimination. The Equation 1 below was used to calculate seeds germination inhibition (I) percentage [13-14].

I (%) = [1 – (SGsample)] × 100/SGcontrol

Where: SGsample is the number of germinated seeds, SGcontrol is the number of germinated seeds in the control sample.

For verification of biological effects, experimental lin-eation was entirely randomized with three repetitions for each one. Bar chart was made in Excel 2013.

Phytotoxic effects of Tephrosia cinerea crude extracts on Seeds germination
- The seeds germination inhibition of each extract (ethyl acetate, hexane and methanolic) on invasive species Mimosa pudica, and Senna obtusifolia are shown in Fig 1. The hexane extract inhibited 48.0% germination of the M. pudica while the ethyl acetate and the methanolic extract inhibited 92.0% and 81.0% respectively. The hexane extract inhibited 3.3% germination of the S. obtusifolia, and the ethyl acetate and the methanolic extract, 81.0% and 32.2% respectively.

Fig. 1: Seed germination inhibitory effects of the crude extracts of Tephrosia cinerea leaves, 1% (m/v) in
relation to the control test, using distilled water

The inhibition of seed germination the weed species M. pudica and S. obtusifolia caused by T. cinerea extracts (hexane, ethyl acetate and methanolic extracts) are shown in Fig 1. The hexane extract inhibited 48.0% of the seed germination of M. pudica, while the ethyl acetate and the methanolic extracts inhibited 92.0% and 81.0%, respectively. Inhibition of 3.3% on the germination of S. obtusifolia seeds was observed when the hexane extract was present, and of 81.0% and 32.2% when the ethyl acetate and the methanolic extracts were tested respectively.

Substance identification by NMR- sSubstances obtained from crude extracts of T. cinerea leaves were identified as (E)-cinnamic acid (1) and rotenone (2). A mixture of triacylglycerol (3), ß-sitosterol fatty esters (4) and trehalose disaccharides (5). Structures for the substances 1-5 are shown in Fig 2.


Fig. 2: Isolated and identified substances in crude extracts from Tephrosia cinerea leaves

Confirmation of the structures of the isolated sub-stances (1 and 2), as well as the substances identified in mixtures (3-5), was given by 1H and 13C NMR spectra analysis and comparison with literature data [15-19]. Identification of substance 2 was also was given by DEPT-135 and correlation maps COSY, HMBC e HSQC analysis and comparison to literature data [16].
Substance 1: 1H NMR (300 MHz, CDCl3), d (mult., J in Hz, H): 6.48 (d, 16.0, H-8), 7.40 (m, H-2/H-6), 7.56 (m, H-3/H-4/H-5), 7.81 (d, 16.0, H-7). 13C NMR (75 MHz, CDCl3) d: 117.3 (C-8), 128.3 (C-3/C-5), 128.8 (C-2/C-6), 130.6 (C-4), 135.9 (C-1), 147.1 (C-7), 172.5 (C-9). These data are in agreement with data found in literature for cinnamic acid [15].
Substance 2: : 1H NMR (300 MHz, CDCl3) d (mult., H): 1.74 (ls, H-8’), 2.95 (dd, 15.8 and 8.1, a H-4’), 3.31 (dd, 15.8 and 8.1), 3.74/3.78 (s, 2 x OCH3), 4.15 (d, 12.0), 4.56 (d, 12.0), 4.90 (d, 3.6, H-6a), 4.91 (ls, H-7’a), 5.05 (m,), 6.46 (s, H-1), 6.75 (ls, H-4). 13C NMR (75 MHz, CDCl3) d: 17.0 (C-8’), 31.1 (C-4’), 44.4 (C-12a), 55.7/56.2 (2 x OCH3), 66.1 (C-6), 72.1 (C-6a), 87.7 (C-5’), 100.8 (C-1), 104.7 (C-1a), 104.8 (C-11), 110.2 (C-4), 112.4 (C-7’) 112.8 (C-8), 113.2 (C-11a), 129.8 (C-10), 142.9 (C-6’), 143.7 (C-2), 147.2 (C-4a), 149.3 (C-3), 157.8 (C-7a), 167.3 (C-9), 188.9 (C-12). These data are in agreement with data found in literature for rotenone [16].
Substance 3: : 1H NMR (300 MHz, CDCl3), d (H): Several signals between 0.84-0.88 ppm (methyl hydrogens), 2.30-2.32 ppm (carbonyl a-hydrogens), 2.34-2.08 ppm (methylene allylic hydrogens), 2.74-2.79 ppm (bis-allylic hydrogens), 1.59 ppm (carbonyl ß-hydrogens), two around 4.3-4.0 ppm (oxidized methylene hydrogens from the glycerol moiety), intense signals between 1.24-1.30 ppm (other equivalent methylene hydrogens), 5.00-5.34 ppm (olefinic hydrogens derived from unsaturated fatty acids and oxidized methine hydrogen derived from glycerol). All 1H and 13C NMR signals were assigned by .comparison with literature data for triacylglicerols [17].
Substances 4: 1H NMR (300 MHz, CDCl3), d (H): Several signals between 0.67-0.86 ppm (methyl hydrogens), (methylene hydrogens of the fatty acid moiety), a multiplet at 4.55 ppm (H-3 the high chemical shift is an evidence that carbon C-3 is esterified), a large doublet at 5.34 ppm with J = 5.7 Hz (olefinic hydrogen H-6). 13C NMR (75 MHz, CDCl3): 73.6 ppm (C-3), 140.7 ppm (C-5), 121.7 (C-6) ppm, among others. 1H and 13C NMR data for ß-sitosterol fatty esters were compared to literature’s [18].
Substance (5): 1H NMR (300 MHz, CD3OD), d (H): Several signals between 3.00-3.93 ppm (oxidized methylene and methine hydrogens of a sugar unit), doublets at 4.51 ppm, J= 8.1 Hz and 5.14 ppm, J = 3.6 Hz (anomeric hydrogens H-1 e H-1’). 13C NMR (75 MHz, CD3OD): signals between 61.7-69.1 ppm (oxidized methylene carbons), signals between 70.9-82.8 ppm (oxidized methine carbons) and signals between 93.7-102.9 ppm (anomeric carbons) that are characteristic of mixtures of the isomers of the disaccharide trehalose (a,a-trehalose; a,ß-trehalose; ß,ß-trehalose). The major component was a,ß-trehalose. The NMR data is in accordance to literature data [19]. The presence of (E)-cinnamic acid (1) and rotenone (2) in both EtOAc and MeOH extracts, justifies the seeds germination inhibition effects on the test plants. Substance (1) was also tested in seeds germination inhibition and radicle and hypocotyl growth experiments for the same test plants and the inhibition was 99% at 0.1% concentration [20]. Rotenone (2), which is commonly found in legumes belonging to Derris and Lonchocarpus genera, as well as Tephrosia genus popularly known as Timbos, also has a potent phytotoxic effect against the same test plants [21].

Chemical study of the crude extracts from Tephrosia cinerea leaves showed that this species is an important source of phenolic acids and rothenoids, classes of substances usually associated with Tephrosia genus. Evaluation of seed germination inhibition using Mimosa pudicaand Senna obtusifolia as test plants shows important inhibition effects were caused by the ethyl acetate and methanol extracts, the hexane extract was less effective on the inhibition. Mimosa pudica was the most sensitive species to all tested extracts. The seed germination inhibition of the ethyl acetate and methanol extracts can be related to the presence of (E)-cinnamic acid (1) and rotenone (2) in these extracts. Therefore, this study allows one to conclude that the crude extracts of T. cinerea leaves have allelopathic potential and can be used in composition of bioherbicides for the tested invasive species seeds germination control. New studies must be carried out as well to evaluate possible phytotoxic effects of these same plants on seedlings (radicle and hypocotyl) development.

The authors would like to thank the Graduate Program in Chemistry (Chemistry Graduation Program) from the Universidade Federal do Para (Federal University of Para), the (Chemistry Academic Department) from the Federal Institute of Education Science and Technology of Maranhao (Science and Technology Federal Institute of Maranhao) and EMBRAPA-PA for the support and infrastructure.

  1. Queiroz R. T., & Tozzi A. M. Tephrosia cinerea. Retrieved from List of Brazilian Flora Species: http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB83839, 2015.
  2. Malcher GT, Arriaga AMC, Gomes TBM, Vasconcelos JN, Rodrigues FEA, Santiago GMP. Tephrosia cinerea Pers. (Fa-baceae) chemical study. Paper presented at 30th Brazilian Chemistry Society Annual Reunion 2007 May 31-Jun 6; Águas de Lindóia, Brazil.
  3. Maldini M, Montoro P, Macchia M, Pizza C, Piacente S. Profiling of Phenolics from Tephrosia cinerea. Planta Médi-ca, 2011; 77(16): 1861-1864.
  4. Inoue MH, Santana DC, Souza Filho APS, Possamai ACS, Silva LE, Pereira MJB, Pereira KM. Allelopathic potential of Annona crassiflora: Effects on weeds. Planta Daninha, 2010; 28(3): 489-498.
  5. Soares LW, Porto FM. Agricultural activity and environmental externality: an analysis of the use of pesticides in the Brazilian savannah. Science and Collective Health, 2007; 12(1): 131-147.
  6. Alves SM, Muller AH, Souza Filho APS.Allelopathy: basic principles and general aspects. Belém, PA: Embrapa Amazônia Oriental; c2002. Chapter 8, Allelopathy and the production of pesticides; p. 205-260.
  7. Vyvyan JR. Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron, 2002; 58(9): 1631-1646
  8. Huang H, Morgan CM, Asolkar RN, Marja E, Koivunen ME, Marrone PG. Phytotoxicity of sarmentine isolated from long pepper (Piper longum) fruit. Journal of agricultural and food chemistry, 2010; 58(18): 9994-10000.
  9. Inderjit, Dakshini KMM. On laboratory biossays in allelopa-thy. The Botanical Review, 1995; 6(1): 28-44.
  10. Macías FA, Castellano D, Molinillo JMG. Search for a standard phytotoxic bioassay for allelochemicals. Selection of standard target species. Journal of Agricultural Food and Chemistry, 2000; 48(6): 2512-2521.
  11. Brazil Ministry of Agriculture. (2009). Rules for seeds’ analysis. Retrieved from the Ministry of Agriculture, Livestock and Food Supply of Brazil website: http://www.agricultura.gov.br/assuntos/insumos-agropecuarios/arquivos-publicacoes-insumos/2946_regras_analise__sementes.pdf/@@download/file/2946_regras_analise__sementes.pdf
  12. Souza Filho APS, Dutra S, Silva MAMM. Methods of determination of plant seed dormancy Cultivated grasslands of Amazonia. Weed plant, 1998; 16(1): 2-11.
  13. Hamdi BA, Inderjit, Olofsdotter M, Streibig JC. Laboratory bioassay for phytotoxicity: an example from wheat straw. Agronomy Journal, 2001; 93(1): 43-48.
  14. Santos LS, Borges FC, Oliveira MN, Arruda AC. Allelo-chemicals isolated from the leaves of Virola michelli Heckel. Allelopathy Journal, 2007; 20(1): 235-242.
  15. Xia LZ, Zhou M, Xiao YH, Li GY, Chen XZ, Zhang G. Chemical constituents from Helwingia japonica.Chinese Journal of Natural Medicine, 2010; 8(1): 16-20.
  16. [16] Leuner O, Havilik J, Budesinsky M, Vrkoslav V, Chu J, Bradshaw TD, Hummelova J, Miksatkova P, Lapcik O, Val-terova I, Kokoska L. Cytotoxic constituents of Pachyrhizus tuberosus from Peruvian Amazon. Natural Products Com-munications, 2013; 8(10): 1423-1426.
  17. Thoss V, Murphy PJ, Marriot R, Wilson T. Triacylglycerol composition of British bluebell (Hyacinthoides non-scripta) seed oil. RSC Advances, 2012; 2(12): 5314-5322. Correia SJ, David JM, Silva EP, David JP, Lopes LMX, Guedes MLS. Flavonoids, norisoprenoids and other terpenes from leaves of Tapirira guianensis. Quimica Nova, 2008; 31(8): 2056-2059.
  18. Roslund MU, Tähtinen P, Niemitz M, Sjöholm R. Complete assignments of the 1H and 13C chemical shifts and JH,H coupling constants in NMR spectra of D-glucopyranose and all D-glucopyranosyl-D-glucopyranosides. Carbohydrate Research, 2008; 343(1): 101-112.
  19. Pereira SG, Guilhon GMSP, Santos LS, Pacheco LC, Can-tanhede Filho AJ, Souza Filho APS. Phytotoxicity of the or-ganic phase and the major compound obtained from fruit pulp of Crescentia cujete L. (Bignoniaceae). Biotemas, 2015; 28(4): 51-59.
  20. Souza Filho APS, Nascimento JLM.Timbó: Botanical aspects and bioactive molecules: Embrapa Eastern Ama-zon; 2012, pp:94.

    How to cite this article:
    Cantanhede Filho AJ, Santos LS, Pinheiro Guilhon GMS, Costa Moraes RP, Araujo dos Santos R, Pedro da Silva Souza Filho A,
    Felizzola JF: Identified Substances from the Leaves of Tephrosia cinerea (Leguminoseae) Crude Extracts and their
    Phytotoxic Effects. Int. J. Life. Sci. Scienti. Res., 2017; 3(4):1137-1141. DOI:10.21276/ijlssr.2017.3.4.6
    Source of Financial Support: Nil, Conflict of interest: Nil