Phytochemical Investigation and Cytotoxic Properties of Tabernaemontana catharinensis A. DC. Cultivated in Brazil
Many laboratories of natural products have entered into their routines various biological assays simple and phytochemical study of plants extracts used popularly in medicine in order to select bioactive substances. Tabernaemontana catharinensis is a medicinal plant used in folk medicine with antidote for snakebites, to relieve toothache and as a vermifuge to eliminate warts. Phytochemical constituents and cytotoxic effect of the plant were investigated using the qualitative analysis and the brine shrimp lethality method, respectively. The phytochemistry of aqueous and hydroalcoholic extracts of T. catharinensis leaves and stem bark revealed the presence of alkaloids, flavonoids, phenols, tannins, sterols, saponins, catechins and coumarins. The aqueous extracts of this plant did not induce the lethality in the brine shrimp (Artemia salina Leach) bioassay (LC50 = 1057.90±0.48 and 1002.5±0.36 μg mL-1 for leaves and stem bark, respectively); however, hydroalcoholic extracts shows cytotoxicity for this crustacean (LC50 = 629.35±0.12 and 504.18±0.15 μg mL-1 for leaves and stem bark, respectively). In this study, it was observed that the toxicity to Artemia salina converged to the extracts containing the highest amount of bioactive compounds (hydroalcoholic extracts), other in vivo tests will be conducted to determine the exact mechanism of action involved in the toxicity described.
to cite this article:
A.A. Boligon and M.L. Athayde, 2012. Phytochemical Investigation and Cytotoxic Properties of Tabernaemontana catharinensis A. DC. Cultivated in Brazil. Research Journal of Phytochemistry, 6: 127-131.
Received: March 21, 2012;
Accepted: June 25, 2012;
Published: December 03, 2012
Plants of the genus Tabernaemontana, also known as Peschiera,
family Apocynaceae, comprising about 44 species widely distributed in America
and is potentially rich in alkaloids which are considered as chemical markers
of this genus, thus contributing to the classification of its species (Chaturvedula
et al., 2003; Pereira et al., 2008).
Antileishmanial, trypanocidal, antiviral, antimicrobial, antitumoral, anti-inflammatory,
antioxidant, analgesic and cardiotonic activities are well described for the
Tabernaemontana species and presence of triterpenoids, steroids and several
indole alkaloids can be responsible for these actions described (Van
Beek et al., 1984; De Almeida et al.,
2004; Pereira et al., 2008; Lim
et al., 2009). Tabernaemontana catharinensis (common known
as snake skin) is a native tree in the southern part of Brazil and in popularly
medicine, it is used for its anti-inflammatory properties, to relieve toothache,
eliminate warts and as antidote for snakebites (Leeuwenberg,
1994; Pereira et al., 2008).
Several researchers working with plant products are adding into their routine
isolation, purification and structural elucidation, several simple biological
assays in order to select and monitor the phytochemical study of plant extracts
in the search for substances bioactive (Ansari and Inamdar,
2010; Boligon et al., 2012). Among these
bioassays is the brine shrimp toxicity (Artemia salina) which is characterized
by being low cost, fast and does not require aseptic techniques (Mojica
and Micor, 2007; Ramachandran et al., 2011).
Numerous bioactive constituents have been obtained from medicinal herbs such
as: flavonoids, phenolic compounds, alkaloids, steroids, tannins, saponins,
catechins, among others; these chemical constituents are responsible for a particular
pharmacological action on the human body (Janovik et
al., 2011; Agrawal et al., 2011; Namrata
et al., 2011; Gill et al., 2012; Das
et al., 2012).
Considering the importance of the discovery of new therapeutic agents from plant products, the aim of this study was phytochemical investigation and evaluation of cytotoxic effect of the aqueous and hydroalcoholic extracts of T. catharinensis leaves, stem and bark.
MATERIALS AND METHODS
Plant collection: Leaves and stem bark of T. catharinensis were collected in Bossoroca (Rio Grande do Sul State of Brazil) in September of 2009 (coordinates 28°6593 S and 55°0127 W). A dried voucher specimen is preserved in the herbarium of the Department of Biology at Federal University of Santa Maria by register number SMBD 12355.
Preparation of extracts: The leaves (1580.76 g) and stem bark (1051.23 g) of the plant were dried at room temperature and powdered in a knife mill (0.86 μm). The powders were macerated separately at room temperature with ethanol 70% for seven days a week with daily shake-up. A portion of the ethanolic extracts were reserved and the other part was filtered and evaporated to remove ethanol to obtain the aqueous extracts remaining for stem, bark and leaves.
Phytochemical screening: Qualitative phytochemical screening analysis
of the hydroalcoholic and aqueous extracts of the leaves and stem bark of T.
catharinensis were carried out to identify the presence of various phytoconstituents
(Moreira, 1979; Gill et al.,
2012). The phytochemicals groups tested were: Alkaloids (Dragendorff, Bouchardat
and Bertrand reactions), Flavonoids (Shinoda reaction), Phenols and Tannins
(Ferric chloride 1%), Steroids (Liebermann-Burchard reaction), Saponins (Foam
test), Catechins (Na2CO3 reaction), Coumarins (NaOH/Ethanol,
UV) and Reducing sugars (Benedict reaction).
Cytotoxicity assay (Brine Shrimp lethality bioassay): Shrimp eggs were
purchased from a local Pet Shop (Santa Maria, RS, Brazil). The eggs were hatched
in a glass flask containing 1 liter of artificial seawater (Meyer
et al., 1982). The flask was aerated with the aid of an air pump
and kept in a water bath at 29-30°C. A bright light source was left on and
the nauplii hatched within 24 h. Each sample to be tested was dissolved in DMSO
and subsequent diluted serially (10, 5, 1, 0.1 and 0.01 mg mL-1)
in seawater. Ten nauplii were collected with a Pasteur pipette and added to
each set of tubes containing the samples. A check count was performed and the
number of alive nauplii after 24 h was noted. The negative control was saline
solution and the positive control was potassium dichromate solution (20 ppm).
LD50 were determined using the Probit analysis method (Finny,
Statistical analysis: The results of cytotoxic effect were presented as mean of triplicate and Standard Deviation (SD).
RESULTS AND DISCUSSION
Hydroalcoholic extracts of the leaves and stem bark of T. catharinensis showed the presence of alkaloids, flavonoids, phenols, tannins, sterols, saponins and catechins. Coumarin was not found in the hydroalcoholic extract leaves and reducing sugars are not present in the hydroalcoholic extract of both parts of the plant. On the other hand, the aqueous extracts of the plant revealed the presence of alkaloids, phenols, tannins, steroids and catechins (Table 1).
The phytochemical screening is an important step in the chemical and pharmacological
study of a medicinal plant. It may suggest possible pharmacological effects
of its extracts or fractions in comparison of identified phytochemicals groups,
highlighting a close relationship with its main therapeutic uses (Jana
and Shekhawat, 2010; Adinortey et al., 2012).
Thereby, results of phytochemical screening are in accordance with previously
published work, Batina et al. (2000) isolated
from T. catharinensis a quaternary base alkaloid which proved to be able
to inhibit the lethal activity of 2 LD50 of Crotalus durissus
terrificus venom, the presence of alkaloids can also be related with anticancer
and antileishmanial activities described for the specie (Van
Beek et al., 1984). The presence of flavonoids and phenols may have
related to the T. catharinensis use as antioxidant and antimycobacterial
(Pereira et al., 2005). Additionally, steroids
were identified for this specie and this group of substances may promote antitumoral,
anti-inflammatory, analgesic properties (Pereira et al.,
2008; Gill et al., 2011).
The Artemia salina lethality assay has been demonstrated to be an effective,
robust and rapid assay method for primary screening of extracts and compounds
for potential cytotoxic activities (Meyer et al.,
1982; Monira et al., 2012). Cytotoxicity
of T. catharinensis extracts were evaluated by brine shrimp lethality
bioassay (Table 2) in view of the fact that Artemia salina
larvae have been used as a target organism to detect bioactive compounds
in plant extracts (Sam, 1993).
|| Phytochemical analysis of the hydroalcoholic and aqueous
extracts of T. catharinensis leaves and stem bark
|-: Absent, Presence; +++: Strong, ++: Average, +: Weak, No:
|| Cytotoxic activities of the T. catharinensis extracts
against Artemia salina larvae
|Results are expressed as Mean±SD of three determinations
Accordingly to Meyer et al. (1982), who classified
crude extracts and pure substances into toxic (LC50 value <1000
μg mL-1) and non-toxic (LC50 value >1000 μg
mL-1), aqueous tested extracts of T. catharinensis may be
considered non-toxic for this crustacean (LC50 = 1057.90±0.48
and 1002.5±0.36 μg mL-1 for leaves and stem bark, respectively).
However, the hydroalcoholic extracts were classified with toxic (LC50 =
629.35±0.12 and 504.18±0.15 μg mL-1 for leaves
and stem bark, respectively) (Table 2). These toxicological
data, can be correlated with tests acute oral toxicity in animals (Parra
et al., 2001). Das et al. (2012) using
the brine shrimp bioassay demonstrated also toxicity for the hydroalcoholic
extract of Cucumis sativus.
It was concluded that the hydroalcoholic extracts of T. catharinensis leaves and stem bark had cytotoxic activity. The present study in vitro of the plant was a preliminary investigation for future research work. So, further phytochemical and pharmacological studies on T. catharinensis are strongly recommended to elucidate the extract chemical compounds and mechanisms involved.
The authors thank the financial support of FAPERGS/CAPES-Brazil.
Adinortey, M.B., J.K. Sarfo, E.T. Quayson, A. Weremfo, C.A. Adinortey, W. Ekloh and J. Ocran, 2012.
Phytochemical screening, proximate and mineral composition of Launaea taraxacifolia
leaves. Res. J. Med. Plant, 6: 171-179.CrossRef | Direct Link |
Agrawal, B., S. Das and A. Pandey, 2011. Boerhaavia diffusa
Linn: A review on its phytochemical and pharmacological profile. Asian J. Applied Sci., 4: 663-684.CrossRef |
De Almeida, L., A.C.O. Cintra, E.L.G. Veronese, A. Nomizo and J.J. Franco et al
Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis
(Apocynaceae). Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol., 137: 19-27.CrossRef | Direct Link |
Ansari, J.A. and N.N. Inamdar, 2010.
The promise of traditional medicines. Int. J. Pharmacol., 6: 808-812.CrossRef | Direct Link |
Batina, M.D.F.C., A.C.O. Cintra, E.L.G. Veronese, M.A.S. Lavrador and J.R. Giglio et al
Inhibition of the lethal and myotoxic activities of Crotalus durissus
terrificus venom by Tabernaemontana catharinensis
: Identification of one of the active components. Planta Med., 66: 424-428.CrossRef | PubMed | Direct Link |
Boligon, A.A., T.F. de Brum, J.K. Frolhich, A.L.F. Froeder and M.L. Athayde, 2012.
HPLC/DAD profile and determination of total phenolics, flavonoids, tannins and alkaloids contents of Scutia buxifolia
reissek stem bark. Res. J. Phytochem., 6: 84-91.CrossRef | Direct Link |
Chaturvedula, V.S.P., S. Sprague, J.K. Schilling and D.G. Kingston, 2003.
New cytotoxic indole alkaloids from Tabernaemontana calcarea
from the Madagascar rainforest. J. Nat. Prod., 66: 528-531.CrossRef | PubMed |
Das, J., A. Chowdhury, S.K. Biswas, U.K. Karmakar, S.R. Sharif, S.Z. Raihan and M. Abdul Muhit, 2012.
Cytotoxicity and antifungal activities of ethanolic and chloroform extracts of Cucumis sativus
linn (cucurbitaceae) leaves and stems. Res. J. Phytochem., 6: 25-30.CrossRef | Direct Link |
Finny, D.J., 1971.
Probit Analysis. Cambridge University Press, London, UK
Gill, N.S., J. Bajwa, K. Dhiman, P. Sharma and S. Sood et al
Evaluation of therapeutic potential of traditionally consumed Cucumis melo
seeds. Asian J. Plant Sci., 10: 86-91.CrossRef | Direct Link |
Gill, N.S., R. Kaur, R. Arora and M. Bali, 2012.
Phytochemical investigation of Caesalpinia crista
seed extract for their therapeutic potential. Res. J. Med. Plant, 6: 100-107.CrossRef |
Janovik, V., A.A. Boligon, R.V. Bandeira and M.L. Athayde, 2011.
HPLC/DAD analysis, determination of total phenolic and flavonoid contents and antioxidant activity from the leaves of Cariniana domestica
(Mart) miers. Res. J. Phytochem., 5: 209-215.CrossRef | Direct Link |
Leeuwenberg, A.J.M., 1994.
A Revision of Tabernaemontana. The New World Species and Stemmadenia. Vol. 2, The Royal Botanic Gardens, Kew, UK.
Lim, K.H., N.F. Thomas, Z. Abdullah and T.S. Kam, 2009.
Seco-tabersonine alkaloids from Tabernaemontana corymbosa
. Phytochemistry, 70: 424-429.CrossRef | PubMed |
Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. McLaughlin, 1982.
Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med., 45: 31-34.CrossRef | PubMed | Direct Link |
Mojica, E.R.E. and R.L. Micor, 2007.
Bioactivity study of Barringtonia asiatica
(Linnaeus) kurz. seed aqueous extract in Artemia salina
. Int. J. Bot., 3: 325-328.CrossRef | Direct Link |
Monira, S., A. Haque, A. Muhit, N.C. Sarker, A.H.M.K. Alam, A.A. Rahman and P. Khondkar, 2012.
Antimicrobial, antioxidant and cytotoxic properties of hypsizygus tessulatus
cultivated in Bangladesh. Res. J. Med. Plant, 6 : 300-308.CrossRef |
Moreira, E.A., 1979.
Contribution to the phytochemical study of Lobelia hassleri
A. ZAHLB e Lobelia stellfeldii
R. braga companulaceae. Tribuna Farmaceutica, 47: 13-39.
Namrata, L. Kumar, D. Ghosh, S.C. Dwivedi and B. Singh, 2011.
Wild edible plants of uttarakhand Himalaya: A potential nutraceutical source. Res. J. Med. Plant, 5: 670-684.CrossRef | Direct Link |
Parra, A.L., R.S. Yhebra, I.G. Sardinas and L.I. Buela, 2001.
Comparative study of the assay of Artemia salina
L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. Phytomedicine, 8: 395-400.CrossRef | PubMed | Direct Link |
Pereira, P.S., S.C. Franca, P.V.A. Oliveira, C.M.S. Breves and S.I.V. Pereira et al
Chemical constituents from Tabernaemontana catharinensis
root bark: A brief NMR review of indole alkaloids and in vitro
cytotoxicity. Quim. Nova, 31: 20-24.CrossRef |
Pereira, C.G., P.F. Leal, D.N. Sato and M.A.A. Meireles, 2005.
Antioxidant and antimycobacterial activities of Tabernaemontana catharinensis
extracts obtained by Supercritical CO2 + cosolvent. J. Med. Foods, 8: 533-538.PubMed |
Ramachandran, S., M. Vamsikrishna, K.V. Gowthami, B. Heera and M.D. Dhanaraju, 2011.
Assessment of cytotoxic activity of agave cantula using brine shrimp (Artemia salina
) lethality bioassay. Asian J. Sci. Res., 4: 90-94.CrossRef |
Sam, T.W., 1993.
Toxicity Using the Brine Shrimp Artemia salina
. In: Bioactive Natural Products: Detection, Isolation and Structural Determinationm, Colegate, S.M and R.J. Molyneux (Eds.). CRC Press, USA., pp: 441
Jana, S. and G.S. Shekhawat, 2010.
Phytochemical analysis and antibacterial screening of in vivo
and in vitro
extracts of Indian medicinal herb: Anethum graveolens
. Res. J. Med. Plant, 4: 206-212.CrossRef | Direct Link |
Van Beek, T.A., R. Verpoorte, A.B. Svendsen, A.J.M. Leeuwenberg and N.G. Bisset, 1984. Tabernaemontana
L. (Apocynaceae): A review of its taxonomy, phytochemistry, ethnobotany and pharmacology. J. Ethnopharmacol., 10: 1-156.CrossRef |