Transcript Casearia sylvestris

PARTIAL PURIFICATION OF CASEARIA SYLVESTRIS Sw. EXTRACT
AND ITS ANTI-PLA2 ACTION.
Márcia H. Borges1, Claudia M. Jamal2, Daniel M. dos Santos1, Délio S. Raslan2 and Maria Elena De Lima1.
1Laboratório de Venenos e Toxinas Animais – Depto. Bioquímica e Imunologia, ICB-UFMG.
2Laboratório de Produtos Naturais – Depto. Química ICEx-UFMG, Belo Horizonte, MG., Brasil.
Introduction:
Inhibition of PLA2 Activity:
The Flacourtiaceae family encompasses 1300 species that are distributed in the tropical
regions of mainly South America (Joly, 1976). The plants of this family are used in the traditional
medicine as anti-inflammatory, anti-diarrhea, anti-feverish and against snakebite (Teske and
Trentini, 1997).
Snake venoms are complex mixtures of proteins, many of which are toxins or enzymes. As
examples, we can include hemorrhagins, proteases, phospholipase A2 (PLA2) and myotoxins that
act by different mechanism. Phospholipases A2 are found in animal tissues, including snake
venoms. These enzymes are related with wide variety of pharmacological effects induced by
snakebite such as neurotoxicity, myotoxicity, edema and inflammatory response. Furthemore
appears that increased levels of PLA2 are found in inflammatory diseases (Lindahl and Tagesson,
1997).
Previous studies showed that aqueous extract from Casearia sylvestris Sw. was able to
neutralize PLA2 activity and others activities induced by crude snake venoms or by purified toxins
(Borges et al., 2000). Snake venom inhibitors have been purified from many plants (Ferreira et al,
1992; Melo et al, 1994; Melo and Ownby, 1999).
In this work, we report the chemical study of Casearia sylvestris Sw leaves and branches
extracts as well as the ability of its components to inhibit the phosholipase A2 activity (PLA2)
induced by Lachesis muta and Bothrops jararacussu venoms.
Casearia sylvestris crude extract inhibited approximately 64% and 48% of PLA2 activity from
Bothrops jararacussu and Lachesis muta venoms, respectively. Fractions C1 (12%), C2 (45%), C3
(22%), C5 (21%), C7 (18%), C9 (30%), C16 (17%), C22 (14%), C23 (30%) and C27 (10%) neutralized
the PLA2 activity from B. jararacussu venom, while for Lachesis muta venom significative inhibition
was evidenced when fractions C2 (19%), C3 (26%), C8 (17%), and C16 (10%) were used. Others
fractions showed a smaller or any inhibition (Figure 2). The percentages of inhibition are in parentheses.
Neither Casearia sylvestris crude extract nor semi-purified fractions induced PLA2 activity. Results
were expressed as mean  standard deviation. One Way Anova (P <0.005) determined the significance
of the differences between the mean values.
26
8
19
18
17
10
6
47
4
2
0
mEq. NaOH/min/mgVb B.jararacussu
10
50
40
10
14
12
22
30
21 18
30
17
30
45
20
64
10
0
Controle
Cscrude
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
Botanical material was collected in Municipal Park of Cachoeira das Andorinhas (Ouro
Preto - MG). The leaves of Casearia sylvestris Sw were submitted to extraction with hot water
and submitted to three-phase partition (hexane:CHCl3: MeCN:H2O - 2:1:3.4) and bio-guided
assay. Extracts from branches of Casearia sylvestris obtained by percolation were submitted to
the usual procedures of fractionating and purification.
Fractionating of the extracts led to the isolation and identification of hydrocarbons,
aliphatic esters of long chain, sterols, alcohols of long chains, and flavonoids mixtures.
60
Controle
Cscrude
C2
C3
C4
C6
C7
C8
C9
C10
C11
C12
C13
C14
C16
C19
C20
C21
C22
C23
Preparation of Casearia sylvestris Extract:
12
mEq. NaOH/min/mgVb Lachesis muta
Material and Methods:
Effect of Casearia sylvestris fractions on PLA2 activity
induced by B. jararacussu venom.
Effect of Casearia sylvestris fractions on PLA2 activity
induced by Lachesis muta venom.
Figure 2: Effect of Casearia sylvestris crude extract/fractions on PLA2 activity of L.muta and B. jararacussu venoms. Bothrops jararacussu (20mg) and
Lachesis muta (50mg) solutions were incubated with Casearia sylvestris extract or its fractions for 30 min at 37oC. Extract/fractions were weighed and
dissolved in deionized water before use. The extract/fractions concentration were expressed in terms of dry weight and the amount used in each test was
estimated according to concentration of the venom in a ratio 1:5 (venom:extract). Numbers on the bars represents inhibition percentage.
PLA2 Activity
Discussions and Conclusions:
Enzymatic activity was assayed according De Haas et al.1968) using egg yolk as
substrate. Lachesis muta (50mg) and Bothrops jararacussu (20mg) venoms were dissolved in
salina and protein concentration was estimated according to Lowry (1952). Fat acids released
were titraed with NaOH 0.12N. PLA2 activity was expressed in mEq.NaOH/min/mg venom.
In this work, we verified that Casearia sylvestris crude extract inhibited significantly both PLA2
activity of Bothrops jararacussu and Lachesis muta venoms. Some fractions were particularly more
effective than others in the inhibition of PLA2 activity induced by these venoms.
Several studies have demonstrated that flavonoids are able to inhibit PLA2 activity (Gil et al., 1994;
Lindahl and Tagesson, 1993). Lindhal and Tagesson (1997) reported that certain flavonoids, such as rutin
and quercetin appear to inhibit selectively phospholipases A2 group-II but not PLA2 group-I. This
selectivity seems to be related to the chemical structure of the flavonoid. Our results show that rutin
(C14), an isolated fraction, neutralized approximately 8% of the PLA2 activity from Bothrops jararacussu
venom, but did not inhibit PLA2 from Lachesis muta venom.
Considering that venoms used in this work are a mixture of many substances, we could suggest
that interactions of the extract/fractions are different for both venoms.
High percentage of inhibition when crude extract was used compared to lower percentages for
Casearia sylvestris fractions may indicate synergism among the fractions.
PLA2 inhibitors have a medical interest because increased levels PLA2 enzymes (mainly group II)
are found in many inflammatory diseases. Although our results are preliminary subsequent studies may
indicate the possible use of this extract to development of new drugs to treatment for inflammatory
diseases.
Inhibition of PLA2 Activity
For inhibition test, Bothrops jararacussu (20mg) and Lachesis muta (50mg) solutions
were incubated with Casearia sylvestris extract or their fractions for 30 min at 37oC.
Extract/fractions were weighed and dissolved in deionized water before use. The extract
concentration was expressed in terms of dry weight and the amount of extract used in each test
was estimated according to the concentration of the venom in a ratio 1:5 (venom:extract).
Results:
Phytochemical study of Casearia sylvestris
Casearia
sylvestris
References:
Leaves
(1038g)
Branches
(450g)
Water
Aqueous
extract*
(92g)
hexane: acetonitrile:
chloroform: water
(2:1:3.4)
Hexane
extract
Acetonitrile/
chloroform
extract (5.0g)
Benzene
extract
(6.01)
Marc
C19; C20; C21
C22; C23*; C24
C25; C26; C27*
and C281
Chloroform
Aqueous extract
(58.0g)
Chloroform
extract (1.8g)
Marc
Ethanol
C12,*
C141,2,*
C2*,2; C3*; C4; C5*;C6 C7*;
C8; C9*; C10;C11 C12;
C13; C14* and C152,*
Ethanol extract
(44.5g)
1.
Borges, M. H., et al., 2000. Effects of aqueous extract of Casearia sylvestris (flacourtiaceae) on actions of snake and
bee venoms and on activity of phospholipases A2. Comparative Biochemistry and Physiology, 127B, 21-30.
2.
de Haas, G. H. et al. (1968) Purification and properties of phospholipase A2 from porcine pancreas. Biochim.
Biophys. Acta 159, 103-117.
3.
Ferreira, L. A. F., 1992. Antivenom and biological effects of ar-tumerone isolated from Curcuma longa
(zingiberiaceae). Toxicon 30, (10) 1211-1218.
4.
Gil, B.et al.(1994) Effects od flavonoids on Naja naja and human recombinant synovial phospholipases A2 and
inflammatory responses in mice. Life Sci. 54:333-338.
5.
Joly A.B.,, 1976 Introdução a taxonomia vegetalEditora Nacional, Rio de Janeiro, pp. 470-472
6.
Lindahl, M. and Tagesson, C. (1993) Selective inhibition of group II phospholipases A2 by quercetin. Inflammation
17:573-582.
7.
Lindahl, M. and Tagesson, C. 1997 Flavonoids as Phospholipase PLA2 inhibitors: Importance of their structure for
selective Inhbition of Group II Phospholipase A2. Inflammation, 21(3), 347-356.
8.
Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R..J.(1951) Protein measurement with phenol reagent. J.
Biol. Chem.193,265-275.
9.
Melo, P. A., et al., 1994. Inhibition of the myotoxic and haemorrhagic activities of crotalid venoms by Eclipta
prostata (Asteraceae) extracts and constituents. Toxicon 32 (5), 595-603.
10.
Melo, P.A. and Ownby, C. L., 1999. Ability of wedelolactone, heparin and para-bromophenacyl bromide to
antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins. Toxicon, 37, 199-215.
11.
Teske M. and Trentini A.M.M. 1997 Compêndio de Fitoterapia 3ed., Curitiba 317p.
C163,* C17; C18;
C28 C29 and C301
Figure 1. Phytochemical study and bio-guided screening of Casearia sylvestris
.
* Active fractions; 1-isolated compounds; 2- identified in the fractions/crude extracts by HPLC; 3- identified in the fractions by gas
chromatography (GC).
Surpport: CNPq, CAPES, FAPEMIG