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:

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.

Material and Methods:

Preparation of Casearia sylvestris Extract:

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.

PLA2 Activity

Enzymatic activity was assayed according De Haas et al.1968) using egg yolk as substrate. Lachesis muta (50g) and Bothrops jararacussu (20g) 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 Eq.NaOH/min/mg venom.

Inhibition of PLA2 Activity

For inhibition test, Bothrops jararacussu (20g) and Lachesis muta (50g) 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).

Inhibition of PLA2 Activity:

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.

Results:Phytochemical study of Casearia sylvestris

Discussions and Conclusions:

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.

References:

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. 470-4726. 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.

Con

trol

e

Csc

rude C

2

C3

C4

C6

C7

C8

C9

C10

C11

C12

C13

C14

C16

C19

C20

C21

C22

C23

0

2

4

6

8

10

12

1017

1826

19

47

Eq

. NaO

H/m

in/m

gV

b

Lac

hes

is m

uta

Effect of Casearia sylvestris fractions on PLA2 activity

induced by Lachesis muta venom.

Con

trole

Csc

rude C

1C

2C

3C

4C

5C

6C

7C

8C

9C

10C

11C

12C

13C

14C

15C

16C

17C

18C

19C

20C

21C

22C

23C

24C

25C

26C

27C

28C

29C

30

0

10

20

30

40

50

60

10

30

1417

301812 2122

45

64

Eq.

NaO

H/m

in/m

gVb

B.ja

rara

cuss

u

Effect of Casearia sylvestris fractions on PLA2 activity

induced by B. jararacussu venom.

Figure 2: Effect of Casearia sylvestris crude extract/fractions on PLA2 activity of L.muta and B. jararacussu venoms. Bothrops jararacussu (20g) and Lachesis muta (50g) 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.

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).

Casearia sylvestris

Leaves(1038g) Branches

(450g)

Aqueous extract*

(92g)

Water

Benzene extract(6.01)

Marc

C19; C20; C21 C22; C23*; C24 C25; C26; C27*

and C281

Chloroform

Chloroform extract (1.8g)

Marc

Ethanol

Ethanol extract(44.5g)

C163,* C17; C18; C28 C29 and C301

hexane: acetonitrile: chloroform: water (2:1:3.4)

Hexane extract

Acetonitrile/chloroform

extract (5.0g)

Aqueous extract (58.0g)

C12,* C141,2,* C2*,2; C3*; C4; C5*;C6 C7*; C8; C9*; C10;C11 C12; C13; C14* and C152,*

Surpport: CNPq, CAPES, FAPEMIG