Journal of Ethnopharmacology, Elsevier
Scientific
Publishers
EFFECTS OF FIG LATEX ON LIPID CCIJNDUCED LIPID PEROXIDATION
ZAINAB
A.F.
AL-BAYATI
215
30 (1990) 215-221 Ireland Ltd.
PEROXIDATION AND IN RAT LIVER
and AL1 H. ALWAN
Pharmacognosy and Pharmacology Department, Biological Research Centre, Scientific Research Council, Jadiriyah, P.O. Box 2371 Baghdad IIraq) (Accepted
May 14,199O)
sllmmary The oral and intraperitoneal effects of fig milk latex on lipid peroxidation and Ccl,-induced lipid peroxidation in liver homogenates of female rats were investigated. Oral treatment had no effect, while i.p. administration produced a significant increase in hepatic lipid peroxidation. When the latex was given before Ccl, treatment: it produced no protective effect against Ccl,-induced hepatotoxicity. Addition of the latex to the incubation mixture produced a dose-dependent increase in lipid autoxidation, while the chloroform and ether extracts of the latex, as well as heated latex, had no effect on hepatic lipid autoxidation.
Introduction
The common fig-tree, Ficus carica L. (Moraceael, is indigenous to most It is cultivated in Asia Minor, the warm and temperate climates. Mediterranean countries, all the milder areas of Europe and in the United State of America (Guest, 1966; Chakaravarty, 19761. The milky juice of freshly-broken buds, stalks and leaves is very acrid and and has been used in some countries for the treatment of warts and blisters on the body (Perdue and Hartwell, 19691. This latex has high coagulating activity and it has been used for the preparation of cheese and junkets, fat extraction from meat and in other food-processing industries and in medicine (Chakaravarty, 1976). The latex contains enzymes such as peroxidase, proteolytic enzymes, diastase, lipase, and catalase (Chakaravarty, 19761. Recent studies have demonstrated that the latex will inhibit [3HJbenzo[a]pyrene binding to rat hepatic microsomes (Alwan and Al-Bayati, 19881. The role of lipid peroxidation in biological systems is of recent research interest due to its involvement in a number of pathological and toxicological 0 1990 Elsevier 037%8741/$03.50 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
216
conditions (Plaa and Witschi, 1976; Tihanyi et al., 1985; Halliwell and Gutteridge, 19861. Furthermore, lipid peroxidation has been shown to be modified by antioxidants present in plants (Stich and Rosin, 1984; Kiso et al., 1985; Hikino et al., 19861. Therefore, we have examined the effect of administering fig milk latex orally and i.p. on lipid peroxidation and on carbon tetrachloride (CClJ-induced hepatic lipid peroxidation in female rats. In addition, the effects of adding milk latex, centrifuged upper and lower layers, organic fractions and heated milk latex on hepatic lipid autoxidation were also investigated. Materials and methods Chemical
sources
Thiobarbituric acid (TBA), Ccl,, KCl, H,PO, and n-butanol from Fluka, AG (Switzerland). Collection and preparation
were purchased
of fig latex
The milky latex of freshly broken stalks and leaves was collected in the early morning. One part of milk latex was extracted with an equal volume of chloroform or ether, mixed and centrifuged for 10 min at room temperature. The residue from each extraction was dissolved in DMSO and used for experimental assay. The milk latex (2 ml) was centrifuged and the upper and lower layers separated. The latex (1 ml1 was heated at 100°C for 1 h to destroy the enzymes. Animal
treatment
Female albino Wistar rats (140- 160 g; bred in our laboratory1 were housed in stainless steel hanging cages and provided free access to feed (Standard chow, Al-Kadhmya, Baghdad) and water. One group of animals was treated orally with 1 ml/kg of milk latex in corn oil for one day. A second group was treated i.p. with 1 ml/kg of milk latex in corn oil for one day. Control animals received corn oil only (1 ml/kg). Half of each milk latex-treated group was given Ccl, (0.5 ml/kg) in corn oil in a single dose orally 1 h after milk latex treatment. Lipid peroxidation
All animals were killed the day after treatment. Livers were removed and 20%~ homogenates were prepared in ice cold 1.15% KCl. The amount of malondialdedhyde (MDA) present was determined by using the procedure of Uchiyama and Mihara (1978). To 0.5 ml of the 20% homogenate were added 3.0 ml 1% H,PO, and 1.0 ml 0.6% TBA solution. Each mixture was heated for 45 min, cooled and extracted with n-butanol. The optical densities of the organic phases were determined at 535 and 520 nm, and MDA was calculated using an extinction coefficient of 1.56 x 105/M cm (Sinnhuber and Yu, 19581. Lipid autoxidation was measured as described by Stocks et al. (19741. The
217
liver homogenate (5 ml) was prepared and incubated at 37OC for 1 h. MDA formation was measured by the TBA reaction. Protein concentrations were determined according to the method of Lowry et al. (1951). Results
When rats were treated orally with 1 ml/kg of latex and killed one day post-treatment, no significant changes in MDA content (as an index of lipid peroxidation) of liver homogenates were observed in the latex-treated group as compared to the control group (Table 1). Ccl, was used to induce hepatic lipid peroxidation and also served as a positive control. Ccl, administration alone increased MDA content three-fold. Latex given together with Ccl, had no effect on MDA content as compared to Ccl,treated rats not receiving the latex. Latex administered i.p. resulted in a five-fold increase in hepatic MDA content as compared to the corn oil control animals (Table 2). This increase in lipid peroxidation was greater than that seen in animals receiving latex plus Ccl, or Ccl, alone. The ability of the latex to increase hepatic lipid autoxidation when added in vitro is presented in Table 3. A dose as low as 10 ~1 of latex was able to increase significantly MDA content two-fold. The effects of the upper and lower layers of milk latex after centrifugation, the organic extracts (chloroform and ether fractions) and heated milk latex on in vitro hepatic autoxidation as determined by MDA formation using liver homogenate from control rats are presented in Table 4. Both upper and lower layers of the milk latex equally stimulated MDA formation. Chloroform extract produced no significant stimulation of MDA formation, while the ether extract produced a slight, but insignificant, increase in MDA content. The aqueous layers resulted from chloroform and
TABLE
1
EFFECT OF ORAL TREATMENT WITH PEROXIDATION IN FEMALE RATS Each
value
is the mean
FIG
+ S.D. of 6 animals.
Treatment
Malondialdehyde (nmol/mg protein)
Control Latex
0.68 0.56 1.92 2.03
Latex CCI,
+ Ccl,
Significant
relative
LATEX
to the control
group:
f 2 f 2
0.21 0.11 0.42* 0.43*
*P < 0.001.
formation
(1 ML/KG)
ON
HEPATIC
LIPID
218
TABLE
2
EFFECT OF i.p. TREATMENT WITH PEROXIDATION IN FEMALE RATS Each value is the mean
FIG
LATEX
(1 ML/KG)
Malondialdehyde formation (nmollmg protein)
Control Latex
0.88 4.20 3.87 2.80
+ Ccl,
Significant
ether 41.
relative
to the control
extractions
HEPATIC
LIPID
-C S.D. of 6 animals,
Treatment
Latex ccl,
ON
produced
f -c + f
0.15 0.29* l.?O* 0.21*
group: *P < 0.001.
significant
increases
in lipid autoxidation
(Table
Discussion
Previous studies in our laboratory have demonstrated that fig latex inhibits [3H]benzo[a]pyrene (3H-B[a]Pl binding to hepatic microsomes. It has been suggested that this activity is due to the presence of enzyme(s). Since it has been hypothetized that the milk latex may act as an antioxidant (Alwan and Al-Bayati, 19881, we examined the effect of the milk latex on lipid peroxidation in vivo and in vitro using rat liver homogenates. The effect of the milk latex on Ccl,-induced hepatic lipid peroxidation was also studied. The latex was adminisiered orally- and iip. (Tables 1 and 2, respectively). In these studies, Ccl, served as a positive control (Recknagel et al., 1976; Stohs et al., 19831. TABLE
3
INCUBATION
EFFECT
Each value is the mean
OF FIG LATEX
ON HEPATIC
Malondialdehyde
W/ml)
(nmol/mg
Control
0.19 f
10 20 30
0.39 -c 0.01* 0.43 + 0.01* 0.46 +: 0.02* relative
IN FEMALE
RATS
2 S.D. of 3 determinations.
Fig milk latex concentration
Significant
AUTOXIDATION
to the control
formation
Increase (%)
protein) 0.00
group: *P < 0.001.
_ + 105 + 126 + 142
219 TABLE
4
INCUBATION EFFECT OF FIG LATEX, UPPER AND LOWER LAYERS AFTER CENTRIFUGATION, ORGANIC EXTRACTS AND HEATED LATEX ON HEPATIC AUTOXIDATION IN FEMALE RATS Each value is the mean f
S.D. of 3 determinations.
Addition (20 &ml)
Malondialdehyde formation (nmol/mg protein)
Increase (0~1
Control Milk latex
0.24 f
0.01
-
0.44 0.44 0.44 0.24
+ f f f
0.01* 0.01* 0.02* 0.00
+83 +83 +83 -
0.42 f
O.Ol*
+75
0.27 f 0.39 f
0.01 0.02*
+ 12 +62
Lower layer Upper layer Chloroform extract Aqueous layer from chloroform extract Ether extract Aqueous layer from ether extract Milk latex (heated)
0.24 r 0.01
-
Significant relative to the control group: *P < 0.001.
Oral administration of the latex neither induced lipid peroxidation nor modified Ccl,-induced lipid peroxidation (Table 11. However, fig latex given i.p. produced a significant increase in hepatic lipid peroxidation as compared to the control (Table 2). The i.p. treatment is toxic and has no scavenging or antioxidant effects since administration of the latex with Ccl, produced no inhibition or decrease in MDA content. The results suggest that activity of the latex may be due to enzyme(s) which are susceptible to destruction in the gastrointestinal tract or which are not absorbed from the tract. It is well known that Ccl, is converted into the Ccl, radical which attacks unsaturated fatty acids in the presence of oxygen to give lipid peroxidation, leading to liver damage (Recknagel et al., 19761. Lipid peroxidation is a destructive process for biological membranes (Wailer et al., 19831. Milk latex may act in similar manner to Ccl, through accumulation of a reactive OXYgen species (Stohs et al., 19861. Reactive oxygen species may cause a parallel increase in enzyme activity with destruction of membrane lipids and induction of lipid peroxidation (Stohs et al., 19841. Our results agree well with previous findings that the latex is toxic to animals when injected, but ineffective when administered orally (Chakaravarty, 19761. The addition of fig latex in vitro has been shown here to enhance the rate of hepatic lipid autoxidation (Table 31. Induction of lipid autoxidation appeared to be proportional to the concentration of milk latex added to the system. After centrifugation of the milk latex, the upper and lower layers
220
were added, respectively, to the incubation mixture (Table 4). Both the upper and lower layers markedly catalysed lipid autoxidation but were equally effective in this regard. Thus, centrifugation has no effect on fig milk latex activity. This result is in contrast to our previous study (Alwan and Al-Bayati, 19881, in which centrifugation decreased fig milk latex activity in causing inhibition of 3H-B[a]P binding to rat liver microsomal protein. However, a combination of active ingredients in fig milk latex may be involved in causing induction of hepatic lipid peroxidation and inhibition of B[a]P binding. Chloroform and ether extracts produced no significant effect on MDA content (Table 4). The aqueous layers resulting from chloroform and ether extraction produced significant increases in lipid autoxidation. Preheating at 100°C for 1 h abolished the activity of the milk latex. These results added to the previous findings indicate that the active components of the latex are water soluble, heat labile and may be enzyme(s). Further fractionating studies are needed to separate and identify the active and toxic components in the milk latex. References Alwan, A.H. and Al-Bayati, Z.A.F. (1988) Effect of milk latex on fig (Ficus carica) on 3H-benzo[u]pyrene binding to rat liver microsomal protein. International Journal of Crude Drug Research, 26, 209-213. Chakaravarty, H.L. (1976) Plant Wealth of Iraq. Ministry of Agriculture and Agrarian Reform, Baghdad, Iraq, pp. 237 - 242. Guest, E. (1966) Flora of Iraq, Vol. 1. Ministry of Agriculture, Baghdad, Iraq, p. 82. Halliwell, B. and Gutteridge, J.M.C. (1986) Oxygen free radicals and iron in relation to biology and medicine: Some problems and concepts. Archives of Biochemistry and Biophysics 246, 501-514. Harvey, M.J. and Klaassen, C.D. (1983) Interaction of metals and carbon tetrachloride on lipid peroxidation and hepatotoxicity. Toxicology and Applied Pharmacology 71,316-322. Hikino, H., Tohkin, M., Kiso, Y., Namiki, T., Nishimura, S. and Takeyama, K. (1986) Antihepatotoxic actions of Allium sativum bulbs. Planta Medicu 163- 168. Kiso, Y., Tohkin, M. and Hikino, H. (1985) Mechanism of antihepatotoxic activity of atractylon. Planta Medica 50, 97 - 100. Lowry, O.H., Rosenbrough, N.J. Farr, W.L. and Randall, R.J. (1951) Protein measurement with the Folin-phenol reagent. The Journal of Biological Chemistry 193, 265-275. Perdue, R.E. Jr. and Hartwell, J.L. (1969) The search for plant sources of anticancer drugs. Morris Arboretum Bulletin 20, 35-53. Plaa, G.L. and Witschi, H. (1976) Chemicals, drugs and lipid peroxidation. Annual Review of Pharmacology
and Toxicology
16, 125-
141.
Recknagel, R.O., Glende, E.A. and Hruszkewycz, A.M. (1976) In: W.A. Pryor (Ed.), Free Radicals in Biology, Vol. 3. Academic Press, New York, pp. 97 - 132. Sinnhuber, R.O. and Yu, T.C. (1958) 2-Thiobarbituric acid method for the measurement of rancidity in fishery products. II: The quantitative determination of malonaldehyde. Food Technology 12,9-
12.
Stich, H.F. and Rosin, M.P. (1984) Naturally occurring phenolics as antimutagenic and anticarcinogenic agents. In: M. Friedman (Ed.), Nutritional and Toxicological Aspects of Food Safety. Plenum Press, New York, pp. 1- 29. Stocks, J., Gutteridge, J.M.C., Sharp, R.J. and Dormandy, T.L. (1974) The inhibition of lipid autoxidation by human serum and its relationship to serum protein and alpha-tocopherol. Clinical Science
of Molecular
Medicine
47, 223-233.
221 Stohs, S.J., Al-Bayati, Z.A.F., Hassan, M.Q., Murray, W.J. and Mohammadpour, H.A., 0986) Glutathione peroxidase and reactive oxygen species in TCDD-induced lipid peroxidation. Advances
in Experimental
Medicine
and Biology
197, 357-365.
Stohs, S.J., Hassan, M.Q. and Murray, W.J. (1983) Lipid peroxidation as a possible cause of TCDD toxicity. Biochemical and Biophysical Research Communications 111, 854-859. Stohs, S.J., Hassan, M.Q. and Murray, W.J. (1984) Induction of lipid peroxidation and inhibition of glutathione peroxidase by TCDD. In: A. Poland and R.D. Kimbrough (Eds.), Biological Mechanisms of Dioxan Action, Banbury Report 18. Cold Spring Harbor Laboratory, New York, pp. 241-253. Tihanyi, K., Gachalyi, B. and Rozsa, I. (1985) Indomethacin inhibits microsomal NADPH-supported lipid peroxidation. IRCS Medical Science 13, 826-827. Uchiyama, M. and Mihara, M. (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271- 278. Wailer, RL Glendi, E.A. Jr. and Recknagel, R.O. (1983) Carbon tetrachloride and bromotrichloromethane toxicity. Biochemical Pharmacology 32, 1613- 1617.
Scientific
Publishers
EFFECTS OF FIG LATEX ON LIPID CCIJNDUCED LIPID PEROXIDATION
ZAINAB
A.F.
AL-BAYATI
215
30 (1990) 215-221 Ireland Ltd.
PEROXIDATION AND IN RAT LIVER
and AL1 H. ALWAN
Pharmacognosy and Pharmacology Department, Biological Research Centre, Scientific Research Council, Jadiriyah, P.O. Box 2371 Baghdad IIraq) (Accepted
May 14,199O)
sllmmary The oral and intraperitoneal effects of fig milk latex on lipid peroxidation and Ccl,-induced lipid peroxidation in liver homogenates of female rats were investigated. Oral treatment had no effect, while i.p. administration produced a significant increase in hepatic lipid peroxidation. When the latex was given before Ccl, treatment: it produced no protective effect against Ccl,-induced hepatotoxicity. Addition of the latex to the incubation mixture produced a dose-dependent increase in lipid autoxidation, while the chloroform and ether extracts of the latex, as well as heated latex, had no effect on hepatic lipid autoxidation.
Introduction
The common fig-tree, Ficus carica L. (Moraceael, is indigenous to most It is cultivated in Asia Minor, the warm and temperate climates. Mediterranean countries, all the milder areas of Europe and in the United State of America (Guest, 1966; Chakaravarty, 19761. The milky juice of freshly-broken buds, stalks and leaves is very acrid and and has been used in some countries for the treatment of warts and blisters on the body (Perdue and Hartwell, 19691. This latex has high coagulating activity and it has been used for the preparation of cheese and junkets, fat extraction from meat and in other food-processing industries and in medicine (Chakaravarty, 1976). The latex contains enzymes such as peroxidase, proteolytic enzymes, diastase, lipase, and catalase (Chakaravarty, 19761. Recent studies have demonstrated that the latex will inhibit [3HJbenzo[a]pyrene binding to rat hepatic microsomes (Alwan and Al-Bayati, 19881. The role of lipid peroxidation in biological systems is of recent research interest due to its involvement in a number of pathological and toxicological 0 1990 Elsevier 037%8741/$03.50 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
216
conditions (Plaa and Witschi, 1976; Tihanyi et al., 1985; Halliwell and Gutteridge, 19861. Furthermore, lipid peroxidation has been shown to be modified by antioxidants present in plants (Stich and Rosin, 1984; Kiso et al., 1985; Hikino et al., 19861. Therefore, we have examined the effect of administering fig milk latex orally and i.p. on lipid peroxidation and on carbon tetrachloride (CClJ-induced hepatic lipid peroxidation in female rats. In addition, the effects of adding milk latex, centrifuged upper and lower layers, organic fractions and heated milk latex on hepatic lipid autoxidation were also investigated. Materials and methods Chemical
sources
Thiobarbituric acid (TBA), Ccl,, KCl, H,PO, and n-butanol from Fluka, AG (Switzerland). Collection and preparation
were purchased
of fig latex
The milky latex of freshly broken stalks and leaves was collected in the early morning. One part of milk latex was extracted with an equal volume of chloroform or ether, mixed and centrifuged for 10 min at room temperature. The residue from each extraction was dissolved in DMSO and used for experimental assay. The milk latex (2 ml) was centrifuged and the upper and lower layers separated. The latex (1 ml1 was heated at 100°C for 1 h to destroy the enzymes. Animal
treatment
Female albino Wistar rats (140- 160 g; bred in our laboratory1 were housed in stainless steel hanging cages and provided free access to feed (Standard chow, Al-Kadhmya, Baghdad) and water. One group of animals was treated orally with 1 ml/kg of milk latex in corn oil for one day. A second group was treated i.p. with 1 ml/kg of milk latex in corn oil for one day. Control animals received corn oil only (1 ml/kg). Half of each milk latex-treated group was given Ccl, (0.5 ml/kg) in corn oil in a single dose orally 1 h after milk latex treatment. Lipid peroxidation
All animals were killed the day after treatment. Livers were removed and 20%~ homogenates were prepared in ice cold 1.15% KCl. The amount of malondialdedhyde (MDA) present was determined by using the procedure of Uchiyama and Mihara (1978). To 0.5 ml of the 20% homogenate were added 3.0 ml 1% H,PO, and 1.0 ml 0.6% TBA solution. Each mixture was heated for 45 min, cooled and extracted with n-butanol. The optical densities of the organic phases were determined at 535 and 520 nm, and MDA was calculated using an extinction coefficient of 1.56 x 105/M cm (Sinnhuber and Yu, 19581. Lipid autoxidation was measured as described by Stocks et al. (19741. The
217
liver homogenate (5 ml) was prepared and incubated at 37OC for 1 h. MDA formation was measured by the TBA reaction. Protein concentrations were determined according to the method of Lowry et al. (1951). Results
When rats were treated orally with 1 ml/kg of latex and killed one day post-treatment, no significant changes in MDA content (as an index of lipid peroxidation) of liver homogenates were observed in the latex-treated group as compared to the control group (Table 1). Ccl, was used to induce hepatic lipid peroxidation and also served as a positive control. Ccl, administration alone increased MDA content three-fold. Latex given together with Ccl, had no effect on MDA content as compared to Ccl,treated rats not receiving the latex. Latex administered i.p. resulted in a five-fold increase in hepatic MDA content as compared to the corn oil control animals (Table 2). This increase in lipid peroxidation was greater than that seen in animals receiving latex plus Ccl, or Ccl, alone. The ability of the latex to increase hepatic lipid autoxidation when added in vitro is presented in Table 3. A dose as low as 10 ~1 of latex was able to increase significantly MDA content two-fold. The effects of the upper and lower layers of milk latex after centrifugation, the organic extracts (chloroform and ether fractions) and heated milk latex on in vitro hepatic autoxidation as determined by MDA formation using liver homogenate from control rats are presented in Table 4. Both upper and lower layers of the milk latex equally stimulated MDA formation. Chloroform extract produced no significant stimulation of MDA formation, while the ether extract produced a slight, but insignificant, increase in MDA content. The aqueous layers resulted from chloroform and
TABLE
1
EFFECT OF ORAL TREATMENT WITH PEROXIDATION IN FEMALE RATS Each
value
is the mean
FIG
+ S.D. of 6 animals.
Treatment
Malondialdehyde (nmol/mg protein)
Control Latex
0.68 0.56 1.92 2.03
Latex CCI,
+ Ccl,
Significant
relative
LATEX
to the control
group:
f 2 f 2
0.21 0.11 0.42* 0.43*
*P < 0.001.
formation
(1 ML/KG)
ON
HEPATIC
LIPID
218
TABLE
2
EFFECT OF i.p. TREATMENT WITH PEROXIDATION IN FEMALE RATS Each value is the mean
FIG
LATEX
(1 ML/KG)
Malondialdehyde formation (nmollmg protein)
Control Latex
0.88 4.20 3.87 2.80
+ Ccl,
Significant
ether 41.
relative
to the control
extractions
HEPATIC
LIPID
-C S.D. of 6 animals,
Treatment
Latex ccl,
ON
produced
f -c + f
0.15 0.29* l.?O* 0.21*
group: *P < 0.001.
significant
increases
in lipid autoxidation
(Table
Discussion
Previous studies in our laboratory have demonstrated that fig latex inhibits [3H]benzo[a]pyrene (3H-B[a]Pl binding to hepatic microsomes. It has been suggested that this activity is due to the presence of enzyme(s). Since it has been hypothetized that the milk latex may act as an antioxidant (Alwan and Al-Bayati, 19881, we examined the effect of the milk latex on lipid peroxidation in vivo and in vitro using rat liver homogenates. The effect of the milk latex on Ccl,-induced hepatic lipid peroxidation was also studied. The latex was adminisiered orally- and iip. (Tables 1 and 2, respectively). In these studies, Ccl, served as a positive control (Recknagel et al., 1976; Stohs et al., 19831. TABLE
3
INCUBATION
EFFECT
Each value is the mean
OF FIG LATEX
ON HEPATIC
Malondialdehyde
W/ml)
(nmol/mg
Control
0.19 f
10 20 30
0.39 -c 0.01* 0.43 + 0.01* 0.46 +: 0.02* relative
IN FEMALE
RATS
2 S.D. of 3 determinations.
Fig milk latex concentration
Significant
AUTOXIDATION
to the control
formation
Increase (%)
protein) 0.00
group: *P < 0.001.
_ + 105 + 126 + 142
219 TABLE
4
INCUBATION EFFECT OF FIG LATEX, UPPER AND LOWER LAYERS AFTER CENTRIFUGATION, ORGANIC EXTRACTS AND HEATED LATEX ON HEPATIC AUTOXIDATION IN FEMALE RATS Each value is the mean f
S.D. of 3 determinations.
Addition (20 &ml)
Malondialdehyde formation (nmol/mg protein)
Increase (0~1
Control Milk latex
0.24 f
0.01
-
0.44 0.44 0.44 0.24
+ f f f
0.01* 0.01* 0.02* 0.00
+83 +83 +83 -
0.42 f
O.Ol*
+75
0.27 f 0.39 f
0.01 0.02*
+ 12 +62
Lower layer Upper layer Chloroform extract Aqueous layer from chloroform extract Ether extract Aqueous layer from ether extract Milk latex (heated)
0.24 r 0.01
-
Significant relative to the control group: *P < 0.001.
Oral administration of the latex neither induced lipid peroxidation nor modified Ccl,-induced lipid peroxidation (Table 11. However, fig latex given i.p. produced a significant increase in hepatic lipid peroxidation as compared to the control (Table 2). The i.p. treatment is toxic and has no scavenging or antioxidant effects since administration of the latex with Ccl, produced no inhibition or decrease in MDA content. The results suggest that activity of the latex may be due to enzyme(s) which are susceptible to destruction in the gastrointestinal tract or which are not absorbed from the tract. It is well known that Ccl, is converted into the Ccl, radical which attacks unsaturated fatty acids in the presence of oxygen to give lipid peroxidation, leading to liver damage (Recknagel et al., 19761. Lipid peroxidation is a destructive process for biological membranes (Wailer et al., 19831. Milk latex may act in similar manner to Ccl, through accumulation of a reactive OXYgen species (Stohs et al., 19861. Reactive oxygen species may cause a parallel increase in enzyme activity with destruction of membrane lipids and induction of lipid peroxidation (Stohs et al., 19841. Our results agree well with previous findings that the latex is toxic to animals when injected, but ineffective when administered orally (Chakaravarty, 19761. The addition of fig latex in vitro has been shown here to enhance the rate of hepatic lipid autoxidation (Table 31. Induction of lipid autoxidation appeared to be proportional to the concentration of milk latex added to the system. After centrifugation of the milk latex, the upper and lower layers
220
were added, respectively, to the incubation mixture (Table 4). Both the upper and lower layers markedly catalysed lipid autoxidation but were equally effective in this regard. Thus, centrifugation has no effect on fig milk latex activity. This result is in contrast to our previous study (Alwan and Al-Bayati, 19881, in which centrifugation decreased fig milk latex activity in causing inhibition of 3H-B[a]P binding to rat liver microsomal protein. However, a combination of active ingredients in fig milk latex may be involved in causing induction of hepatic lipid peroxidation and inhibition of B[a]P binding. Chloroform and ether extracts produced no significant effect on MDA content (Table 4). The aqueous layers resulting from chloroform and ether extraction produced significant increases in lipid autoxidation. Preheating at 100°C for 1 h abolished the activity of the milk latex. These results added to the previous findings indicate that the active components of the latex are water soluble, heat labile and may be enzyme(s). Further fractionating studies are needed to separate and identify the active and toxic components in the milk latex. References Alwan, A.H. and Al-Bayati, Z.A.F. (1988) Effect of milk latex on fig (Ficus carica) on 3H-benzo[u]pyrene binding to rat liver microsomal protein. International Journal of Crude Drug Research, 26, 209-213. Chakaravarty, H.L. (1976) Plant Wealth of Iraq. Ministry of Agriculture and Agrarian Reform, Baghdad, Iraq, pp. 237 - 242. Guest, E. (1966) Flora of Iraq, Vol. 1. Ministry of Agriculture, Baghdad, Iraq, p. 82. Halliwell, B. and Gutteridge, J.M.C. (1986) Oxygen free radicals and iron in relation to biology and medicine: Some problems and concepts. Archives of Biochemistry and Biophysics 246, 501-514. Harvey, M.J. and Klaassen, C.D. (1983) Interaction of metals and carbon tetrachloride on lipid peroxidation and hepatotoxicity. Toxicology and Applied Pharmacology 71,316-322. Hikino, H., Tohkin, M., Kiso, Y., Namiki, T., Nishimura, S. and Takeyama, K. (1986) Antihepatotoxic actions of Allium sativum bulbs. Planta Medicu 163- 168. Kiso, Y., Tohkin, M. and Hikino, H. (1985) Mechanism of antihepatotoxic activity of atractylon. Planta Medica 50, 97 - 100. Lowry, O.H., Rosenbrough, N.J. Farr, W.L. and Randall, R.J. (1951) Protein measurement with the Folin-phenol reagent. The Journal of Biological Chemistry 193, 265-275. Perdue, R.E. Jr. and Hartwell, J.L. (1969) The search for plant sources of anticancer drugs. Morris Arboretum Bulletin 20, 35-53. Plaa, G.L. and Witschi, H. (1976) Chemicals, drugs and lipid peroxidation. Annual Review of Pharmacology
and Toxicology
16, 125-
141.
Recknagel, R.O., Glende, E.A. and Hruszkewycz, A.M. (1976) In: W.A. Pryor (Ed.), Free Radicals in Biology, Vol. 3. Academic Press, New York, pp. 97 - 132. Sinnhuber, R.O. and Yu, T.C. (1958) 2-Thiobarbituric acid method for the measurement of rancidity in fishery products. II: The quantitative determination of malonaldehyde. Food Technology 12,9-
12.
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