115
Cancer Letters, 66 (1992) 115- 121 Elsevier Scientific Publishers Ireland Ltd.
Reversal of aflatoxin induced liver damage curcumin
by turmeric and
K.B. Soni, A. Rajan and R. Kuttan Amala Cancer Research
Centre,
(Received 18 June 1991) (Revision received 14 September (Accepted 15 July 1992)
Amala Nagar,
Trichur-680
553, Kerala State (India)
1991)
Summary
Introduction
The effect of certain food additives on aflatoxin production bb Aspergillus parasiticus has been studied in vitro. Extracts of turmeric (Curcuma longa), garlic (Allium sativum) and asafoetida (Ferula asafoetida) inhibited the aflatoxin production considerably (more than 90%) at concenhaGons of 5 - 10 mg/ml. Similar results were also seen using butylated hydrbxytoluene, butylated hydroxyanisole and ellagic acid at concentration 0.1 mM. Curcumin, the antioxidant principle from Curcuma longa did not have any effect on aflatoxin production. Turmeric and curcumin were also found to reverse the aflatoxin induced liver damage produced by feeding aflatoxin BI (AFBJ (5 pg/day per 14 days) to ducklings. Fatty changes, necrosis and biliary hyperplasia produced by AFBI were considerably reversed r5y these food additives.
Mycotoxins such as aflatoxins pose a major health hazard to the population especially in the developing countries [l]. Although mutagenicity of aflatoxin has been clearly demonstrated, its role in hepatocarcinoma formation in human beings is still not clearly understood [2,3]. However, the aflatoxins have been shown to produce severe liver damage in various species of animals including primates [4,5]. Poultry is especially susceptible to the toxic effects of aflatoxins [6]. Aflatoxin was shown to be converted into its epoxide and this derivative produces DNA adducts causing DNA strand breaks and point mutations [7,8]. Antioxidants were reported to inhibit the aflatoxin induced DNA adduct formations [9,10]. Turmeric (Curcuma longa) and its active ingredient curcumin have been shown to scavenge the free radicals and thereby act as good antioxidants [ll]. Moreover, because of their non-toxicity the use of these agents in preventing the aflatoxin induced liver damage in the ducklings has been studied.
Keywords: aflatoxin; turmeric; antioxidants
food
additives;
Materials Correspondence
Research India.
Centre,
to: Ramadasan Kuttan, Amala Cancer Amala Nagar, Trichur-680 553, Kerala State,
0304-3835/92/$05.00 Printed and Published
@ 1992 Elsevier Scientific Publishers in Ireland
and Methods
Aflatoxin-Bl was Chemical Company, Ireland Ltd.
bought from Sigma USA and 5 mg/ml
116
solution was prepared in dimethyl sulfoxide. Cultures of the fungus Aspergillus parasiticus were kindly given by Dr. Mariamma, Veterinary College, Mannuthy. Curcumin was a gift from Bombay Oil Industries Ltd., Angamali. Butylated hydroxy toluene and butylated hydroxy anisole were purchased from Central Drug House, Delhi and ellagic acid was obtained as a gift from Dr. Conney, USA. All other chemicals obtained locally were of analytical reagent grade.
aflatoxin was extracted by chloroform using Pon’s method [13]. The amount of the toxin in the extract was estimated using fluorotoxin meter (Velasco) in which aflatoxin was previously separated on a silica gel-alumina column after appropriate dilution and estimation by absorption on flurosil. Amount of aflatoxin produced is expressed as parts per billion in the fluorotoxin meter and the amount produced in the medium was calculated. All the experiments were repeated twice.
Culture of Aspergillus parasiticus A. parasiticus was originally grown on Sabouraud’s dextrose agar. After 7 days of growth at 30°C the cultures were uniformly suspended in glucose (5%) ammonium nitrate (0.24%) medium (5 ml). A 100~~1 quantity of the suspension was inoculated into 2 ml of glucose-ammonium nitrate medium containing zinc sulphate (26 mg/l) and cobalt nitrate (2.6 mg/l) as mineral supplements. These mineral supplements have been shown to increase the toxin production by A. parasiticus [12]. Garlic (A/hum satiuum) asafoetida (Ferula asafoetida) and turmeric (Curcuma longa) were purchased from a local market and were authenticated by a qualified botanist. Alcoholic extracts of garlic and turmeric were prepared by extracting 1 g of fresh garlic or 1 g of turmeric powder in 50 ml of 75% methanol overnight at room temperature. It was then filtered and the filtrate was evaporated and made up to 1 ml with methanol. The aqueous extracts of asafoetida was prepared by boiling 2 g of asafoetida in 10 ml distilled water. Aqueous extract of garlic was prepared by extracting 1 g of crushed garlic in 10 ml of distilled water. The aqueous extract of turmeric was prepared by boiling 1 g of turmeric powder in 100 ml water and after concentration was made up to 10 ml.
Determination of the effect of food additives on the aflatoxin induced toxicity in ducklings Three-day-old ducklings (40 - 50 g) were used for determining the aflatoxin induced toxicity. They were purchased from the Veterinary College, Mannuthy and brought to the laboratory just prior to the experiment. Groups of 10 ducklings were used for each set of experiments. Aflatoxin Bl was dissolved in a minimum volume of acetone (0.2 - 0.4 ml) and mixed with hot duckling feed composed of boiled wheat powder in milk. Each animal received aflatoxin concentration of 5 pg per day for 14 days. The food additives were mixed with the feed containing aflatoxin-Bl at the following concentrations: turmeric 50 mg and curcumin 10 mg per day animal. After 14 days the animals were sacrificed and blood was collected by heart puncture and liver was stored at -9OOC. The following estimations were undertaken: (a), total WBC using haemocytometer; (b), haemoglobin using the Drabkin’s cyanomet-haemoglobin method [14]; (c), liver glutamate pyruvate transaminase [ 151; (d), alkaline phosphatase [16]; (e), serum glutamate pyruvate transaminase 1151; (0, liver peroxides by thiobarbituric acid method [17]. A small section of the liver was fixed in 10% formalin and histopathological analysis was carried out after staining with haematoxylineosin.
Estimation of toxin production in in vitro cultures of A. parasiticus Food additives at various concentrations were added to the above cultures and incubated for 6 days at 30°C. After incubation mycelium was crushed in the medium and
Results Effect of food additiues in aflatoxin production by A. parasiticus culture The effect of food additives in the aflatoxin
117 Table 1.
Effect of food additives
on aflatoxin production
Food additives
Garlic Asafoetida
Alcoholic Aqueous
The basal level of toxin production
Effect of artioxidants
Antioxidants
Curcumin BHA BHT Ellagic acid The basal level of toxin production
(%)
10 mg
5 mg
2.5 mg
99.41 f 0.075 86.12 + 1.68 No growth
76.92 + 1.42 50.80 + 2.36 No growth 0 88.62 f 0.83
49.95
90.13 in the medium
0 f 0.70 under the conditions
production by A. parasiticus is shown in Table I. The basal level of toxin production in the medium under the conditions studied here was 5.1 * 2 pg/ml of the medium, which is similar to that reported earlier 1181. Addition of aqueous extract of turmeric (10 mg/ml) inhibited the toxin production by 99 % . Similarly asafoetida inhibited the toxin production at the same concentration by 90%. Aqueous extract of garlic inhibited the growth of the fungus at a higher concentration and inhibited the toxin production at a lc’wer concentration. While alcoholic extract of turmeric inhibited the toxin production to a much lower level compared to aqueous extract, alcoholic extract of garlic did not inhibit the toxin production even at high concentrations. The concentration of the extracts needed for 50% inhibition of toxin production was found to be approximately 2.5 mg/ml.
Table II.
parasiticus
Inhibition of toxin production
Aqueous Alcoholic Aqueous
Turmeric
by A.
on aflatoxin production
55.14
studied was 5.1 f
2.0 pg/ml.
The effect of antioxidants on aflatoxin production by A. parasiticus The antioxidants such as BHA, BHT and ellagic acid inhibited the toxin production at concentrations of 0.1 mM, or more (Table II). The inhibition of toxin production at 0.1 mM for BHA was 7 1.56% ; BHT 76% and ellagic acid 65%. However, at higher concentrations such as 1 mM, BHA and ellagic acid inhibited the growth of the organism. The active ingredient from the turmeric, i.e. curcumin, did not inhibit the toxin production even at 10 mM concentration. The effect of turmeric and curcumin on the hepatotoxicity induced by AFBl in ducklings The effects of food additives such as turmeric and curcumin on the toxicity induced by AFBl in ducklings is shown in Table III. The control animals gained weight during the
by A.
Inhibition of toxin production
parositicus.
(9)
10 mM
1 mM
0.1 mM
0 ND ND ND
0 No growth 85.52 ztz 10.94 No growth
0 71.56 76.11 65.28
in the medium
f 5.90 0 f 0.52 0 0
under the conditions
studied was 5.1 f
f 6.1 zt 4.46 zt 13.80
2.0 pg/ml.
1. 2. 3. 4.
Normal AFB, AFB, + turmeric AFB, + curcumin
and curcumin
61.2 35.5 42.5 47.5
(9)
+ 16.0 f 5.9 +z 10.8 +z 1.85
Weight gain
Table 111. Effect of turmeric
50 46 76 58
000 840 520 200
f 7 133 +z 12 285 f 7 297 f. 5 283
WBC counts/ mm3
on the hepatotoxicity
18.5 17.18 19.64 18.78
+ 3.8 zt 3.7 z+z3.8 +z 4.8
98.0 150.6 250.6 269.4
zt 37.8 f 46.6 ziz 123.7 f 225.0
(pg of formed/ml)
GPT
Serum
(SW)
by AFB, in ducklings
Haemoglobin
induced
1.8 2.3 1.7 1.6
zrz 0.47 f 0.55 f 0.24 z!z 0.29
Liver GPT (fimoles of pyruvate formed/mg protein)
0.27 0.24 0.08 0.07
f +z zt f
0.1 0.04 0.05 0.03
Liver alkaline phosphatase (pmoles of phenol formed/ mg protein)
119
120
course of the experiment (61.25 g) whereas the AFBl treated animals gained only 35.5 g. When treated with turmeric and curcumin there was an increased weight change, i.e. 42.5 and 47.5 g, respectively. In the case of curcumin the value was found to be statistically significant (P < 0.01). The total WBC and haemoglobin did not significantly alter in the case of AFBl treated animals. Liver alkaline phosphatase was found to be significantly decreased after treatment with turmeric and curcumin. Serum GPT on the other hand increased significantly after AFBl treatment and remained elevated even after treatment with turmeric and curcumin. Similarly the lipid peroxides in the liver did not alter significantly after AFBl treatment, but remained lower than the control after treatment with turmeric and curcumin (data not shown). The pathological observations of liver after 14 days of treatment with AFBl showed extensive fatty changes, granular degeneration, necrosis and bile duct hyperplasia (Fig. la,b). There was almost complete reversal of necrosis and fatty changes in the case of animals treated with AFBl in the presence of curcumin (Fig. lc). In animals treated with AFBl in the presence of turmeric necrosis was almost completely reversed and there was only moderate fatty changes (Fig. Id). In both these cases there was no marked reversal of bile duct hyperplasia which was shown after AFBl treatment. Discussion The present results indicate that the food additives such as turmeric and asafoetida when added to the medium containing A. parasiticus inhibited the toxin production, without inhibiting the growth of mycelium. The concentration needed for 50% inhibition was approximately 2.5 mg/ml of the medium. Garlic on the other hand inhibited the growth of the organism. The inhibition of the aflatoxin production was also seen in the presence of antioxidants such as BHA, BHT and ellagic acid. However, curcumin did not inhibit the
toxin production even at 10 mM concentration. Tumeric has been shown to inhibit the in Salmonella typhimurium mutagenesis strains induced by polycyclic hydrocarbons and also was found to be anticarcinogenic [19]. Although the major antioxidant component present in turmeric is curcumin, recently a small peptide with antioxidant and anticlastogenie properties has been reported [20]. Similarly asafoetida and garlic contain several antioxidant sulphur containing compounds such as diallyl sulphide and umbelliferone which are anticarcinogenic in experimental animals [21,22]. The property of the extracts to inhibit the aflatoxin production could be mainly attributed to their antioxidant activity as several oxidation steps are involved in the aflatoxin biosynthesis by A. parasiticus. Similar results were also seen with antioxidants such as BHA, BHT and ellagic acid. The inability of curcumin to inhibit aflatoxin production may be related to its decreased permeability. I Both turmeric and curcumin inhibited the aflatoxin induced toxicity in experimental ducklings. The weight change induced by the toxin is partially reversed by the simultaneous administration of turmeric or curcumin. The such as WBC and other parameters haemoglobin, GPT and alkaline phosphatase in the liver remained unaltered in the condition studied. Turmeric and curcumin alone has not been found to have any inhibitory effect on alkaline phosphatase. The most remarkable feature is the histopatholgical findings in which the aflatoxin induced injury is remarkably reduced in animals treated with curcumin and turmeric. Both turmeric and curcumin are non-toxic [35], non-mutagenic [24] and are being consumed as spices in several parts of the world. The use of these cost effective food additives in the amelioration of aflatoxicosis needs further serious consideration. References 1
Shank, Dietary
R.C., Wogan. G.N. and Gibson, aflatoxins and human liver cancer.
J.B. (1972) I. Toxigenic
121
2
moulds in foods and food stuffs of tropical South-East Asia.
tions of Pons method
Food Cosmet.
gorund nut cake. J. Food Sci. Technol.,
Garner,
Toxiccll.,
B.C.
(1980)
10, 51-
60.
Carcinogenesis
by fungal products.
4
Mutagenicity
of carcinogenic
typhimurium.
Cancer Res., 38, 536-542.
Garner,
R.C.
and Matrin,
C.N.
15
Bergmeyer,
1, pp. 287-225,
Editor: P.L.
G.N.
Methods
in Cancer
Stolman,
A. (1974)
(1982)
Aflatoxin
Research,
carcinogenesis.
Vol.
VII,
pp. 309-
R.G.,
R.A.
60,
1036-
Firozi, P.F.
by aflatoxin B1 in vitro. Carcinogenesis, Firozi, P.F.,
Aboobaker,
V.S.
Action of vitamin-A
5, 1359-
catalysed reaction.
and 181112
O.P.
related
Antioxidant
22
compounds.
Pharmacoi.,
23
Picretti, S., Finotti., S. and Passi,
on
aflatoxin
production.
and free radical Mycotoxin
Res.,
K.K.
and Kuttan,
carcinogenesis
by
R.
(1989)
curcumin.
Inhibition J.
of
Ethnophar-
27, 227-233.
Shalini, V.K.
and Srinivas, L. (1987) action of turmeric
and
longa).
J.
4, 82-89.
Wargovich,
M.J.,
Woods,
C.L.
Gray,
K.
and
Anti-mutagenic (Curcumo
C.,
(1988)
Eng., V.W.S.,
Stephens,
Chemoprevention
of
N-
induced esophageal cancer in diallyl sulphide.
Unnikrishnan,
M.C. and Kuttan, R. (1990) Tumour reduc-
51, 85-89.
Abraham,
S.K. and Kesavan, P.C. (1984)
Genotoxicity
of
85-88. In: The Genus Aspergillus,
B. Raper
Williams Wilkins Company, Rati, E.R.,
18,
garlic, turmeric and asafoetida in mice. Mutat. Res., 136,
25,
1812.
Brain et al. (1961) Editors: Kennth
13
Biochem.
Lipid
ing and anti-carcinogenic activity of selected spices. Cancer
Cancer
activity of curcumin
(1971)
Neurochem.,
Effect of different antioxidants
Soudamini,
Len,
(1976:
A.S. J.
Cancer Res., 48, 6872 - 6875.
R.K.
Lett., 34, 213-220. Sharma,
A.H.
London.
rats by the naturally occurring thioether,
1362.
on DNA adduct formation by
aflatoxin B, in a microsome
in rat brain.
nitrosomethyl benzyl-amine
adduct
and Bhattacharya,
London.
Editors: B. Varley.
S. and Balasubramanian, formation
Nutr. Growth Cancer,
V.S.
Factors modul.ating the formation of DNA
Practical
(1980) In: Practical Clinical
5th Edn. p. 850.
anti-carcinogenic 21
1043.
and Aboobaker,
and M. Bell. Heinmann,
Fanelli, C., Fabbri, A.A.,
macol., 20
liver microsomes in vitro. Biochem.
R.K.,
Bishayee,
chemical
evidence for its formation in rat liver in
Biophys. Res. Commun., Bhattacharya,
19
13, 581-602. (1974) Afla-
In:
65-69.
removal and excretion in relation
Dreg Metab. Rev.,
(1980)
and M. Bell. Heinmann,
S. (1985)
and Wogan,
Miller, E.C. and Miller, J.A.
vivo and by human
11
Gowenlock
scavengers
Bennet,
E.
909 - 920. 18
Press, Inc., New York.
13, 168. Bernt,
King, E.J. and Amstrong, A.R.
peroxide
304.
Metabolic activation of aflatoxin B. pattern of
toxin Bl 2,3-oxide:
(1987)
In:
In: Progress in Chemical Toxicology,
Croy,
Swenson, D.H.,
(1984)
CRC
Press, New York.
to carcinogenesis.
10
16
17
(1973)
Essigman, J.M.
Glover.
F:_.
Wogan,
DNA adduct formation,
9
toxins,
Gowenlock
G.N.
8
Fungal
and
Clinical Biochemistry, 5th Edn. p. 685. Editors: B. Varley, A.H.
(1979)
H.U.
Biochemistry,
Vol.
Vol. 5, Academic 7
in Salmonella
Br. J. Haematol.,
aflatoxins and nucleic acids. In: Chemical Carcinogens and
Editor: H. Bush. Academic 6
mycotoxins
Y. (1978)
DNA,
Press, Boca-Raton, 5
T. and Nakamura,
and
International committee for standardisation in haematology (1967)
, ho,
Ueno, Y., Kubota, K
in Corn
24, 90-91.
14
Br. Med. Bull., 36, 47-52. 3
of estimating AFBr
Prema,
and Dorothy Baltimore,
V. and Shantha,
pp. 109.
I. Fennel.
The
Nagabhushan,
M.
and
Bhide,
S.V.
Modifica-
(1986)
Non-
mutagenicity of curcumin and its anti-mutagenic action versus chilli and capsaicin. Nutr. Cancer,
MD. T. (1987)
24
8, 201-210.
Cancer Letters, 66 (1992) 115- 121 Elsevier Scientific Publishers Ireland Ltd.
Reversal of aflatoxin induced liver damage curcumin
by turmeric and
K.B. Soni, A. Rajan and R. Kuttan Amala Cancer Research
Centre,
(Received 18 June 1991) (Revision received 14 September (Accepted 15 July 1992)
Amala Nagar,
Trichur-680
553, Kerala State (India)
1991)
Summary
Introduction
The effect of certain food additives on aflatoxin production bb Aspergillus parasiticus has been studied in vitro. Extracts of turmeric (Curcuma longa), garlic (Allium sativum) and asafoetida (Ferula asafoetida) inhibited the aflatoxin production considerably (more than 90%) at concenhaGons of 5 - 10 mg/ml. Similar results were also seen using butylated hydrbxytoluene, butylated hydroxyanisole and ellagic acid at concentration 0.1 mM. Curcumin, the antioxidant principle from Curcuma longa did not have any effect on aflatoxin production. Turmeric and curcumin were also found to reverse the aflatoxin induced liver damage produced by feeding aflatoxin BI (AFBJ (5 pg/day per 14 days) to ducklings. Fatty changes, necrosis and biliary hyperplasia produced by AFBI were considerably reversed r5y these food additives.
Mycotoxins such as aflatoxins pose a major health hazard to the population especially in the developing countries [l]. Although mutagenicity of aflatoxin has been clearly demonstrated, its role in hepatocarcinoma formation in human beings is still not clearly understood [2,3]. However, the aflatoxins have been shown to produce severe liver damage in various species of animals including primates [4,5]. Poultry is especially susceptible to the toxic effects of aflatoxins [6]. Aflatoxin was shown to be converted into its epoxide and this derivative produces DNA adducts causing DNA strand breaks and point mutations [7,8]. Antioxidants were reported to inhibit the aflatoxin induced DNA adduct formations [9,10]. Turmeric (Curcuma longa) and its active ingredient curcumin have been shown to scavenge the free radicals and thereby act as good antioxidants [ll]. Moreover, because of their non-toxicity the use of these agents in preventing the aflatoxin induced liver damage in the ducklings has been studied.
Keywords: aflatoxin; turmeric; antioxidants
food
additives;
Materials Correspondence
Research India.
Centre,
to: Ramadasan Kuttan, Amala Cancer Amala Nagar, Trichur-680 553, Kerala State,
0304-3835/92/$05.00 Printed and Published
@ 1992 Elsevier Scientific Publishers in Ireland
and Methods
Aflatoxin-Bl was Chemical Company, Ireland Ltd.
bought from Sigma USA and 5 mg/ml
116
solution was prepared in dimethyl sulfoxide. Cultures of the fungus Aspergillus parasiticus were kindly given by Dr. Mariamma, Veterinary College, Mannuthy. Curcumin was a gift from Bombay Oil Industries Ltd., Angamali. Butylated hydroxy toluene and butylated hydroxy anisole were purchased from Central Drug House, Delhi and ellagic acid was obtained as a gift from Dr. Conney, USA. All other chemicals obtained locally were of analytical reagent grade.
aflatoxin was extracted by chloroform using Pon’s method [13]. The amount of the toxin in the extract was estimated using fluorotoxin meter (Velasco) in which aflatoxin was previously separated on a silica gel-alumina column after appropriate dilution and estimation by absorption on flurosil. Amount of aflatoxin produced is expressed as parts per billion in the fluorotoxin meter and the amount produced in the medium was calculated. All the experiments were repeated twice.
Culture of Aspergillus parasiticus A. parasiticus was originally grown on Sabouraud’s dextrose agar. After 7 days of growth at 30°C the cultures were uniformly suspended in glucose (5%) ammonium nitrate (0.24%) medium (5 ml). A 100~~1 quantity of the suspension was inoculated into 2 ml of glucose-ammonium nitrate medium containing zinc sulphate (26 mg/l) and cobalt nitrate (2.6 mg/l) as mineral supplements. These mineral supplements have been shown to increase the toxin production by A. parasiticus [12]. Garlic (A/hum satiuum) asafoetida (Ferula asafoetida) and turmeric (Curcuma longa) were purchased from a local market and were authenticated by a qualified botanist. Alcoholic extracts of garlic and turmeric were prepared by extracting 1 g of fresh garlic or 1 g of turmeric powder in 50 ml of 75% methanol overnight at room temperature. It was then filtered and the filtrate was evaporated and made up to 1 ml with methanol. The aqueous extracts of asafoetida was prepared by boiling 2 g of asafoetida in 10 ml distilled water. Aqueous extract of garlic was prepared by extracting 1 g of crushed garlic in 10 ml of distilled water. The aqueous extract of turmeric was prepared by boiling 1 g of turmeric powder in 100 ml water and after concentration was made up to 10 ml.
Determination of the effect of food additives on the aflatoxin induced toxicity in ducklings Three-day-old ducklings (40 - 50 g) were used for determining the aflatoxin induced toxicity. They were purchased from the Veterinary College, Mannuthy and brought to the laboratory just prior to the experiment. Groups of 10 ducklings were used for each set of experiments. Aflatoxin Bl was dissolved in a minimum volume of acetone (0.2 - 0.4 ml) and mixed with hot duckling feed composed of boiled wheat powder in milk. Each animal received aflatoxin concentration of 5 pg per day for 14 days. The food additives were mixed with the feed containing aflatoxin-Bl at the following concentrations: turmeric 50 mg and curcumin 10 mg per day animal. After 14 days the animals were sacrificed and blood was collected by heart puncture and liver was stored at -9OOC. The following estimations were undertaken: (a), total WBC using haemocytometer; (b), haemoglobin using the Drabkin’s cyanomet-haemoglobin method [14]; (c), liver glutamate pyruvate transaminase [ 151; (d), alkaline phosphatase [16]; (e), serum glutamate pyruvate transaminase 1151; (0, liver peroxides by thiobarbituric acid method [17]. A small section of the liver was fixed in 10% formalin and histopathological analysis was carried out after staining with haematoxylineosin.
Estimation of toxin production in in vitro cultures of A. parasiticus Food additives at various concentrations were added to the above cultures and incubated for 6 days at 30°C. After incubation mycelium was crushed in the medium and
Results Effect of food additiues in aflatoxin production by A. parasiticus culture The effect of food additives in the aflatoxin
117 Table 1.
Effect of food additives
on aflatoxin production
Food additives
Garlic Asafoetida
Alcoholic Aqueous
The basal level of toxin production
Effect of artioxidants
Antioxidants
Curcumin BHA BHT Ellagic acid The basal level of toxin production
(%)
10 mg
5 mg
2.5 mg
99.41 f 0.075 86.12 + 1.68 No growth
76.92 + 1.42 50.80 + 2.36 No growth 0 88.62 f 0.83
49.95
90.13 in the medium
0 f 0.70 under the conditions
production by A. parasiticus is shown in Table I. The basal level of toxin production in the medium under the conditions studied here was 5.1 * 2 pg/ml of the medium, which is similar to that reported earlier 1181. Addition of aqueous extract of turmeric (10 mg/ml) inhibited the toxin production by 99 % . Similarly asafoetida inhibited the toxin production at the same concentration by 90%. Aqueous extract of garlic inhibited the growth of the fungus at a higher concentration and inhibited the toxin production at a lc’wer concentration. While alcoholic extract of turmeric inhibited the toxin production to a much lower level compared to aqueous extract, alcoholic extract of garlic did not inhibit the toxin production even at high concentrations. The concentration of the extracts needed for 50% inhibition of toxin production was found to be approximately 2.5 mg/ml.
Table II.
parasiticus
Inhibition of toxin production
Aqueous Alcoholic Aqueous
Turmeric
by A.
on aflatoxin production
55.14
studied was 5.1 f
2.0 pg/ml.
The effect of antioxidants on aflatoxin production by A. parasiticus The antioxidants such as BHA, BHT and ellagic acid inhibited the toxin production at concentrations of 0.1 mM, or more (Table II). The inhibition of toxin production at 0.1 mM for BHA was 7 1.56% ; BHT 76% and ellagic acid 65%. However, at higher concentrations such as 1 mM, BHA and ellagic acid inhibited the growth of the organism. The active ingredient from the turmeric, i.e. curcumin, did not inhibit the toxin production even at 10 mM concentration. The effect of turmeric and curcumin on the hepatotoxicity induced by AFBl in ducklings The effects of food additives such as turmeric and curcumin on the toxicity induced by AFBl in ducklings is shown in Table III. The control animals gained weight during the
by A.
Inhibition of toxin production
parositicus.
(9)
10 mM
1 mM
0.1 mM
0 ND ND ND
0 No growth 85.52 ztz 10.94 No growth
0 71.56 76.11 65.28
in the medium
f 5.90 0 f 0.52 0 0
under the conditions
studied was 5.1 f
f 6.1 zt 4.46 zt 13.80
2.0 pg/ml.
1. 2. 3. 4.
Normal AFB, AFB, + turmeric AFB, + curcumin
and curcumin
61.2 35.5 42.5 47.5
(9)
+ 16.0 f 5.9 +z 10.8 +z 1.85
Weight gain
Table 111. Effect of turmeric
50 46 76 58
000 840 520 200
f 7 133 +z 12 285 f 7 297 f. 5 283
WBC counts/ mm3
on the hepatotoxicity
18.5 17.18 19.64 18.78
+ 3.8 zt 3.7 z+z3.8 +z 4.8
98.0 150.6 250.6 269.4
zt 37.8 f 46.6 ziz 123.7 f 225.0
(pg of formed/ml)
GPT
Serum
(SW)
by AFB, in ducklings
Haemoglobin
induced
1.8 2.3 1.7 1.6
zrz 0.47 f 0.55 f 0.24 z!z 0.29
Liver GPT (fimoles of pyruvate formed/mg protein)
0.27 0.24 0.08 0.07
f +z zt f
0.1 0.04 0.05 0.03
Liver alkaline phosphatase (pmoles of phenol formed/ mg protein)
119
120
course of the experiment (61.25 g) whereas the AFBl treated animals gained only 35.5 g. When treated with turmeric and curcumin there was an increased weight change, i.e. 42.5 and 47.5 g, respectively. In the case of curcumin the value was found to be statistically significant (P < 0.01). The total WBC and haemoglobin did not significantly alter in the case of AFBl treated animals. Liver alkaline phosphatase was found to be significantly decreased after treatment with turmeric and curcumin. Serum GPT on the other hand increased significantly after AFBl treatment and remained elevated even after treatment with turmeric and curcumin. Similarly the lipid peroxides in the liver did not alter significantly after AFBl treatment, but remained lower than the control after treatment with turmeric and curcumin (data not shown). The pathological observations of liver after 14 days of treatment with AFBl showed extensive fatty changes, granular degeneration, necrosis and bile duct hyperplasia (Fig. la,b). There was almost complete reversal of necrosis and fatty changes in the case of animals treated with AFBl in the presence of curcumin (Fig. lc). In animals treated with AFBl in the presence of turmeric necrosis was almost completely reversed and there was only moderate fatty changes (Fig. Id). In both these cases there was no marked reversal of bile duct hyperplasia which was shown after AFBl treatment. Discussion The present results indicate that the food additives such as turmeric and asafoetida when added to the medium containing A. parasiticus inhibited the toxin production, without inhibiting the growth of mycelium. The concentration needed for 50% inhibition was approximately 2.5 mg/ml of the medium. Garlic on the other hand inhibited the growth of the organism. The inhibition of the aflatoxin production was also seen in the presence of antioxidants such as BHA, BHT and ellagic acid. However, curcumin did not inhibit the
toxin production even at 10 mM concentration. Tumeric has been shown to inhibit the in Salmonella typhimurium mutagenesis strains induced by polycyclic hydrocarbons and also was found to be anticarcinogenic [19]. Although the major antioxidant component present in turmeric is curcumin, recently a small peptide with antioxidant and anticlastogenie properties has been reported [20]. Similarly asafoetida and garlic contain several antioxidant sulphur containing compounds such as diallyl sulphide and umbelliferone which are anticarcinogenic in experimental animals [21,22]. The property of the extracts to inhibit the aflatoxin production could be mainly attributed to their antioxidant activity as several oxidation steps are involved in the aflatoxin biosynthesis by A. parasiticus. Similar results were also seen with antioxidants such as BHA, BHT and ellagic acid. The inability of curcumin to inhibit aflatoxin production may be related to its decreased permeability. I Both turmeric and curcumin inhibited the aflatoxin induced toxicity in experimental ducklings. The weight change induced by the toxin is partially reversed by the simultaneous administration of turmeric or curcumin. The such as WBC and other parameters haemoglobin, GPT and alkaline phosphatase in the liver remained unaltered in the condition studied. Turmeric and curcumin alone has not been found to have any inhibitory effect on alkaline phosphatase. The most remarkable feature is the histopatholgical findings in which the aflatoxin induced injury is remarkably reduced in animals treated with curcumin and turmeric. Both turmeric and curcumin are non-toxic [35], non-mutagenic [24] and are being consumed as spices in several parts of the world. The use of these cost effective food additives in the amelioration of aflatoxicosis needs further serious consideration. References 1
Shank, Dietary
R.C., Wogan. G.N. and Gibson, aflatoxins and human liver cancer.
J.B. (1972) I. Toxigenic
121
2
moulds in foods and food stuffs of tropical South-East Asia.
tions of Pons method
Food Cosmet.
gorund nut cake. J. Food Sci. Technol.,
Garner,
Toxiccll.,
B.C.
(1980)
10, 51-
60.
Carcinogenesis
by fungal products.
4
Mutagenicity
of carcinogenic
typhimurium.
Cancer Res., 38, 536-542.
Garner,
R.C.
and Matrin,
C.N.
15
Bergmeyer,
1, pp. 287-225,
Editor: P.L.
G.N.
Methods
in Cancer
Stolman,
A. (1974)
(1982)
Aflatoxin
Research,
carcinogenesis.
Vol.
VII,
pp. 309-
R.G.,
R.A.
60,
1036-
Firozi, P.F.
by aflatoxin B1 in vitro. Carcinogenesis, Firozi, P.F.,
Aboobaker,
V.S.
Action of vitamin-A
5, 1359-
catalysed reaction.
and 181112
O.P.
related
Antioxidant
22
compounds.
Pharmacoi.,
23
Picretti, S., Finotti., S. and Passi,
on
aflatoxin
production.
and free radical Mycotoxin
Res.,
K.K.
and Kuttan,
carcinogenesis
by
R.
(1989)
curcumin.
Inhibition J.
of
Ethnophar-
27, 227-233.
Shalini, V.K.
and Srinivas, L. (1987) action of turmeric
and
longa).
J.
4, 82-89.
Wargovich,
M.J.,
Woods,
C.L.
Gray,
K.
and
Anti-mutagenic (Curcumo
C.,
(1988)
Eng., V.W.S.,
Stephens,
Chemoprevention
of
N-
induced esophageal cancer in diallyl sulphide.
Unnikrishnan,
M.C. and Kuttan, R. (1990) Tumour reduc-
51, 85-89.
Abraham,
S.K. and Kesavan, P.C. (1984)
Genotoxicity
of
85-88. In: The Genus Aspergillus,
B. Raper
Williams Wilkins Company, Rati, E.R.,
18,
garlic, turmeric and asafoetida in mice. Mutat. Res., 136,
25,
1812.
Brain et al. (1961) Editors: Kennth
13
Biochem.
Lipid
ing and anti-carcinogenic activity of selected spices. Cancer
Cancer
activity of curcumin
(1971)
Neurochem.,
Effect of different antioxidants
Soudamini,
Len,
(1976:
A.S. J.
Cancer Res., 48, 6872 - 6875.
R.K.
Lett., 34, 213-220. Sharma,
A.H.
London.
rats by the naturally occurring thioether,
1362.
on DNA adduct formation by
aflatoxin B, in a microsome
in rat brain.
nitrosomethyl benzyl-amine
adduct
and Bhattacharya,
London.
Editors: B. Varley.
S. and Balasubramanian, formation
Nutr. Growth Cancer,
V.S.
Factors modul.ating the formation of DNA
Practical
(1980) In: Practical Clinical
5th Edn. p. 850.
anti-carcinogenic 21
1043.
and Aboobaker,
and M. Bell. Heinmann,
Fanelli, C., Fabbri, A.A.,
macol., 20
liver microsomes in vitro. Biochem.
R.K.,
Bishayee,
chemical
evidence for its formation in rat liver in
Biophys. Res. Commun., Bhattacharya,
19
13, 581-602. (1974) Afla-
In:
65-69.
removal and excretion in relation
Dreg Metab. Rev.,
(1980)
and M. Bell. Heinmann,
S. (1985)
and Wogan,
Miller, E.C. and Miller, J.A.
vivo and by human
11
Gowenlock
scavengers
Bennet,
E.
909 - 920. 18
Press, Inc., New York.
13, 168. Bernt,
King, E.J. and Amstrong, A.R.
peroxide
304.
Metabolic activation of aflatoxin B. pattern of
toxin Bl 2,3-oxide:
(1987)
In:
In: Progress in Chemical Toxicology,
Croy,
Swenson, D.H.,
(1984)
CRC
Press, New York.
to carcinogenesis.
10
16
17
(1973)
Essigman, J.M.
Glover.
F:_.
Wogan,
DNA adduct formation,
9
toxins,
Gowenlock
G.N.
8
Fungal
and
Clinical Biochemistry, 5th Edn. p. 685. Editors: B. Varley, A.H.
(1979)
H.U.
Biochemistry,
Vol.
Vol. 5, Academic 7
in Salmonella
Br. J. Haematol.,
aflatoxins and nucleic acids. In: Chemical Carcinogens and
Editor: H. Bush. Academic 6
mycotoxins
Y. (1978)
DNA,
Press, Boca-Raton, 5
T. and Nakamura,
and
International committee for standardisation in haematology (1967)
, ho,
Ueno, Y., Kubota, K
in Corn
24, 90-91.
14
Br. Med. Bull., 36, 47-52. 3
of estimating AFBr
Prema,
and Dorothy Baltimore,
V. and Shantha,
pp. 109.
I. Fennel.
The
Nagabhushan,
M.
and
Bhide,
S.V.
Modifica-
(1986)
Non-
mutagenicity of curcumin and its anti-mutagenic action versus chilli and capsaicin. Nutr. Cancer,
MD. T. (1987)
24
8, 201-210.
