WO2003080081A2 - Synergistic composition of trans-tetracos-15-enoic acid and apocynin and use thereof - Google Patents

Synergistic composition of trans-tetracos-15-enoic acid and apocynin and use thereof Download PDF

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WO2003080081A2
WO2003080081A2 PCT/IB2002/001212 IB0201212W WO03080081A2 WO 2003080081 A2 WO2003080081 A2 WO 2003080081A2 IB 0201212 W IB0201212 W IB 0201212W WO 03080081 A2 WO03080081 A2 WO 03080081A2
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composition
mammals
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WO2003080081A3 (en
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Sukhdev Swami Handa
Om Parkash Suri
V.N. Gupta
Krishan Avtar Suri
Naresh Kumar Satti
Vikram Bhardwaj
Bupinder Singh
Bal Krishan Chandan
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Council of Scientific and Industrial Research CSIR
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Priority to EP02722566A priority patent/EP1509285B1/en
Priority to AT02722566T priority patent/ATE348648T1/en
Priority to JP2003577907A priority patent/JP4299147B2/en
Priority to DE60216979T priority patent/DE60216979T2/en
Priority to PCT/IB2002/001212 priority patent/WO2003080081A2/en
Priority to AU2002253445A priority patent/AU2002253445A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • the present invention relates to a synergistic hepatoprotective composition comprising trans-tetracos-15-enoic acid (TCA) and Apocynin (APO).
  • TCA trans-tetracos-15-enoic acid
  • APO Apocynin
  • the present invention also relates to use in the treatment of hepatotoxicity in mammals.
  • BACKGROUND ART Apocynin a cardiotonic constituent of the rhizomes of Apocyanum cannabium [Finnemore Horace, J. Chem. Soc. 93 (1908) 1513-1520] and A.
  • Roots of Picrorhiza kurroa are used therapeutically in traditional medicine of almost all Asian countries to treat a manifold of conditions of illness including liver, lung & spleen ailments [Rajaram, D. (1976) Bomb. Hosp. Journal, 18, 66-69; Pandey, G.S. (1979) Indian Materia Medica, Chaukhamba Sanskrit Sansthan, Varanasi pp 70-71; Langar, J.G., Gupta, O.P. and Atal, C.K. (1981), Ind. J. Pharm. 13, 98-99; Handa, S.S., Sharma, A.
  • the mixture of Apocynin, trans-tetracos-15-enoic acid prepared in many other proportions by weight have not shown any enhancement in hepatoprotective action and in some experiments the biological activity of the mixture is much less than the individual constituents.
  • Yet another object of the present invention is to provide a composition having broader spectrum of hepatoprotective activity than the established herbal product in use viz., Silymarin.
  • Still another object of the invention is to provide a composition having potential therapeutic application in obstructive and viral hepatitis.
  • the present invention relates to a synergistic hepatoprotective composition containing trans-tetracos-15-enoic acid (TCA) and Apocynin (APO).
  • TCA trans-tetracos-15-enoic acid
  • APO Apocynin
  • the present invention also relates to use of treatment for hepatotoxicity in mammals.
  • the present invention provides a synergistic pharmaceutical composition having enhanced hepatoprotective activity on subjects, obtained from the plant Indigofera tinctoria, said composition comprising an effective amount of:
  • TCA trans-tetracos-15-enoic acid
  • Apocynin (APO) obtained from the plants Apocyanum cannabium and A. androsaemifolium;
  • composition is used for both preventive and curative properties.
  • composition is used systemically, orally or by any clinically/medically accepted uses.
  • composition is used to treat hepatic disorders that are clinically, biochemically and histologically similar to that of viral hepatitis, chronic hepatitis, fatty liver, cirrhosis and several vascular lesions of the liver.
  • composition is used to treat the liver damage induced by hepatotoxins.
  • hepatotoxins are selected from the group consisting of Galactosamine, Paracetamol and Carbon tetrachloride.
  • Still another embodiment of the present invention wherein the subject is selected from the group consisting of mammals. Yet another embodiment of the present invention, wherein the dosage of said composition for the treatment of CC1 induced hepatotoxicity in mammals is 50 mg/kg-body weight.
  • composition having the enhanced hepatoprotective activity in CCL induced hepatotoxic mammals upto 92%.
  • compositions for the treatment of acetaminophen induced hepatotoxicity in mammals is 50mg/kg-body weight.
  • composition having the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals upto 86% wherein said composition having the dosage of said composition for the treatment of Galactosamine induced hepatotoxicity in mammals 50 mg/kg of body weight.
  • composition having the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals upto 75%.
  • the dosage of said composition for chloretic activity in mammals to control bile flow and bile solids is 50 mg/kg of body weight.
  • Still another embodiment of the present invention wherein the enhanced chloretic activity is upto 39%.
  • the present invention also provides use in treating subjects with liver disorders with an effective amount of synergistic pharmaceutical composition to induce enhanced hepatoprotective activity, said composition comprising:
  • trans-tetracos-15-enoic acid obtained from the plant Indigofera tinctoria
  • APO Apocynin obtained from the plants Apocyanum cannabium and A. androsaemifolium
  • the ratio of APO and TCA is in the range of 3:1 to 1:3.
  • Still another embodiment of the present invention wherein said use is used to treat liver disorders caused by Galactosamine, Paracetamol and Carbon tetrachloride.
  • a use wherein the dosage for the treatment of CC1 4 induced hepatotoxicity in mammals is about 50-mg/kg-body weight.
  • a use wherein the enhanced hepatoprotective activity in CC1 4 induced hepatotoxic mammals is upto 92%.
  • a use wherein the dosage for the treatment of acetaminophen induced hepatotoxicity in mammals is 50mg/kg-body weight.
  • Still another embodiment of the present invention a use wherein the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals is upto
  • Still another embodiment of the present invention a use wherein the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals is upto 75%.
  • a use wherein the dosage for chloretic activity in mammals to control the bile flow and bile solids is 50 mg/kg of body weight.
  • Still another embodiment of the present invention a use wherein the enhanced cholretic activity in mammals is upto 39%. Yet another embodiment of the present invention, a use wherein the composition is used either singly or in combination with pharmaceutically acceptable earners.
  • composition is administered to a subject in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binder or stabilizers.
  • desired dosage is administered for both preventive and curative properties.
  • composition is administered systemically, orally or by any clinically/medically accepted uses.
  • Yet another embodiment of the present invention a use wherein the subject is selected from animals, mammals.
  • Hepatotoxins It is emphasized that hepatotoxin that causes acute hepatitis should have close resemblance with the viral hepatitis, clinically, biochemically and histologically.
  • Drugs are also causes of chronic hepatic disease as chronic hepatitis, fatty liver, cirrhosis and several vascular lesions of the liver. In many instances drug induced hepatitis proves indistinguishable from viral hepatitis.
  • Chemically induced hepatic injury for experimental studies should be severe enough to cause cell death or to modify hepatic functions. The mechanism of acute hepatic injury depends upon the chemical compound and the species of animals used. Many chemicals produce parenchymal damage, arrest bile flow and cause jaundice (chloretic injury). Hepatoprotective activity against CC1 4 , paracetamol and, D- galactosamine induced hepatotoxicity are studied.
  • CC1 4 Carbon tetrachloride (CC1 4 ): CC1 4 is one of the most powerful hepatotoxins (in term of severity of injury). It causes toxic necrosis, which leads to biochemical changes having clinical features similar to those of acute viral hepatitis (Nogel, 1977, Bramanti et. al., 1978, Kumar et. al., 1992). Liver injury is produced by administration of CC1 4 mixed with liquid paraffin. Animals are given single dose of CC1 4 (50 ⁇ l.kg "1 , p.o.) in acute single treatment and (0.5 ml.kg "1 , p.o.) in case of multitreatment with drug. It is . administered orally (p.o) by gastric intubation.
  • the control animals received the equal volume of liquid paraffin.
  • Paracetamol APAP, acetaminophen: It is a therapeutic agent widely used as analgesic/antipyretic drug. When taken in large doses it causes hepatic necrosis which leads to biochemical changes having clinical features similar to those of acute viral hepatitis. The similar effect is observed in animals. The toxic effect can be potentiated if it is given several hours after the anesthetic ether inhalation (Wells et. al, 1986).
  • Liver injury is induced by injecting paracetamol (200 mg.kg "1 ) interaperitoneally in normal saline (pH 9.4) six hour after inhalation of anesthetic ether (4ml/4min/6animals) in a closed chamber.
  • the control animals received the equal volume of vehicle.
  • D-Galactosamine It is one of the toxins that induce hepatic inflammatory conditions in the rat liver that clinically resembles to viral hepatitis.
  • the mechanism of Gal ⁇ induced liver injury has been extensively examined and this model is now accepted as one of the authentic systems of liver damage (Bauer et. al., 1974, Al-Tuwaijiri et. al., 1981).
  • Treatment of test material before and after hepatotoxin The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of rats at 48 hours, 24 hours and 2 hours before and 6 hours after hepatotoxin (CCL , 0.5 ml.kg "1 , p.o.) intoxication. Blood is collected from orbital sinus in all the animals 18 hours after last treatment and serum separated for different estimations.
  • Paracetamol induced hepatotoxicity Treatment of test material before and after hepatotoxin: The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of mice at 72 hours, 48 hours and 24 hours, 1 hour before diethyl ether inhalation and lhour after hepatotoxin (paracetamol, 200 mg.kg " , i.p.) given 6 hours after exposure to diethyl- ether. Blood is collected from orbital sinus in all the animals 18 hours after last treatment and serum separated for different estimations. A portion of the liver is processed for histopathological studies. (Table 2) D-Galactosamine induced hepatotoxicity:
  • mice The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of mice at 48 hours, 24 hours and 2 hours before and 6 hours after hepatotoxin (GalN, 300 mg.kg "1 , s.c.) intoxication. Blood is collected from orbital sinus in all the animals 18 h after last treatment and serum separated for different estimations. All the animals are then killed by decapitation, their livers are quickly excised, cleaned of adhering tissue, weighed and homogenised in phosphate buffer saline for the analysis of hepatic parameters. A portion of the liver is processed for histopathological studies (Table 1)
  • GPT and GOT Pyruvate formed by transamination reaction is determined spectrophotometrically after reaction with 2,4-dinitrophenylhydrazine (Reitman and
  • ALP /7-nitrophenol formed in alkaline medium is measured spectrophotometrically using p-nitrophenyl phosphate as substrate (Walter and Schutt, 1974).
  • Bilirubin Total bilirubin is measured by diazotization reaction with NaNO 2 (Malloy and
  • Triglycerides Triglycerides from serum are extracted with isopropanol and sopanified with KOH. The liberated glycerol is converted to formaldehyde by periodate and determined after reaction with acetyl acetone. Triolein is used as standard (Neri and
  • Glutathione It is determined after deproteination by reaction with DTNB (Ellman 1959 as modified by David 1987) .
  • Lipid peroxidation Thiobarbituric acid reacting substances are determined spectrophotometrically at 535 nm. Buege. and Aust. (1978).
  • Hepatoprotective activity is calculated by the following equation :
  • H [ 1- ( TC - V / VC -V ) ] x 100
  • TC, VC, and V are drug + toxin, vehicle + toxin and vehicle treated groups of animals respectively.
  • the liver by producing bile, plays an important role in digestion.
  • the presence of bile in the intestine is necessary to accomplish the digestion and absorption of fats as well as absorption of the fat-soluble vitamins- A, D, E & K.
  • Bile is also an important vehicle of excretion. It removes many drugs, toxins, bile pigments and various inorganic substances either derived from the diet or synthesized by the body as cholesterol or as cholic acid.
  • Increase in the bile flow is suggestive of stimulating action of liver microsomal enzymes. Effect on the liver bile flow of test drug and that of vehicle is carried out after cannulating the bile duct in normal anaesthesied rats.
  • Bile collected is from each animal from 0-5 hours (Klaassen, 1969, Donal et al.1953). (Table 5) Histopathological studies: Hitopathological studies: A portion of the liver after treatment of hepatotoxin (GalN, CCl j and paracetamol) and test material is processed for histopathological studies by routine hematoxyline and eosin stained sections (Krajian, A. A., 1963). General behaviour and acute toxicity: Using different doses (10, 30, 100, 1200, 1400, 1600, 1800 and 2000 mg.kg "1 ) of said composition given orally to the groups of 10 mice for each dose, while one group with same number of mice served as control.
  • mice are observed continuously for 1 hour and then half hourly for 4 hours for any gross behavioral changes and general motor activity, writhing, convulsion, response to tail pinching, gnawing, piloerection, pupil size, fecal output, feeding behaviour etc. and further up to 72 hours for any mortality.
  • Acute LD 50 values in mice are calculated by the use of Miller and Tainter, (1944). Mortality of animals in all the groups used in different models for determining hepatoprotective activity during the period of treatment is also recorded as a rough index of subacute toxicity.
  • Statistical analysis The data obtained are subjected to statistical analysis using ANOVA for comparing different groups (Armitage, 1987) and Dunnett's t test for control and test groups (Dunnett, 1964).
  • the regression coefficient (Slope b) correlation coefficient (r) with its p value and ED 5 o with 95% confidence limit (CL) are determined by regression analysis using log dose and percent effect of adaptogenic activity (Swinscow, 1980).
  • the two tailed paired student t test for comparing means before and after treatment and one tailed unpaired student t test for comparing control and drug treated groups (Ghosh, 1984) are used.
  • the ? value of ⁇ 0.05 or less is taken as the criterion of significance.
  • Table 1 Hepatoprotective activity (in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (pre-treatment fed at 48 h, 24h, before and 6 h after hepatotoxin ) against the D-Galactosamine (GalN) [(300 mg kg "1 in normal saline, sub cutaneously (s.c.)] induced hepatic injury rats"
  • GalN D-Galactosamine
  • a Values represent the mean ⁇ S.E. and within parentheses hepatoprotective activity percent of six animals in each group, Rats: Wistar, (150-1 g) male.
  • each unit is ⁇ ole pyruvate/min L.
  • b is ⁇ mole of -nitrophenol formed min L
  • c is n moles MDA/g liver.
  • d is ⁇ mole GSH g liver
  • Table 2 Hepatoprotective activity ⁇ in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (Pre-treatment) fed at 72 h, 48 h, 24 h, lh befo inhalation of diethyl-ether and I h after 'Acetaminophen' ((APAP) 200 mg kg "1 ) given i. p. 6 h after exposure to diethyl-ether in mice 3
  • APAP 'Acetaminophen'
  • Unit each unit is ⁇ mole pyruvate/min L.
  • b is ⁇ mole of 7-nitrophenol formed/min/ L
  • c is n moles MDA/g liver.
  • d is ⁇ mole GSH/g liver
  • Table 3 Hepatoprotective activity (in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (pre- treatment fed at 48h, 24h, 2h before 6h after hepatotoxin) against CCL-. (0.5 ml kg "1 , p.o.) induced hepatic injury in rats a
  • a Values represent the mean ⁇ S.E. and within parentheses hepatoprotective activity percent of six animals in each group, Rats: Wistar, (150-1 g) male.
  • each unit is ⁇ mole pyruvate/min/L.
  • b is ⁇ mole ofp-nitrophenol formed/min/ L
  • c is n moles MDA/g liver.
  • d is ⁇ mole GSH/g liver
  • Table 4 Hepatoprotective activity (in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (pre- treatment fed at 48h, 24h, 2h before a 6h after hepatotoxin) against CC1 4 (0.5 ml kg "1 , p.o.) induced hepatic injury in rats"
  • a Values represent the mean ⁇ S. E. and within parentheses hepatoprotective activity percent of six animals in each group, Rats: Wistar, (150- 175 male.
  • each unit is ⁇ mole pyruvate/min/L.
  • b is ⁇ mole ofp-nitrophenol formed/mm/ L
  • c is n moles MDA/g liver.
  • d is ⁇ mole GSH/g liver
  • Table- 5 Chloretic activity of TCA, APO, Mixture of TCA & APO (1:1) and Dehydrocholic acid (DHC) as percent increase in bile flow and bile solids when compared to normal values in rats a .
  • DHC Dehydrocholic acid

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Abstract

The present invention relates to a synergistic hepatoprotective pharmaceutical composition comprising an effective amount of trans-tetracos-15-enoic acid (TCA) and Apocynin (APO), the present invention also relatesto use in the treatment for hepatotoxicity.

Description

SYNERGISTIC COMPOSITION OF TRANS-TETRACOS-15-ENOIC ACID AND
APOCYNLN AND USE THEREOF
TECHNICAL FIELD The present invention relates to a synergistic hepatoprotective composition comprising trans-tetracos-15-enoic acid (TCA) and Apocynin (APO). The present invention also relates to use in the treatment of hepatotoxicity in mammals. BACKGROUND ART Apocynin, a cardiotonic constituent of the rhizomes of Apocyanum cannabium [Finnemore Horace, J. Chem. Soc. 93 (1908) 1513-1520] and A. androsaemifolium [Naves Yves-Rene, Helv Chim Acta 32 (1949) 1351] and essential oils of the rhizomes of Iris species have been reported to be responsible for choleretic activity of Picrorhiza kurroa [Basu, K., Dasgupta B., Bhattacharya, S.K. and Debnath, P.K. Curr Sci, 40 (1971) 603]. The constituent has been synthesized and pharmacologically evaluated [Suri, O.P., Bindra, R.S., Satti, N.K. and. Khajuria, R.K. Indian Journal of Chemistry, 26B (1987), p 587-88]. Apocynin has also been evaluated for antioxidant and free radical scavenging activities. Roots of Picrorhiza kurroa are used therapeutically in traditional medicine of almost all Asian countries to treat a manifold of conditions of illness including liver, lung & spleen ailments [Rajaram, D. (1976) Bomb. Hosp. Journal, 18, 66-69; Pandey, G.S. (1979) Indian Materia Medica, Chaukhamba Sanskrit Sansthan, Varanasi pp 70-71; Langar, J.G., Gupta, O.P. and Atal, C.K. (1981), Ind. J. Pharm. 13, 98-99; Handa, S.S., Sharma, A. and Chakraborty, K.K. (1986), Fitotherapia, 58, 307-351; Ansari, R.R., Kapoor, N.K., Kulshreshta, D.K., Mehta, H., Mehrotra, B.N., Patnaik, G.K. and Sharma, S.K. (1988), Indian J. Med. Plants, 87, 401-404] and inflammatory disorders [Nadkarni, K.M. (1954), Indian Materia Medica, vol.l, Popular Book Depot, Bombay pρ.25-27, 619-622, 634-651, 953-955, 1220-1221, 1252-53; Dey, A.C. (1981), Indian Medicinal Plants used in Ayurvedic Preparations, Bishan Singh & Mahendra Pal Singh, Dehra Dun, India pp 81- 82; Jayaweera, D.M.A. (1982) Medicinal Plants used in Ceylon, The National Science Council of Sri Lanka, Colombo, Sri Lanka, part 5, pp 76]. Hepatoprotective, Immunostimulant & Immunorestorative formulations based on P. Kurroa chemical constituents, mainly iridoid glycosides, have been developed at CDRI Lucknow & RRL Jammu [450/DEL/89 & 845/DEL/92].
Literature survey revealed that earlier reports showed the presence of trans-tetracos-15- enoic acid in Jojoba oil ex. Simmondsia chinensis seeds (0.62-1.11%) and cis isomer of the acid is reported in fatty acids of the seed oil of Microula sikkimensis (1.2% [Wang Huiying, Yu Xuefian, Yi Yuanfen and Ding Jingkai Yunnan Zhiwu-Yanjiu 1989, 11 (1), 60-4 (Ch.), L. Jing Jingmin, Wang Jingping, Yu Fenglan. Zhiwu Xuebao, 1989, 31 (1), 50- 3 (Ch.) These reports do not mention isolation of the constituent and the content estimation based on GLC data. Indigofera tinctoria has been in use in indigenous system of medicine in epilepsy, nervous disorders & bronchitis [Wealth of India, vol.5. (Council of Scientific & industrial Research, New Delhi) 182, (1959)]. The plant is also used as ointment in sores, old ulcers and haemorrhoids [R.N. Chopra, S.L. Nayar and LC. Chopra, Glossary of Indian Medicinal Plants, 141 (1956)]. The leaves of the plants have been used in liver ailments [Nadkarni, K.M., Indian Materia Medica, vol. 1 (Popular Book Depot, Bombay, 680 (1954)]. Extract of the leaves of the plant has exhibited marked hepatoprotective effect against CC1 induced hepatic injury in rabbits, rats and mice at Regional Research Laboratory (RRL) Jammu. [Anand, K.K., Chand Dewan and Ghatak, B.J. Ray, Indian J. Exp. Biol, 17, 685 (1979); Anand, K.K., Chand Dewan, Ghatak, B.J. Ray and Arya, R.K., Indian J. Expl. Biol., 19, 298 (1981)].
Recent study in RRL Jammu for hepatoprotective effect of the plant extract and further bioactivity-guided fractionation has resulted in identification of trans-tetracos-15-enoic acid as the active principle. The constituent has been synthesized and observed to possess dose related hepatoprotective effect against galactosamine, paracetamol and CC14 as hepatotoxins using commercially available silymarin as reference material. Activity of the formulation being described in the invention is not exactly equal to the sum of the activities of the two individual constituents and activity enhancement does not occur simply due to the mixing of the two compounds. This has been verified by mixing the formulation with RLJ-NE-299A, a standardized mixture of iridoid glycosides from Picrorhiza kurroa possessing hepatoprotective, immunostimulant and immunorestorative effects [Indian patent no. 178866].
The mixture of Apocynin, trans-tetracos-15-enoic acid prepared in many other proportions by weight have not shown any enhancement in hepatoprotective action and in some experiments the biological activity of the mixture is much less than the individual constituents.
OBJECTS OF THE INVENTION
The primary object of the invention is to provide a synergistic composition of trans- tetracos-15-enoic acid and Apocynin. Another object of the present invention is to provide use in the treatment for hepatotoxicity.
Yet another object of the present invention is to provide a composition having broader spectrum of hepatoprotective activity than the established herbal product in use viz., Silymarin.
Still another object of the invention is to provide a composition having potential therapeutic application in obstructive and viral hepatitis.
SUMMARY OF THE INVENTION
The present invention relates to a synergistic hepatoprotective composition containing trans-tetracos-15-enoic acid (TCA) and Apocynin (APO). The present invention also relates to use of treatment for hepatotoxicity in mammals.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a synergistic pharmaceutical composition having enhanced hepatoprotective activity on subjects, obtained from the plant Indigofera tinctoria, said composition comprising an effective amount of:
(a) trans-tetracos-15-enoic acid (TCA) obtained from the plant Indigofera tinctoria;
(b) Apocynin (APO) obtained from the plants Apocyanum cannabium and A. androsaemifolium; and
(c) the ratio of APO and TCA is in the range of 3 : 1 to 1 :3. An embodiment of the present invention, wherein the said composition is used either singly or in combination with pharmaceutically acceptable additives.
An another embodiment of the present invention, wherein the pharmaceutically acceptable additives are selected from the group consisting of carriers, diluents, solvents, filters lubricants, excipients, binder and stabilizers. Yet another embodiment of the present invention, wherein the said composition is used for both preventive and curative properties.
Still another embodiment of the present invention, wherein the said composition is used systemically, orally or by any clinically/medically accepted uses.
Yet another embodiment of the present invention, wherein the said composition is used to treat hepatic disorders that are clinically, biochemically and histologically similar to that of viral hepatitis, chronic hepatitis, fatty liver, cirrhosis and several vascular lesions of the liver.
Still another embodiment of the present invention, wherein the said composition is used to treat the liver damage induced by hepatotoxins. Yet another embodiment of the present invention, wherein the hepatotoxins are selected from the group consisting of Galactosamine, Paracetamol and Carbon tetrachloride.
Still another embodiment of the present invention, wherein the subject is selected from the group consisting of mammals. Yet another embodiment of the present invention, wherein the dosage of said composition for the treatment of CC1 induced hepatotoxicity in mammals is 50 mg/kg-body weight.
Still another embodiment of the present invention, wherein said composition having the enhanced hepatoprotective activity in CCL induced hepatotoxic mammals upto 92%.
Yet another embodiment of the present invention, wherein the dosage of said composition for the treatment of acetaminophen induced hepatotoxicity in mammals is 50mg/kg-body weight.
Still another embodiment of the present invention, wherein said composition having the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals upto 86%. i Yet another embodiment of the present invention, wherein said composition having the dosage of said composition for the treatment of Galactosamine induced hepatotoxicity in mammals 50 mg/kg of body weight.
Still another embodiment of the present invention, wherein said composition having the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals upto 75%.
Yet another embodiment of the present invention, the dosage of said composition for chloretic activity in mammals to control bile flow and bile solids is 50 mg/kg of body weight.
Still another embodiment of the present invention, wherein the enhanced chloretic activity is upto 39%.
The present invention also provides use in treating subjects with liver disorders with an effective amount of synergistic pharmaceutical composition to induce enhanced hepatoprotective activity, said composition comprising:
(a) trans-tetracos-15-enoic acid (TCA) obtained from the plant Indigofera tinctoria; (b) Apocynin (APO) obtained from the plants Apocyanum cannabium and A. androsaemifolium; and (c) the ratio of APO and TCA is in the range of 3:1 to 1:3.
Still another embodiment of the present invention, wherein said use is used to treat liver disorders caused by Galactosamine, Paracetamol and Carbon tetrachloride. Yet another embodiment of the present invention, a use wherein the dosage for the treatment of CC14 induced hepatotoxicity in mammals is about 50-mg/kg-body weight.
Still another embodiment of the present invention, a use wherein the enhanced hepatoprotective activity in CC14 induced hepatotoxic mammals is upto 92%. Yet another embodiment of the present invention, a use wherein the dosage for the treatment of acetaminophen induced hepatotoxicity in mammals is 50mg/kg-body weight.
Still another embodiment of the present invention, a use wherein the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals is upto
86%. Yet another embodiment of the present invention, a use wherein the dosage for the treatment of Galactosamine induced hepatotoxicity in mammals is 50mg/kg of body weight.
Still another embodiment of the present invention, a use wherein the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals is upto 75%.
Yet another embodiment of the present invention, a use wherein the dosage for chloretic activity in mammals to control the bile flow and bile solids is 50 mg/kg of body weight.
Still another embodiment of the present invention, a use wherein the enhanced cholretic activity in mammals is upto 39%. Yet another embodiment of the present invention, a use wherein the composition is used either singly or in combination with pharmaceutically acceptable earners.
Still another embodiment of the present invention, a use wherein the composition is administered to a subject in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binder or stabilizers. Yet another embodiment of the present invention, a use wherein the desired dosage is administered for both preventive and curative properties.
Still another embodiment of the present invention, a use wherein the composition is administered systemically, orally or by any clinically/medically accepted uses.
Yet another embodiment of the present invention, a use wherein the subject is selected from animals, mammals.
The invention is further explained in the form of preferred embodiments. i. Animals: The pharmacological studies are conducted on Wistar albino rats (150-180g) and Swiss albino mice (25-30g) of either sex, colony - bred in the Institute's animal house.
After procurement, all the animals are divided into different groups and are left for one week for acclimatization to experimentation room and are maintained on standard conditions (23±2°C, 60-70 % relative humidity and 12 h photo period). The animals are fed with standard rodents pellet diet and water ad libitum. There are six animals in each group except for general behaviour and acute toxicity studies where ten animals are used in each group. ii. Hepatotoxins: It is emphasized that hepatotoxin that causes acute hepatitis should have close resemblance with the viral hepatitis, clinically, biochemically and histologically. Drugs are also causes of chronic hepatic disease as chronic hepatitis, fatty liver, cirrhosis and several vascular lesions of the liver. In many instances drug induced hepatitis proves indistinguishable from viral hepatitis. Chemically induced hepatic injury for experimental studies should be severe enough to cause cell death or to modify hepatic functions. The mechanism of acute hepatic injury depends upon the chemical compound and the species of animals used. Many chemicals produce parenchymal damage, arrest bile flow and cause jaundice (chloretic injury). Hepatoprotective activity against CC14, paracetamol and, D- galactosamine induced hepatotoxicity are studied.
Carbon tetrachloride (CC14): CC14 is one of the most powerful hepatotoxins (in term of severity of injury). It causes toxic necrosis, which leads to biochemical changes having clinical features similar to those of acute viral hepatitis (Nogel, 1977, Bramanti et. al., 1978, Kumar et. al., 1992). Liver injury is produced by administration of CC14 mixed with liquid paraffin. Animals are given single dose of CC14 (50 μl.kg"1, p.o.) in acute single treatment and (0.5 ml.kg"1, p.o.) in case of multitreatment with drug. It is. administered orally (p.o) by gastric intubation. The control animals received the equal volume of liquid paraffin. (Table 3, 4). Paracetamol (APAP, acetaminophen): It is a therapeutic agent widely used as analgesic/antipyretic drug. When taken in large doses it causes hepatic necrosis which leads to biochemical changes having clinical features similar to those of acute viral hepatitis. The similar effect is observed in animals. The toxic effect can be potentiated if it is given several hours after the anesthetic ether inhalation (Wells et. al, 1986). Liver injury is induced by injecting paracetamol (200 mg.kg"1) interaperitoneally in normal saline (pH 9.4) six hour after inhalation of anesthetic ether (4ml/4min/6animals) in a closed chamber. The control animals received the equal volume of vehicle. (Table 2) D-Galactosamine: It is one of the toxins that induce hepatic inflammatory conditions in the rat liver that clinically resembles to viral hepatitis. The mechanism of GalΝ induced liver injury has been extensively examined and this model is now accepted as one of the authentic systems of liver damage (Bauer et. al., 1974, Al-Tuwaijiri et. al., 1981). (Tablel) Hepatic damage is produced by injecting GalN (300 mg.kg"1) subcutaneously in normal saline. The control animals received the equal volume of vehicle. iii. Treatment with bio-active compound and silymarin: Freshly prepared suspension (1 %, w/v) in 0.2% gum acacia in normal saline is used for all the experiments except for toxicity studies where (10 %, w/v) suspension is used. Silymarin suspension (1 %, w/v) in 0.2 % gum acacia is used as a reference standard (positive control). iv. Experimental models: Effect on serum and hepatic biochemical parameters: CC14 induced hepatotoxicity :
Treatment of test material before and after hepatotoxin: The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of rats at 48 hours, 24 hours and 2 hours before and 6 hours after hepatotoxin (CCL , 0.5 ml.kg"1, p.o.) intoxication. Blood is collected from orbital sinus in all the animals 18 hours after last treatment and serum separated for different estimations. All the animals are then killed by decapitation, their livers are quickly excised, cleaned of adhering tissue, weighed and homogenised in phosphate buffer saline for the analysis of hepatic parameters (Agarwal and Mehendale, 1983, Klingensmith and Mehendale, 1982, Zimmerman, 1973, Edmondson and Peter, 1985, Mitchell, et al, 1973). (Table 3-4).
Paracetamol induced hepatotoxicity: Treatment of test material before and after hepatotoxin: The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of mice at 72 hours, 48 hours and 24 hours, 1 hour before diethyl ether inhalation and lhour after hepatotoxin (paracetamol, 200 mg.kg" , i.p.) given 6 hours after exposure to diethyl- ether. Blood is collected from orbital sinus in all the animals 18 hours after last treatment and serum separated for different estimations. A portion of the liver is processed for histopathological studies. (Table 2) D-Galactosamine induced hepatotoxicity:
(a) Treatment of test material before and after hepatotoxin:
The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of mice at 48 hours, 24 hours and 2 hours before and 6 hours after hepatotoxin (GalN, 300 mg.kg"1 , s.c.) intoxication. Blood is collected from orbital sinus in all the animals 18 h after last treatment and serum separated for different estimations. All the animals are then killed by decapitation, their livers are quickly excised, cleaned of adhering tissue, weighed and homogenised in phosphate buffer saline for the analysis of hepatic parameters. A portion of the liver is processed for histopathological studies (Table 1)
Parameters studied:
GPT and GOT: Pyruvate formed by transamination reaction is determined spectrophotometrically after reaction with 2,4-dinitrophenylhydrazine (Reitman and
Frankel, 1957). ALP : /7-nitrophenol formed in alkaline medium is measured spectrophotometrically using p-nitrophenyl phosphate as substrate (Walter and Schutt, 1974).
Bilirubin : Total bilirubin is measured by diazotization reaction with NaNO2 (Malloy and
Evelyn, 1937)
Triglycerides : Triglycerides from serum are extracted with isopropanol and sopanified with KOH. The liberated glycerol is converted to formaldehyde by periodate and determined after reaction with acetyl acetone. Triolein is used as standard (Neri and
Firings, 1973).
Glutathione: It is determined after deproteination by reaction with DTNB (Ellman 1959 as modified by David 1987) . Lipid peroxidation: Thiobarbituric acid reacting substances are determined spectrophotometrically at 535 nm. Buege. and Aust. (1978).
Hepatoprotective activity:
Hepatoprotective activity (H) is calculated by the following equation :
H = [ 1- ( TC - V / VC -V ) ] x 100 Where TC, VC, and V are drug + toxin, vehicle + toxin and vehicle treated groups of animals respectively.
Effect on Bile flow and bile solids
The liver, by producing bile, plays an important role in digestion. The presence of bile in the intestine is necessary to accomplish the digestion and absorption of fats as well as absorption of the fat-soluble vitamins- A, D, E & K. Bile is also an important vehicle of excretion. It removes many drugs, toxins, bile pigments and various inorganic substances either derived from the diet or synthesized by the body as cholesterol or as cholic acid. Increase in the bile flow is suggestive of stimulating action of liver microsomal enzymes. Effect on the liver bile flow of test drug and that of vehicle is carried out after cannulating the bile duct in normal anaesthesied rats. Bile collected is from each animal from 0-5 hours (Klaassen, 1969, Donal et al.1953). (Table 5) Histopathological studies: Hitopathological studies: A portion of the liver after treatment of hepatotoxin (GalN, CCl j and paracetamol) and test material is processed for histopathological studies by routine hematoxyline and eosin stained sections (Krajian, A. A., 1963). General behaviour and acute toxicity: Using different doses (10, 30, 100, 1200, 1400, 1600, 1800 and 2000 mg.kg"1) of said composition given orally to the groups of 10 mice for each dose, while one group with same number of mice served as control. The animals are observed continuously for 1 hour and then half hourly for 4 hours for any gross behavioral changes and general motor activity, writhing, convulsion, response to tail pinching, gnawing, piloerection, pupil size, fecal output, feeding behaviour etc. and further up to 72 hours for any mortality. Acute LD50 values in mice are calculated by the use of Miller and Tainter, (1944). Mortality of animals in all the groups used in different models for determining hepatoprotective activity during the period of treatment is also recorded as a rough index of subacute toxicity. Statistical analysis. The data obtained are subjected to statistical analysis using ANOVA for comparing different groups (Armitage, 1987) and Dunnett's t test for control and test groups (Dunnett, 1964). The regression coefficient (Slope b) correlation coefficient (r) with its p value and ED5o with 95% confidence limit (CL) are determined by regression analysis using log dose and percent effect of adaptogenic activity (Swinscow, 1980). The two tailed paired student t test for comparing means before and after treatment and one tailed unpaired student t test for comparing control and drug treated groups (Ghosh, 1984) are used. The ? value of < 0.05 or less is taken as the criterion of significance.
Table 1: Hepatoprotective activity (in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (pre-treatment fed at 48 h, 24h, before and 6 h after hepatotoxin ) against the D-Galactosamine (GalN) [(300 mg kg"1 in normal saline, sub cutaneously (s.c.)] induced hepatic injury rats"
Figure imgf000011_0001
a: Values represent the mean ± S.E. and within parentheses hepatoprotective activity percent of six animals in each group, Rats: Wistar, (150-1 g) male.
Unit: each unit is μ ole pyruvate/min L. b: is μ mole of -nitrophenol formed min L, c: is n moles MDA/g liver., d: is μ mole GSH g liver
Table 2:Hepatoprotective activity {in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (Pre-treatment) fed at 72 h, 48 h, 24 h, lh befo inhalation of diethyl-ether and I h after 'Acetaminophen' ((APAP) 200 mg kg"1) given i. p. 6 h after exposure to diethyl-ether in mice3
Figure imgf000012_0001
a: Values represent the mean ± S.E. and within parentheses hepatoprotective activity percent of six animals in each group Mice: Swiss albino (2
30 g) male.
Unit: each unit is μmole pyruvate/min L. b: is μ mole of 7-nitrophenol formed/min/ L, c: is n moles MDA/g liver., d: is μ mole GSH/g liver
Table 3: Hepatoprotective activity (in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (pre- treatment fed at 48h, 24h, 2h before 6h after hepatotoxin) against CCL-. (0.5 ml kg"1 , p.o.) induced hepatic injury in ratsa
Figure imgf000013_0001
a: Values represent the mean ± S.E. and within parentheses hepatoprotective activity percent of six animals in each group, Rats: Wistar, (150-1 g) male.
Unit: each unit is μmole pyruvate/min/L., b: is μ mole ofp-nitrophenol formed/min/ L, c: is n moles MDA/g liver., d: is μ mole GSH/g liver
Table 4: Hepatoprotective activity (in vivo) of TCA, APO, Mixture of TCA & APO (1:1) and silymarin (pre- treatment fed at 48h, 24h, 2h before a 6h after hepatotoxin) against CC14 (0.5 ml kg"1 , p.o.) induced hepatic injury in rats"
Figure imgf000014_0001
a: Values represent the mean ± S. E. and within parentheses hepatoprotective activity percent of six animals in each group, Rats: Wistar, (150- 175 male.
Unit: each unit is μmole pyruvate/min/L., b: is μ mole ofp-nitrophenol formed/mm/ L, c: is n moles MDA/g liver., d: is μ mole GSH/g liver
Table- 5: Chloretic activity of TCA, APO, Mixture of TCA & APO (1:1) and Dehydrocholic acid (DHC) as percent increase in bile flow and bile solids when compared to normal values in ratsa.
Figure imgf000015_0001
a :Values represent mean ±SE of six animals in each group, Rats: Wistar 150-175 g) male b : Values represent mean ± SE of eight animals in each group. o
I
I
ADVANTAGES
Most of the hepatoprotective preparations/formulations available on the market are herbal based and hence are unstandardised chemically as well as biologically. Efficacy of the herbal formulations are known to be dependent upon secondary metabolites and reliability of these can only be assured if batch to batch standardization (chemical and pharmacological) are carried out. In the present invention a) Chemical composition of the formulation is well described, hence reproducible biological activity is assured. b) Activity parameters are of broader spectrum and hence effectiveness of the formulation in obstructive and viral hepatitis, c) Pharmacological evaluation data of the formulation clearly indicates synergistic action of the constituents of the formulation.

Claims

1. A synergistic pharmaceutical composition having enhanced hepatoprotective activity on subjects, said composition comprising an effective amount of :
(a) trans-tetracos-15-enoic acid (TCA) obtained from the plant Indigofera tinctoria; (b) Apocynin (APO) obtained from the plants Apocyanum cannabium and A. androsaemifolium; and (c) the ratio of APO and TCA is in the range of 3:,1 to 1:3.
2. A composition according to claim 1 wherein said composition is used either singly or in combination with pharmaceutically acceptable additives.
3. A composition according to claim 1 wherein the pharmaceutically acceptable additives are selected from the group consisting of carriers, diluents, solvents, filters lubricants, excipients, binder and stabilizers.
4. A composition according to claim 1 wherein the said composition is used for both preventive and curative properties.
5. A composition according to claim 1 wherein said composition is administered systemically, orally or by any clinically/medically accepted uses.
6. A composition according to claim 1 wherein the composition is used to treat hepatic disorders that are clinically, biochemically and histologically similar to that of viral hepatitis, chronic hepatitis, fatty liver, cirrhosis and several vascular lesions of the liver.
7. A composition according to claim 1 wherein said composition is used to treat the liver damage induced by hepatotoxins.
8. A composition according to claim 1 wherein the hepatotoxins are selected from the group consisting of Galactosamine, Paracetamol and Carbon tetrachloride.
9. A composition according to claim 1, wherein the subjects is selected from the group consisting of mammals.
10. A composition according to claim 1 wherein the dosage for the treatment of CC1 induced hepatotoxicity in mammals is 50 mg/kg-body weight.
11. A composition according to claim 1 wherein the enhanced hepatoprotective activity in CC14 induced hepatotoxic mammals is upto 92%.
12. A composition according to claim 1 wherein the dosage for the treatment of acetaminophen induced hepatotoxicity in mammals is 50mg/kg-body weight.
13. A composition according to claim 1 wherein the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals is upto 86%.
14. A composition according to claim 1 wherein the dosage for the treatment of Galactosamine induced hepatotoxicity in mammals is 50 mg/kg of body weight.
15. A composition according to claim 1 wherein the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals is upto 75%.
16. A composition according to claim 1 wherem the dosage for chloretic activity in mammals to control bile flow and bile solids is 50 mg/kg of body weight.
17. A composition according to claim 1 wherein the enhanced chloretic activity is upto 39%.
18. A use of synergistic pharmaceutical composition in treating subjects with liver disorders with an effective amount of synergistic pharmaceutical composition to induce enhanced hepatoprotective activity, said composition comprising:
(a) trans-tetracos-15-enoic acid (TCA) obtained from the plant Indigofera tinctoria;
(b) Apocynin (APO) obtained from the plants Apocyanum cannabium and A. androsaemifolium; and
(c) the ratio of APO and TCA is in the range of 3:1 to 1:3.
19. A use according to claim 19 wherein said composition is used to treat liver disorders caused by Galactosamine, Paracetamol and Carbon tetrachloride.
20. A use according to claim 19 wherein the dosage for the treatment of CC14 induced hepatotoxicity in mammals is about 50-mg/kg-body weight.
21. A use according to claim 19 wherein the enhanced hepatoprotective activity in CC14 induced hepatotoxic mammals is upto 92%.
22. A use according to claim 19 wherein the dosage for the treatment of acetaminophen induced hepatotoxicity in mammals is 50mg/kg-body weight.
23. A use according to claim 19 wherein the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals is upto 86%.
24. A use according to claim 19 wherein the dosage for the treatment of Galactosamine induced hepatotoxicity in mammals is 50mg/kg of body weight.
25. A use according to claim 19 wherein the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals is upto 75%.
26. A use according to claim 19 wherein the dosage for chloretic activity in mammals to control the bile flow and bile solids is 50 mg/kg of body weight.
27. A use according to claim 19 wherein the enhanced cholretic activity in mammals is upto 39%.
28. A use according to claim 19 wherein the composition is used either singly or in combination with pharmaceutically acceptable carriers.
29. A use according to claim 19 wherein the composition is administered to a subject in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binder or stabilizers.
30. A use according to claim 19 wherein the desired dosage is administered for both preventive and curative properties.
31. A use according to claim 19 wherein said composition is administered systemically, orally or by any clinically/medically accepted uses.
32. A use according to claim 19 wherein the subject is selected from animals and mammals.
PCT/IB2002/001212 2002-03-25 2002-03-25 Synergistic composition of trans-tetracos-15-enoic acid and apocynin and use thereof Ceased WO2003080081A2 (en)

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