introducing a herbal remedy for hepatoprotection
TRANSCRIPT
INTRODUCING
A HERBAL REMEDY for Hepatoprotection
Andrographis paniculata Nees
Aerial Part Flower
Family : Acanthaceae
English name : Creat, Green chirayta, King of bitters
Part used : Leaves
• Known in Ayurveda since 5000 B.C
• Used widely in India
• Standardised from “Seed to Shelf”
• Broad range of pharmacological effects : promotes healthy
immune system, normal liver functions and others
• Safe for human administration
• Advisable and suitable as a dietary supplement
Background
The main bioactive constituents of Andrographis paniculata are diterepene lactones,
andrographolide, neoandrographolide, 14-Deoxy-11,12-dihydroandrographolide,
andrographiside and andrograpanin.
Phytochemistry
O
CH2
OHCH2OH
O
CH3
CH3
HO
O
OH
HOCH2
OH
OH
O
O
Me
OH2C MeH
CH2
Andrographolide Neoandrographolide
O
OHCH3
CH2
O
CH2OH
14 Deoxy-11,12-dihydroandrographolide
Pharmacokinetics of Andrographis paniculata
Rapid metabolism of drug in the body
After oral administration
Bioavailability : 44.06%Peak absorption at : 30.75 minutesPeak plasma concentration : 16μg/ml
Crosses brain barrier rapidly
Peak drug concentration seen in gall bladder, stomach, liver & small intestine after 30 minutes
Excreted through urine and feces.
Ref: Chang HM, But PPH, (Ed), Pharmacology and applications of Chinese Materia Medica (Vol. 1&2), 1987, World Scientific, Singapore
Protection Against Paracetamol Induced Damage in Rat Hepatocytes
Animals were given a single oral toxic dose of paracetamol 2g/kg
Andrographolide dose: 0.75, 1.5, 3, 6, 12 mg/kg for 7 days
Silymarin dose: 3, 6, 12, 20 mg/kg for 7 days
• Dose dependent increase in viability of isolated hepatocytes
• Dose dependent increase in oxygen uptake by isolated hepatocytes.
• The increase in both parameters was more with Andrographolide than
with silymarin
Both Andrographolide and Silymarin pretreatment as compared to paracetamol treated animals showed
• In isolated hepatocytes the levels of GOT, GPT and ALP were
significantly lower
• In serum GOT, GPT and ALP levels decreased significantly.
• The decrease in both parameters was more with Andrographolide than
with silymarin
Biochemical parameters
Ref: Visen et. al. (1993) J. Ethnopharmacol. 40: 131-136
“Andrographolide was found to be more potent than Silymarin (Milk thistle)
a standard hepatoprotective agent”
0
2
4
6
8
10
12
3mg/kg 6mg/kg 12mg/kg
Andrographolide Silymarin
Effect of Andrographolide and Silymarin on Oxygen uptake
0
20
40
60
80
100
120
3mg/kg 6mg/kg 12mg/kg
Andrographolide Silymarin
Effect of Andrographolide and Silymarin on percent viability of hepatocytes
% V
iab
ilit
y
% V
iab
iity
Hepatoprotective effects against Carbon Tetrachloride induced Liver damage
Liver damage was caused by 1 ml/kg CCl4 subcutaneously twice weekly for eight weeks.
300 mg/kg alcoholic extract of Andrographis intragastric daily for 8 weeks in rats caused
• significant decrease in SGPT & Alkaline phosphatase levels as compound to
CCl4 treated control group
• significant decrease in hypnosis [(sleeping time) (improvement in the functioning
of liver cells)]
Ref. Rana AC, Avadhoot Y (1991) Arch Pharm Res 14(1); 93-95
0
100
200
300
400
500
600
SGPT ALP
Normal Control CCI4 Control AAP Treated
Effect of Alcoholic extract A. paniculata on biochemical parameters in rats
Choleretic effect of Andrographolide in Rats and Guinea pigs
Andrographolide at a dose ranging (1.5 – 12mg/kg) orally produced significant
dose dependent choleretic effect as evidenced by ;
• Increase in bile flow
• Increase in bile salt
• Increase in bile acids
Paracetamol induced decrease in volume and content of bile was prevented by
Andrographolide
“Effect was more potent than silymarin”
Ref: Shukla et al 1992, Planta medica, 58,146 – 149.
Choleretic effect of Andrographolide and silymarin in Rats.
0
10
20
30
40
50
60
70
80
Bile flow Bile salts Cholic acids deoxycholic acid
Per
cen
t in
crea
se
Andrographolide Silymarin
0
10
20
30
40
50
60
70
80
Bile flow Bile salts Cholic acids deoxycholic acid
Per
cen
t In
crea
se
Andrographolide Silymarin
Choleretic effect of Andrographolide and silymarin in Guinea pigs.
Hepatoprotection against Carbon tetrachloride induced toxicity
Liver damage was caused by CCl4, 2ml/kg bw sc in equal vol of olive oil on 2nd and 3rd days.
Rats were treated for 4 days with
• Andrographolide :100 mg/kg i.p.
• Methanolic extract extract: 861.33 mg/kg ip
• Andrographolide free met. ext. : 761.33 mg/kg ip
Ref. Handa SS, Sharma A (1990) Ind. J. Med. Res. [B] 92: 276-283
Andrographolide treatment
• exerted max. inhibition against CCl4 induced increase in five biochemical
parameters - SGOT, SGPT ALP, Bilirubin and Hepatic triglycerides
• Significantly ameliorates toxin induced histopathological changes in liver.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
110.00
120.00
SGOT SGPT ALP Bilirubin Hepatic Triglycerides
Andrograholide MeOH Extract Andrograholide Free MeOH Ext.
Control
Effect of different fractions of Andrographis on biochemical parameters in CCL4 intoxicated rats
Hepatoprotection against galactosamine induced toxicity
Acute hepatitis induced in rats by single dose of :
• Galactosamine 800 mg/kg i.p.
Treatment of rats with 400 mg/kg i.p. or 800 mg/kg (orally) 48, 24 & 2 h before galactosamine administration leads to
• Complete normalization of toxin induced increase of all five biochemical
parameters - SGOT, SGPT ALP, Bilirubin and Hepatic triglycerides
• Significantly ameliorates toxin induced histopathological changes in liver.
Ref. Handa SS, sharma A (1990) Ind. J. Med. Res. [B] 92: 284-292
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
110.00
120.00
SGOT SGPT SALP SBRN HTG
Andrographolide 50mg/kg Andrographolide 200mg/kg Andrographolide 400mg/kg
Control
Andrographolide was administed 48,24 and 2 hours prior to GaiN
Effect of pretreatment with different doses of Andrographolide on galatosamine intoxicated rats
Hepatoprotection against Paracetamol induced toxicity
Acute hepatitis induced in rats by single dose of Paracetamol 3 g/kg p.o.
Treatment of rats with 200 g/kg i.p. 1, 4 & 7 h after paracetamol administration leads to
Ref. Handa SS, sharma A (1990) Ind. J. Med. Res. [B] 92: 284-292
• Complete normalization of toxin induced increase of all five biochemical
parameters - SGOT, SGPT ALP, Bilirubin and Hepatic triglycerides
• Significantly ameliorates toxin induced histopathological changes in liver.
0.00
20.00
40.00
60.00
80.00
100.00
120.00
SGOT SGPT SALP SBRN HTG
Andrographolide 50mg/kg Andrographolide 200mg/kg Andrographolide 400mg/kg
Control
Effect of pretreatment with different doses of Andrographolide on Paracetamol intoxicated rats
Antihepatotoxic effects of major diterpenoid constituents of A. paniculata
Hepatotoxicity was induced in mice by CCl4 or tert-butylhydroperoxide (tBHP)
Pretreatment of mice with the diterpenes-
• Andrographolide,
• Andrographiside,
• Neoandrographolide
(100 mg/kg, i.p.) for 3 consecutive days in either group of the toxin-treated animals produced
• significant reduction in malondialdehyde formation,
• reduced glutathione (GSH) depletion and
• enzymatic leakage of glutamic-pyruvate transaminase (GPT) and alkaline
phosphatase (AP)
Comparison with silymarin revealed that
Andrographolide exhibited a lower protective potential than andrographiside and
neoandrographolide which were as effective as silymarin with respect to their
effects on
Contd
The greater protective activity of Andrographiside and Neoandrographolide could be due to their glycosidic nature which may act as strong antioxidants.
Ref: Kapil et. al. (1993) Biochem Pharmacol. Jul 6;46(1):182-5.
• the formation of the degradation products of lipid peroxidation
• release of GPT and AP in the serum.
In vivo effects of Andrographis leaf extract and Andrographolide on Hepatic Lipid peroxidation
CCl4 (5ml/kg) induced hepatic microsomal Lipid peroxidation was decreased
when;
Rats were pretreated (4 hr) but only with singe dose and not long term
administration of both Andrographis extract(500mg/kg) or Andrographolide
(5mg/kg)
Ref: Choudhury et. al. (1984) Methods Find Exp Clin Pharmacol. 6(9):481-5
Singe or repeated dose(15 days) of Andrographis extract(500mg/kg) or
Andrographolide (5mg/kg) in rats produced
• No significant change in NADPH induced microsomal Lipid peroxidation
In vitro effects of Andrographis leaf extract and Andrographolide on Hepatic Lipid peroxidation
Ref: Choudhury et. al. (1984) Methods Find Exp Clin Pharmacol. 6(9):481-5
In vitro CCl4 (1 ul) induced hepatic microsomal Lipid peroxidation was completely
normalized by both
• Andrographis leaf extract (0.5 and 5 mcg/mg protein)
• Andrographolide (0.5 and 5 mcg/mg protein)
• significantly increased in presence of Andrographolide (0.5mcg/mg pr.)
• but not in presence of Andrographis leaf extract (0.5 mcg/mg protein)
At higher concentration of CCl4 (2ul)
Hepatic microsomal Lipid peroxidation remained
Effect of Andrographis extract on mouse hepatic drug metabolising enzymes
Dose – 50 and 100mg/kg of 80% hydroalc extract for 14days in mice.
Both dose levels cause significant increase in levels of ;
• Acid soluble sulphydryl (-SH) content
• Cytochrome P450
• Cytochrome P450 reductase
• Cytochrome b5 reductase
• Glutathione S-Transferase
• DT-diaphorase
• Superoxidase dismutase
Ref: Singh et al, (2001) Phytoether. Res. 15,382-390
In vivo effect on Hepatic Microsomal Drug metabolizing Enzymes in rats
Single oral doses of
Kalmegh extract = 0.5 and 1.0 g/Kg Andrographilide = 5 and 10 mg/kg
Inhibited
• Aniline hydroxylase
• N-demethylase
• O-demethylase
Repeated doses for 7 to 30 days produced induction of all the above enzymes.
Ref: Choudhury et al, (1987), Planta medica, 135-140
In vitro effect on Hepatic Microsomal Drug metabolizing Enzymes in rats
Kalmegh extract : 50 and 500µg/mg protein Andrographilide : 0.5 and 5 µg/mg protein
did not cause the inhibition of the • Aniline hydroxylase activity
Not produced any appreciable effect on
• N-demethylase activity
• O-demethylase activity
Ref: Choudhury et al, (1987), Planta medica, 135-140
Effect of Andrographis extract on liver tumor
Liver damage ultimately leading to tumor was induced in albino mice by
hexachloro cyclohexane (BHC)(500 ppm/kg of food for 1 to 8 month)
In animals supplemented with AP extract, compared to BHC control animals.
Ref. Trivedi A, Rawal UM (1998) IJP 30; 318-322
This confirms hepatoprotective property of AP and its probable role in delaying hepatic tumorogenic condition.
• The liver marker enzymes viz. SGOT, SGPT & ALP were lowered
• The protein levels were increased
Hepatoprotection of Andrographis extract against BHC induced severe liver damage
Liver damage was caused by BHC (500 ppm/kg food for 1 to 8 months)
Mice supplemented with Andrographis extract 12 mg/kg orally showed
compared to BHC control group
• Significant decline in ALT and AST
• Significant decrease in ALP & gama glutamyl transpeptidase
• Significant decrease in lipid peroxidase activity as compared to BHC
• Significant increase in GSH levels
Ref. Trivedi N, Raval UM (2000) Indian Journal of Pharmacology 32: 288-293
Hepatoprotective and Antioxidant property of AP
Liver damage was caused by BHC (500 ppm/kg food for 1 to 8 months) in mice.
Animals supplemented with Andrographis extract, 12 mg/kg b.w./day orally.
• Increased activity of Glutathione reductase (GR) with parallel increase
in GSH levels• Significant decrease in activity of lipid peroxidation• Less of GST activity• Reduction in GPT activity• Increase in SOD, CAT and GSH-PX
Ref. Trivedi NP, Rawal UM (2001) Ind. J. Exp. Biol. 39; 41-46
Place SS Hospital, BHU, Varanasi, India
Patients 20
Dose 60 ml of Kalmegh decoction / day in 3 divided doses
Duration 24 days.
Results Clinical Improvement
• Yellow color of urine and conjunctiva – Normal in 3 wks in all patients (100% )
• Fever (19 patients)– Subsided on 7th day in all patients. (100%)
• Tender hepatic enlargement(18 patients) – Relieved in 24 days in 16 patients (88.8%)
• Loss of appitite – Improved in 19 patients (90%)
Clinical Study
Effect of Kalmegh in patients with Infective Hepatitis
Results Biochemical Improvement
• Highly significant reduction in -
Bilirubin, Alkaline phosphatase, SGOT, SGPT.
• Highly significant increase in
Serum protein and Albumin Globulin ratio
Total response
!6 patients (80%)were reported in cured group
4 patients (20)were reported in relieved group
Reference Chaturvedi et al 1983, J. Int. Inst. Ayurveda. 2, 208-215
Safety study - 1
Male Fertility Study
Animal Male Albino Wistar rats
Dosage 20 mg Andrographis powder daily
Duration 60 days
Results Anti-spermatogenic effect
Anti-androgenic effect
Reference Akbarsha MA et al., Indian J. Exp. Biol. 1990, 28: 421 – 426
Subchronic Testicular Toxicity Study
Animal Male Sprague Dawley rats
Dosage 20, 200 & 1000 mg / kg b.wt
(5.6% Andrographolide)
Duration 60 days
Results No testicular toxicity, no morphological and functional changes in Leydig cells.
Reference Burgos et al., J. Ethanopharmacol, 1997, 58: 219-224.
Safety study - 2
Safety study - 3
Reproductive Toxicity Study
Animal Albino wistar rats
Dosage 0, 20, 200 & 1000 mg / kg b.wt (10% w/w Andrographolide)
Duration 86 days
Results Administration of 95% ethanolic extract of A. paniculata did not affect the fertility rate in male rats.
Reference Pharmacology Report No. 03.1473, Intox, 2003.
Biological Activity Reference
Antibiotic activity Gupta et al., Int. J. Pharmacog. 1993, 31(3): 198
Antihepatotoxic activity Rana et al, Arch. Pharmcol. Res. 1991, 14(1): 93
Antiinflammatory activity Tajuddin et al., Nagarjun, 1983, 27: 13
Antimalarial activity Misra et al., Int. J. Pharmacog. 1992, 30(4): 263
Antiulcer activityViswanathan et al., Ind. J Pharmaceutical Sci. 1981,
43(5): 159
Blood purification effects Vohora, Hamdard Med.1985, 28(1): 72
Hepatostimulation effects Tripathi et al, Phytother. Res. 1991, 5(2): 176
Immunostimulation effects Puri et al., J. Natural Prod. 1993,56(7): 995
Stomachic effectsChoudhury et al, Methods Finding in Exp. Clin.
pharmacol. 1985, 7(12): 617
Leishmaniasis Sinha et al., Drug Delivery, 2000, 7(4): 209
Other biological activities
Analytical Specification
Tests METHOD1. Description 2. Physico-chemical analysis
a. Moisture (%w/w) b. Acid insoluble Ash (%w/w)
3. Particle Sizea. Bulk Density (g/cc) b. Tapped bulk density (g/cc) c. Material Passing through 30# BS/35 ASTM (%w/w)
4. Heavy metal analysisa. Lead b. Cadmium c. Arsenic
5. Microbiological analysis As per FIP Guidelinesa. Total Viable Aerobic count b. Total Enterobacteriaceae c. Total Fungal Count
6. Test for Specific Pathogen As per FIP Guidelinesa. E. coli (1g) b. Salmonella Sp. (10g) c. S.aureus (1g)
7. Mycotoxin analysis
Aflatoxins (B1 +B2 +G1 + G2)
8. Residual solvent analysis As per ICH Guidelinesa. Methanol
b. Ethyl acetate (%w/w) 9. Pesticide residue analysis As per USP & BP Limits
a. Organochlorine Pesticidesb. Organophosphorus Pesticidesc. Synthetic Pyrethroids
10. Phytochemical AnalysisAndrographolide (%w/w) Total Andrographolides (%w/w)Calculated as sum of Andrographolide,Neoandrographolide, Isoandrographolide,Andrograpanin and 14-Deoxy 11,12 Didehydroandrographolide
Protocol: As per USP, AOAC
Sample detail1 : Andrographis paniculata (Kalmegh extract)
Adsorbant : Silica gel 60 F254
Solvent system : Chloroform : Acetone: Benzene 20 : 20 : 10
Sample preparation : Known amount of Andrographis paniculata extract was dissolved in methanol. The solution is applied on TLC plate.
Solvent front run upto : 8 cms
Application : CAMAG Linomat IV
Detection : Under UV 254 nm Under UV 366 nm
Identification of Extract by TLC
254nm 366nm Visible
Estimation of markers by HPLC
Dosage:
A.paniculata standardized to 30% andrographolides
: 250mg - 500 mg per day per adult human
A.paniculata standardized to 40% andrographolides
: 170mg - 350 mg per day per adult human
Thank You for your time
For Details Please Contact:
NATURAL REMEDIES PVT. LTD.,BANGALORE, INDIAE-MAIL: [email protected]