Download Ref: Choudhury et al

INTRODUCING
A HERBAL REMEDY for Hepatoprotection
Andrographis paniculata Nees
Family
English name
: Acanthaceae
: Creat, Green chirayta, King of bitters
Part used
: Leaves
Aerial Part
Flower
Background
•
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
Phytochemistry
The main bioactive constituents of Andrographis paniculata are diterepene lactones,
andrographolide,
neoandrographolide,
14-Deoxy-11,12-dihydroandrographolide,
andrographiside and andrograpanin.
O
HO
O
O
O
O
CH3
O
CH2
Me
CH2
CH2
HO
H3C
CH 2OH
HOCH2
H
OH2C
Me
O
OH
OH
HO
H3C
CH2OH
OH
Andrographolide
Neoandrographolide
14 Deoxy-11,12-dihydroandrographolide
Pharmacokinetics of Andrographis paniculata

Rapid metabolism of drug in the body

After oral administration
Bioavailability
Peak absorption at
Peak plasma concentration
:
:
:
44.06%
30.75 minutes
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:
Silymarin dose:
0.75, 1.5, 3, 6, 12 mg/kg for 7 days
3, 6, 12, 20 mg/kg for 7 days
Both Andrographolide and Silymarin pretreatment as compared to
paracetamol treated animals showed
• 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
Biochemical parameters
• 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
“Andrographolide was found to be more potent than Silymarin (Milk thistle)
a standard hepatoprotective agent”
Ref: Visen et. al. (1993) J. Ethnopharmacol. 40: 131-136
Effect of Andrographolide and Silymarin on
percent viability of hepatocytes
120
80
60
40
20
0
3mg/kg
6mg/kg
Andrographolide
12mg/kg
Silymarin
Effect of Andrographolide and Silymarin on
Oxygen uptake
12
10
% Viabiity
% Viability
100
8
6
4
2
0
3mg/kg
6mg/kg
Andrographolide
12mg/kg
Silymarin
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
Effect of Alcoholic extract A. paniculata on
biochemical parameters in rats
600
500
400
300
200
100
0
SGPT
Normal Control
ALP
CCI4 Control
AAP Treated
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.
80
70
Percent increase
60
50
40
30
20
10
0
Bile flow
Bile salts
Andrographolide
Cholic acids
Silymarin
deoxycholic acid
Choleretic effect of Andrographolide and
silymarin in Guinea pigs.
80
70
60
Percent Increase
50
40
30
20
10
0
Bile flow
Bile salts
Andrographolide
Cholic acids
Silymarin
deoxycholic acid
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
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.
Ref. Handa SS, Sharma A (1990) Ind. J. Med. Res. [B] 92: 276-283
Effect of different fractions of Andrographis on biochemical
parameters in CCL4 intoxicated rats
120.00
110.00
Control
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
SGOT
SGPT
Andrograholide
ALP
MeOH Extract
Bilirubin
Andrograholide Free MeOH Ext.
Hepatic Triglycerides
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
Effect of pretreatment with different doses of Andrographolide
on galatosamine intoxicated rats
120.00
110.00
Control
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
SGOT
SGPT
Andrographolide 50mg/kg
SALP
Andrographolide 200mg/kg
SBRN
HTG
Andrographolide 400mg/kg
Andrographolide was administed 48,24 and 2 hours prior to GaiN
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
• 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
Effect of pretreatment with different doses of Andrographolide
on Paracetamol intoxicated rats
120.00
Control
100.00
80.00
60.00
40.00
20.00
0.00
SGOT
SGPT
Andrographolide 50mg/kg
SALP
Andrographolide 200mg/kg
SBRN
HTG
Andrographolide 400mg/kg
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)
Contd
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
•
the formation of the degradation products of lipid peroxidation
•
release of GPT and AP in the serum.
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.
In vivo effects of Andrographis leaf extract and
Andrographolide on Hepatic Lipid peroxidation
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
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
In vitro effects of Andrographis leaf extract and
Andrographolide on Hepatic Lipid peroxidation
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)
At higher concentration of CCl4 (2ul)
Hepatic microsomal Lipid peroxidation remained
•
significantly increased in presence of Andrographolide (0.5mcg/mg pr.)
•
but not in presence of Andrographis leaf extract (0.5 mcg/mg protein)
Ref: Choudhury et. al. (1984) Methods Find Exp Clin Pharmacol. 6(9):481-5
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.
•
The liver marker enzymes viz. SGOT, SGPT & ALP were lowered
•
The protein levels were increased
This confirms hepatoprotective property of AP and its probable role in
delaying hepatic tumorogenic condition.
Ref. Trivedi A, Rawal UM (1998) IJP 30; 318-322
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
Clinical Study
Effect of Kalmegh in patients with Infective Hepatitis
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%)
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
Safety study - 2
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 - 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.
Other biological activities
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 activity
Viswanathan 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 effects
Choudhury et al, Methods Finding in Exp. Clin.
pharmacol. 1985, 7(12): 617
Leishmaniasis
Sinha et al., Drug Delivery, 2000, 7(4): 209
Analytical Specification
Tests
METHOD
1. Description
2. Physico-chemical analysis
a. Moisture (%w/w)
b. Acid insoluble Ash (%w/w)
3. Particle Size
a. Bulk Density (g/cc)
b. Tapped bulk density (g/cc)
c. Material Passing through 30# BS/35 ASTM (%w/w)
4. Heavy metal analysis
a. Lead
b. Cadmium
c. Arsenic
5. Microbiological analysis
As per FIP Guidelines
a. Total Viable Aerobic count
b. Total Enterobacteriaceae
c. Total Fungal Count
6. Test for Specific Pathogen
a. E. coli (1g)
b. Salmonella Sp. (10g)
c. S.aureus (1g)
As per FIP Guidelines
7. Mycotoxin analysis
Aflatoxins (B1 +B2 +G1 + G2)
8. Residual solvent analysis
As per ICH Guidelines
a. Methanol
b. Ethyl acetate (%w/w)
9. Pesticide residue analysis
As per USP & BP Limits
a. Organochlorine Pesticides
b. Organophosphorus Pesticides
c. Synthetic Pyrethroids
10. Phytochemical Analysis
Andrographolide (%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
Identification of Extract by TLC
254nm
366nm
Visible
Sample detail1
: Andrographis paniculata (Kalmegh extract)
Adsorbant
: Silica gel 60 F254
Solvent system
: Chloroform : Acetone: Benzene 20 : 20
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
: 10
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
For Details Please Contact:
NATURAL REMEDIES PVT. LTD.,
BANGALORE, INDIA
E-MAIL: [email protected]
Thank You for your time