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

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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

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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

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70

80

Bile flow Bile salts Cholic acids deoxycholic acid

Per

cen

t in

crea

se

Andrographolide Silymarin

0

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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.

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90.00

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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

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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: extracts@naturalremedy.com