Inhibitory Effect of Andrographolide From Andrographis

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Introduction

Andrographolide is an active bitter-tasting diterpenelactone of Andrographis paniculata, (Burn) f. Nees oneof the worlds most important medicinal plants, used inChinese and Ayurvedic medicine for gastric disorders,colds, influenza and other infectious diseases (Benskyand Gamble, 1993; Cava et al., 1965; Chakravati andChakravati, 1952; Chang and But, 1987; Dymock etal., 1890; Tang and Eisenbrand, 1992; White, 1901).

An extract of A. paniculata standardized for its con-

tent of andrographolide and deoxyandrographolide,called Kan Jang, was developed by the Swedish Her-bal Institute and used in Scandinavia for the commoncold (Caceras et al., 1997; Hancke et al., 1995; Melchi-or et al., 1997).

Clinical studies conducted elsewhere indicate thatAndrographis paniculata is of help in epidemics. Dur-ing the influenza epidemic of 1919 in India, a tinctureof Andrographis was said to have arrested theepidemics spread. Since Andrographis paniculata was

able to inhibit the growth of Staphilococcus aurePseudomonas aeroginosa, Proteus vulgaris, Shigedysenteriae and Escherichia coli, it was recommendfor the treatment of different diseases ranging frbacterial dysentery, gastrointestinal disorders, tonlitis, pneumonia, pyelonephritis to abscesses (Chaand But, 1987).

Several randomized placebo-controlled double-blclinical trials performed with Kan Jang showed tha

has preventive effects against common colds, and the capacity to significantly shorten the course/dution of the disease (Caceras et al., 1997; Hancke et 1995; Melchior et al., 1997).

Andrographis paniculata has been demonstratedbe effective for the treatment of pharyngotonsillitisadults (Thamlikitkul et al., 1991). These clinical fiings which require further validation using rigorotesting were associated with the stimulation of bantigen specific and nonspecific immune response (P

Inhibitory effect of andrographolide fromAndrographis

paniculata on PAF-induced platelet aggregationE. Amroyan1, E. Gabrielian1, A. Panossian2, G. Wikman3, and H. Wagner4

1Yerevan State Medical University, Yerevan, Armenia2Guelbenkian Research Laboratory, Drug and Medical Technology Agency, Yerevan, Armenia3Swedish Herbal Institute, Gothenburg, Sweden4Institute of Pharmaceutical Biology, University of Munich, Munich, Germany

Summary

Andrographolide, an active principle of the Chinese drugAndrographis paniculata, used for preventiand treatment of common cold in Scandinavia and known as an antiinflammatory, antiviral, antithrobotic, hypotensive and antiatherosclerotic drug, was investigated for its suggested influence on the bsynthesis of eicosanoids and the platelet-activating factor (PAF). Whereas in isolated human polymorpnuclear leukocytes (PMNL) no influence on the biosynthesis was found, it could be shown that andrgrapholide inhibits PAF-induced human blood platelet aggregation in a dose dependent manner (IC55 M). These results indicate that andrographolide has a mechanism of action different from thatnon-steroidal antiinflammatory drugs (NSAID) and most likely associated with the cardiovascular aantithrombotic activity described ofAndrographis paniculata.

Key words: Andrographis paniculata, andrographolide, inhibition of PAF induced human blood plateaggregation, eicosanoids.

0944 7113/99/06/01 027 $ 12 0

Phytomedicine, Vol. 6(1), pp. 2731 Gustav Fischer Verlag 1999http://www.urbanfischer.de/journals/phytomed


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ri et al., 1993) and its antiviral activity (Chang et al.,1991; Mehrotra, 1990).

Andrographolide and related compounds have beeninvestigated for their pharmacological activities in sev-eral animal studies, showing antipyretic and antiin-flammatory activity in animals with experimentally in-duced fever, edema and inflammation (Tang and Eisen-brand, 1992; Chang and But, 1987; Madav et al.,1996).

The anti-inflammatory effect of andrographolidesdisappeared in adrenal-ectomized animals. High dos-age of the andrographolides caused thymic atrophy inyoung mice suggesting a possible involvement of the pi-tuitary and adrenal systems (Chang and But, 1987).

It was also shown that andrographolide reduces theintensity of the peritoneal inflammation produced byacetic acid in mice, indicating its ability to inhibit thepermeability of small blood vessels. Andrographolide,at oral dose up to 300 mg/kg, was very well toleratedboth after acute or sub-acute administration. These re-sults suggest that andrographolide has moderate anti-

inflammatory effects in acute, sub-acute and chronicmodels of inflammation, without side effect on gastricmucosa (Madav et al., 1996). The anti-inflammatorymechanism of action is supposed to be different fromthat of conventional anti-inflammatory drugs.

In this report we show how andrographolide affectsthe most important inflammatory mediators, such aseicosanoids and platelet-activating factor (PAF). Inhibi-tion of the biosynthesis of eicosanoids is characteristicfor NSAID, while PAF antagonists are used as potentialagents in inflammation, asthma, cardiac anaphylaxis,thrombosis, gastrointestinal ulceration, endotoxic

shock, allergy etc. (Braquet et al., 1985;. Pinckard etal., 1988; OFlaherty and Wykle, 1983).

In this study, we examined the effect of andrograph-olide on the biosynthesis of eicosanoids in isolated hu-man polymorphnuclear leukocytes (PMNL) and PAFinduced platelet aggregation of healthy donors.

Material and Methods

Plant Material

Andrographis paniculata Nees (Acantaceae) leaf ex-tract (Batch No. Ex 20 112) air-dried powder stan-dardized for andrographolide content of 3.6% wassupplied by Swedish Herbal Institute (Caceras et al.,1997).

Reagents and analytical standards

Andrographolide (AND) supplied by SHI, 4-hydroxy-benzoic acid propyl ester (Fluka) Arachidonic acid(AA), leukotriene B4 (LTB4), 6E-LTB4, 5S,6E-LTB4,

OH-LTB4, COOH-LTB4, 5S,6S-DHETE, 5S,6DHETE, 5-,12-,15-HETEs, and Prostaglandin (PGB2), PAF-C16 were obtained from Cascade Bchem LTD; 12-hydroxyheptatrienoic acid (HHT)from ICN Biomedicals, Inc., Ionophore A-23187from Boehringer Mannheim Biochemica, Ficoll (n1.077 g/ml) from Gibco, PBS medium solutionsfrom Biochrom KG.

Chromatographic system

Beckman GOLD HPLC system consisting of: Doupump Programmable Solvent Module mod. 125; Sinchannel UV detector module mod. 166; PS/1 Compu486 DX-33 with management software supplied Beckman; Epson FX-800 printer: Column: UltrasphODS, 125 4 mm, 5 (Beckman); Injection valRheodyne mod. 7725l with 20 l loop.

Chromatographic conditions

Mobile phase: methanol-water, 60:40, v/v, flow ra

0.7 ml/min, detection at UV = 229 nm.

Test solution

To an analytical sample of Andrographis paniculata etract powder (about 100 mg, precisely weighed) in Fcon centrifuge tube 20 ml methanol and 5 ml of meanolic solution of propyl p-hydroxybenzoate (1 mg/mInternal standard), were added, left for 30 min in ultsonic bath and centrifuged at 300 g for 10 min. Tsupernatant was used for HPLC analysis.

Human granulocytesThey were isolated by means of dextran sedimentatihypotonic lysis of erythrocytes and density gradientdescribed (Boyum, 1987).

Granulocyte preparations contain 8595% ntrophils of a 95% viability as determined by trypblue exclusion.

Incubation procedure, isolation and HPLC-analysisof AA metabolites

To 10 l of tested compound (different concentratioin duplicate) 1 ml of suspension of PMNL (40

cells/ml) was added. After 10 min of pre-incubatiat 37 C 10 l solutions of calcium chloride, calciuionophore A-23187 and arachidonic acid were addat the final concentrations 2 mM, 5 M and 33 respectively. Incubations were stopped in a 5 min the addition of 1,5 ml methanol containing PG(Internal standard, 25 pg/106 cells). The mixture wcentrifuged (600 g, 10 min), the supernatant diled with water to 10 ml, acidified by 2N HCl to p3.0, and applied to Chromabond C18 (100 m

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Machery-Nagel) cartridge, preliminary washed bymethanol and water. Elution was performed stepwisewith 3 ml methanol-water (15:85, v/v) and 5 ml ofacetonitril. The acetonitril fraction was evaporated todryness, redissolved in 100 l of MeOH and analyzedby HPLC.

HPLC was performed using a Lichro CART HPLCcartridge with LiChrospher RP 18, 5 mm reversedphase column (Merk, Darmstadt) and linear gradient

elution with water-methanol solvent system containing0.01% acetic acid (65%100% methanol, 35 min) atthe flow rate 1.0 ml/min. (Beckman Gold System withProgrammable solvent module 125 and programmabledetection module 166 or Hewlett-Packard with diodearray detection at 235 and 270 nm.) Identification ofeicosanoids was performed on the basis of Rfvalues bycomparison with authentic standards (Cascade Bio-chemical Ltd.).

Quantification was done by comparison of peakheights of the compounds and of the internal standard.The results are the mean SE of three experiments.

Probability value for the difference from the controlwhere significant (p


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However, andrographolide and Andrographis pani-culata (Kan Jang) inhibit PAF-induced human bloodplatelet aggregation in a dose dependent manner (IC50~ 5 M) (Table 1, Fig. 1). This effect is comparablewith the effect of Ginkgo extract (Ginkgo biloba) con-taining Ginkgolides well known PAF-antagonists(Braquet et al., 1985). Pharmacokinetical studies ofKan Jang tablets on humans show that concentrationof andrographolide in blood varies in the concentrationrange 0.31.6 g/ml after administration of two tablets

containing 12 mg of andrographolide (data not pub-lished). This corresponds to 0.9 M4.6 M concen-tration of andrographolide in blood which accordingto the graph in Fig. 1 below gives an inhibitory PAF ef-fect in the range of 20%40%.

These results let us assume that the beneficial proper-ty of Andrographis paniculata (Kan Jang) in commoncold and other inflammatory diseases could be due toinhibitory effect of andrographolide on PAF mediatedinflammatory response (Braquet et al., 1985; Pinckard

et al., 1988; OFlaherty and Wykle, 1983). This effmight be also associated with a cardiovascular aantithrombotic activity of Andrographis panicula(Borhanuddin, 1994; Guo, 1994; Guo et al., 199Wang, 1994; Zhang, 1996; Zhang et al., 1994; Zhaand Tan, 1997; Zhao, 1990; Zhao and Fang, 1991)

References

Bensky, D., Gamble, A. (eds): Chinese Herbal Medicine Mteria Medica, revised edition, Eastland Press, p. 95, 199

Borhanuddin, M.: Hypoglycemic effects of Andrograppaniculata Nees on non-diabetic rabbits. Bangladesh MRes. Counc. Bull. 20 (1): 2426, 1994.

Born, G. V. R.: Aggregation of blood platelets by adenosdiphosphate and its reversal. Nature (London). 1926927, 1962.

Boyum, A.: Isolation of lymphocytes, granulocytes and m

rophages. Scand. J. Immunol. 5: 915, 1987.Braquet, P. G., Spinnewyn, B., Braquet, M., Bougain, P.

Taylor, J. E., Etienne, A., Drien, K.: BN 52021 and relacompounds: a new series if Highly specific PAF Acether tagonists isolated from Ginkgo biloba L., Blood Vessel. 558572, 1985.

Caceras, D. D., Hanke, J. L., Burgos, R. A., Wikman, G. Prevention of common colds with Andrographis panicuta dried extract: A pilot double blind trial. Phytomedic4 (2): 101104, 1997.

Cava, M. P., Chan, W. R., Stein, R. P., Willis, C. R.: Andgrapholide. Further transformations and stereochemievidence the structure of isoandrographolide. Tetrahedr

21: 26172624, 1965.Chakravati, D., Chakravati, R. N.: Andrographolide. Part

J. Chem. Soc. 16971700, 1952.Chang, H. M., But, P. P.-H.: Pharmacology and Applicatio

of Chinese Materia Medica, Vol. 2. Transl. by S.-S. YeuS. C. Yao, L. L. Wang, pp. 918928, 1987.

Chang, R. S., Ding, L., Chen, G. Q., Pan, Q. C., Zhao, Z. Smith, K. M.: Dehydroandrographolide succinic amonoester as an inhibitor against the human immunodeciency virus. Proc. Soc. Exp. Biol. Med. 197 (1): 591991.

E. Amroyan et al.30

Table 1. Effects of Andrographolide, A. paniculata (Kan Jangr) and Ginkgo biloba (Tanakanr) on PAF-induced platelet agggation in healthy donors.

Aggregation, %

concentration Andrographolide, A. paniculata, g/ml, Ginkgo, g/mlM

1 76.8 9.3 81.8 23.7 56.9 3.5*10 32.5 3.2* 76.7 25.8 18.9 4.2*

100 5.4 3.2* 32.0 11.5* 5.0 2.8*

0 (control) 100 12.0 100 17.8 100 16.3

Students t-test, marked differences from control are significant at p


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Dymock, W., Warden, C. J. H., Hooper, D.: Pharmacograph-ica Indica. A history of the Principal Drugs of VegetableOrigin met tith in British India Part III, publ. in London, p.4647, 1890.

Guo, Z. L.: The effect of Andrographis paniculata Nees(APN) in alleviating the myocardial ischemic reperfusioninjury.J. Tongji Med. Univ. 14 (1): 4951, 1994.

Guo, Z. L., Zhao, H. Y., Zheng, X. H.: An experimentalstudy of the mechanism of Andrographis paniculata Nees(APN) in alleviating the Ca(2+)-overloading in the processof myocardial ischemic reperfusion. J. Tongji Med. Univ.15 (4): 205208, 1995.

Hancke, J., Burgos, R., Caceres, D., Wikman, G.: A Double-blind study with a new monodrug Kan Jang: Decrease ofsymptoms and improvements in the recovery from com-mon colds, Phytotherapy Res. 9: 559562, 1995.

Madav, S., Tandan, S. K., Lal, J., Tripathi, H. C.: Anti-inflam-matory activity of andrographolide. Fitoterapia 67 (5):452458, 1996.

Mehrotra, R.: In vitro studies on the effect of certain naturalproducts against hepatitis B virus. Indian J. Med. Res. 92:133138, 1990.

Melchior, J., Palm, S., Wikman, G.: Controlled clinical study

on the efficacy of Kan Jang Tablets in common cold a pi-lot trial. Phytomedicine 4: 101104, 1997.

OFlaherty, J. T., Wykle, R. T.: Biology and biochemistry ofplatelet-activating factor. Clin. Rev. Allergy 1: 357367,1983.

Puri, A., Saxena, R., Saxena, R. P., Saxena, K. C., Srivastava,V., Tandon, J. S.: Immunostimulant agents from Androgra-phis paniculata. J. Nat. Prod. 56 (7): 995999, 1993.

Pinckard, R. N., Ludwig, J. C., McManus, L. M.: Platelet-Ac-tivating Factors. In: Inflammation. Basic principles andclinical correlates. Eds. J. I. Gallin, I. M. Goldstein, R. Sny-derman. Raven Press, New York: 139167, 1988.

Tang, W., Eisenbrand, G.: Andrographis paniculata (Burn.f.)

Nees., In: Chinese Drugs of Plant Origin, Chemistry, Phar-macology and Use in Traditional and Modern Medicine,Springer Verlag Berlin, pp. 97103, 1992.

Thamlikitkul, V., Theerapong, S., Boonroj, P., EkpalakoW., Taechaiya, S., Orn-Chom-Jan, T., Pradipasena, S., Tsard, S., Dechatiwongse, T., Chantrakul, C., Punkrut, WBoontarng, N., Petcharoen, S., Riewpaiboon, W., Riewpboon, A., Tenambergenm E.: Efficacy of Andrograppaniculata, Nees for Pharyngotonsillitis in adults.J. MAssoc. Thai, Oct, 437442, 1991.

Wang, D. W.: Prevention of atherosclerotic arterial stenoand restenosis after angioplasty with Andrographis panilata Nees and fish oil. Experimental studies of effects amechanisms. Chin. Med. J. (Engl) 107 (6): 464470, 19

White, W. H.: Text-Book of Pharmacology and Therapeutp. 115, 1901.

Zhao, H. Y.: Protective effects of Andrographis paniculNees on post-infarction myocardium in experimental doJ Tongji Med Univ 10 (4): 212217, 1990.

Zhao, H. Y., Fang, W. Y.: Antithrombotic effects of Andrraphis paniculata nees in preventing myocardial infarctiChin Med J (Engl) 104 (9): 770775, 1991.

Zhang, C. Y.: Hypotensive activity of aqueous extract of Adrographis paniculata in rats. Clin Exp Pharmacol. Phiol. 23 (8): 675678, 1996.

Zhang, C. Y., Tan, B. K.: Mechanisms of cardiovascular

tivity of Andrographis paniculata in the anaesthetized J. Ethnopharmacol. 56 (2): 97101, 1997.

Zhang, Y. Z., Tang, J. Z., Zhang, Y. J.: Study of Androgphis paniculata extracts on antiplatelet aggregation andlease reaction and its mechanism. Chung Kuo Chung HChieh Ho Tsa Chih 14 (1): 2830, 1994.

Address

A. Panossian, Guelbenkian Research and Drug QualControl Laboratories of the Drug Agency, 49/4 Komtas str., 375051, Armenia

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Inhibitory effect of andrographolide from Andrographis paniculata on PAF-induced platelet aggregation

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Inhibitory Effect of Andrographolide From Andrographis