Andrographis paniculata

19 Andrographis paniculata

and the Cardiovascular System

Benny Kwong-Huat Tan National University of Singapore

Singapore, Republic of Singapore Amy C. Y. Zhang

Epitomics, Inc. Burlingame, California, U.S.A.

I. INTRODUCTION Andrographis paniculata (Burm. f ) Nees (Acanthaceae) is a well-known bitter

medicinal herb found in the Far East (specifically in India,Southeast Asia,the northern parts of the Malayan peninsula,Java,and southern parts of China). It is also cultivated commercially. It is an erect,stiff herb,growing up to 1 m tall,with quadrangular stems that are thickened above the nodes. The leaves are opposite,lanceolate (3–12 cm by 1–3 cm) while flowers are narrow,white, tube-like,and appear as laxly branched terminal or axillary inflorescences (Fig. 1).

The herb is known by several other names—Justicia paniculata Burm. f.; Hempedu bumi,Sambiloto,Sambiroto (Javanese/Malay),Chuan xin lian (Chinese).

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F IGURE 1 Andrographis paniculata (Burm. f) Nees (Acanthaceae). (Courtesy of Dr. Andrew Wee Kien Han.)

A. paniculata and Cardiovascular System 443

II. CHEMICAL CONSTITUENTS OF A. PANICULATA The chemical constituents of A. paniculata are mainly diterpenoids (which

all contain hydroxyl, a,h-unsaturated-g lactone,and exomethylene groups in their chemical structures) and flavonoids. The diterpenoids include andrographolide (1),14-deoxyandrographolide (DA),14-deoxy-11,12-dide- hydroandrographolide (DDA),14-deoxy-11-oxoandrographolide (2),neo- andrographolide (3),andrographiside (di-deoxyandrographolide),deoxy- andrographoside (andropanoside),andrograpanin,deoxyandrographolide

19- D -glucoside,and 14-deoxy-12-methoxy-andrographolide (4–7).

F IGURE 2 Chemical structures of DA (1), DDA (2), andrographolide (3) andro- graphiside (4), and neoandrographolide (5).

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Matsuda et al. (8) reported the isolation of six new diterpenoids of the ent-labdane type,viz. 14-epi-andrographolide,isoandrographolide,14-de- oxy-12-hydroxyandrographolide,and 14-deoxy-11-hydroxyandrographolide and the diterpene glucosides 14-deoxy-11,12-didehydrographiside,and 6Vacetyl-eoandrographolide. Four new diterpene dimers,bis-andrographo- lides,A,B,C,and D,from the aerial parts (including seeds,stem,and leaves) of A. paniculata were also isolated and their structure determined by chemical and spectral analysis (8).

From the root of the plant,a new flavonone glucoside,andrographidine (A),and five new flavone glucosides,andrographidine B,C,D,E,and F,were isolated along with 5-hydroxy-7,8,2V-,3V-tetramethoxyflavone,and 7,8- dimethoxy-5-hydroxyflavone. They have uncommon O-substitution pattern including 5,7-8-,2V-,3V- and 4VO-substituents (9). Tang and Eisenbrand (10) reported that the main constituent of A. paniculata was andrographolide.

DA (Fig. 2,1), C 20 H 30 O 4 ,is a colorless plate crystal (from methanol) with hydroxyl, a,h-unsaturated-g-lactone,and exo-methylene groups in its chemical structure. It was reported to possess antipyretic and anti-inflamma- tory effects (10,11).

DDA (Fig. 2,2), C20H28O4, is a colorless needle crystal (from meth- anol) with hydroxyl, a,h-unsaturated-g-lactone,and exo-methylene groups present in its chemical structure (2). This is very similar to that of 14- deoxyandrographolide,with the exception of a double bond at C-11 and 12. So far,there has been no literature report on the biological activities of DDA.

III. BIOLOGICAL EFFECTS OF A. PANICULATA

A. paniculata has been reported to have multiple pharmacological activities, including the lowering of blood pressure. Anecdotally,it is taken as a bitter infusion from six to seven leaves for lowering very high blood pressure. A published report by Ahmad and Asmawi (12) indicated that the extract of this plant was used among Malays in Malaysia for the treatment of hypertension. Wang and Zhao (13) reported that the extract could alleviate atherosclerotic artery stenosis induced by both deendothelialization and a high-cholesterol diet. This chapter reviews the authors’ investigations into the cardiovascular effects of a crude water extract of A. paniculata,its semipurified fractions,and some of its diterpenoid compounds.

The effects of an intraperitoneally administered aqueous extract of A. paniculata on systolic blood pressure (SBP),plasma and lung angiotensin- converting enzyme (ACE) activities,and also the free radical content in the

A. paniculata and Cardiovascular System 445

kidneys of male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were also evaluated.

It is well known that ACE plays an important role in blood pressure regulation by (a) catalyzing the conversion of angiotensin I to angiotensin II, which has potent vasoconstrictor effects,and (b) promoting inactivation of the natriuretic vasodilator bradykinin. These actions lead to an elevation in blood pressure. Cushman and Cheung (14) reported that the lungs and kidneys had large amounts of this enzyme.

Suryaprabha et al. (15) reported that in hypertensive states,there is an increased amount of free radicals. Elevated levels of free radicals have in turn been associated with increased amounts of ACE in the kidneys (16). Free radicals,especially superoxide ions,can also inactivate the vasodilators nitric

oxide (NO) and prostacyclin (PGI 2 ) (17). All these factors can potentially aggravate the hypertension. We decided to evaluate whether the extract of

A. paniculata could affect these parameters and also the blood pressure of the experimental rats. Chronic intraperitoneal infusion of three different doses of the extract (0.7,1.4,and 2.8 mg/kg) was administered by osmotic pumps (ALZA Corp., USA) to SHR over 14 days. The extract produced significant dose-dependent reductions in the SBP of SHR compared with vehicle-treated controls. Peak reductions in SBP occurred on day 2 with all three doses (Fig. 3). The lowest of the three doses was thus chosen as the optimum hypotensive dose of the extract.

A follow-up study was done using this optimum dose in SHR and WKY rats. We showed that the extract significantly lowered SBP in both these strains of rats. Plasma but not lung ACE activity in extract-treated SHR was found to be significantly lower than that in vehicle-treated SHR. However,no significant difference was found in plasma and lung ACE activities between extract and vehicle-treated WKY rats.

Interestingly,the kidney TBARS value in vehicle-treated SHR was significantly higher than that in vehicle-treated WKY rats. This appears to be consistent with the expectation that free-radical levels are elevated in hyper- tension. The level of lipid peroxidation products in the kidneys [as reflected by estimation of thiobarbituric-acid-reacting substance (TBARS)] was also found to be significantly lower in extract-treated SHR but not WKY rats when compared to their corresponding vehicle-treated controls. More recent- ly,it was reported that A. paniculata has antioxidant properties by production of reactive oxygen species (18) or increasing the levels of antioxidant enzymes (19). It will thus have the capacity to reduce free-radical activity. However,the extract was found to lower TBARS content in SHR but not WKY rats. This may be because there are higher levels in the SHR.

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F IGURE 3 Changes in mean systolic blood pressure (SBP) of SHR and WKY rats over a 13-day intraperitoneal infusion of an aqueous extract of A. paniculata (0.7 g/kg) or distilled water. Values shown are the mean F SEM. SBP of seven animals in each group (except extract-treated SHR, n = 8). *Significantly lower than the vehicle-treated SHR ( p = 0.0001, two-way ANOVA). **Significantly lower than the vehicle-treated WKY ( p = 0.0001, two-way ANOVA).

It appeared from this initial study that the hypotensive responses to A. paniculata were related neither to the basal level of SBP nor to the strain of rat. The finding that ACE activity was depressed in the plasma but not lungs of extract-treated SHR suggests that the extract may exert its hypotensive effect in SHR by selectively inhibiting the activity of the circulating renin-angioten- sin system. However,no change in either plasma or lung ACE activities oc- curred in the extract-treated WKY rats,which also had a significant fall in SBP. This suggests that in the normotensive rat, A. paniculata extract may have

a different mode of hypotensive action,such as calcium channel blockade, resulting in vascular smooth muscle relaxation,or interaction with the sym- pathetic nervous system and its receptors at either central or peripheral levels.

Captopril,an ACE inhibitor that is widely used in the treatment of human hypertension,has the ability to scavenge free radicals (20). This could result in the sparing of PGI 2 and NO degradation,thus indicating the possibility of a second,and possibly indirect,mode of hypotensive action. Our finding that the hypotensive effect of the A. paniculata extract was associated with reduced ACE activity and reduced kidney lipid peroxidation level indicates some similarity to the effects of captopril.

A. paniculata and Cardiovascular System 447

IV. FURTHER STUDIES WITH FRACTIONS OF A. PANICULATA EXTRACT AND SOME DITERPENOID DERIVATIVES

A. paniculata extract was fractionated with solvents to obtain three fractions with different polarity—FA (ethyl acetate fraction),FB (butanol fraction), and FC (aqueous fraction). These were tested in the anesthetized normoten- sive Sprague-Dawley (SD) rat to evaluate their effects on the mean arterial blood pressure (MAP). MAP is the average systolic pressure that drives blood through the systemic organs and is thus a critical cardiovascular parameter. We found that FA did not reduce MAP in the anesthetized SD rat,while the crude aqueous extract of A. paniculata (WE), FB, and FC produced a significant fall in MAP in a dose-dependent manner without significant

decrease in heart rate,the ED 50 values for WE,FB,and FC being 11.4,5.0, and 8.6 mg/kg,respectively (Fig. 4). These findings suggested that the hypotensive substance(s) in the crude water extract was concentrated in FB.

The lack of significant change in the heart rate suggests also that the hypotensive compound(s) in the FB fraction of A. paniculata extract may not have a direct action on the heart.

F IGURE 4 The effects of a crude water extract of A. paniculata (WE), its semi- purified butanol fraction (FB), and aqueous fraction (FC) on MAP of anaes- thetized SD rats. Points represent the mean percent change in MAP of six ani- mals in each group; bars indicate the SEM.

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Pharmacological antagonist studies were performed using the FB fraction (5 mg/kg) to further evaluate the mechanism(s) of hypotensive action. The findings showed that the a-adrenoceptor,muscarinic cholinergic receptor,and ACE were not involved in the hypotensive action of FB. This was because this action was not affected by propranolol,atropine,and captopril,respectively (Fig. 5). Furthermore,in the presence of hexametho- nium,pyrilamine,and cimetidine,the decreases in MAP induced by FB were significantly attenuated,suggesting that the hypotensive action of FB might involve the autonomic ganglion and histaminergic systems. In addition,the data also indicated that the a-adrenoceptors are involved,since phentolamine almost completely abolished the hypotensive effect.

The following diterpenoids from A. paniculata were tested for their effects on the MAP of anesthetized SD rats: DA,DDA,andrographolide, andrographiside,neoandrographolide. We found that andrographolide, andrographiside,and neoandrographolide were without effect on the MAP

F IGURE 5 The effects of ganglionic, a- and h-adrenergic, muscarinic cholinergic, and histaminergic receptor blocking agents, and captopril on the hypotensive action of butanol fraction (FB) from crude water extract of A. paniculata. Columns represent the mean percent change in MAP of six animals; bars indicate the SEM. *Denotes that hypotensive responses of FB were significantly reduced from those of control.

A. paniculata and Cardiovascular System 449

F IGURE 6 Effects of DA or DDA on MAP and HR of anesthetized SD rats. Each point represents the mean percentage decrease in MAP or HR of eight animals; bars indicate the SEM.

of the anesthetized rat. DDA dose-dependently decreased MAP and heart rate while DA had a weaker effect on these parameters than DDA (Fig. 6). It thus appeared that the hypotensive effect of A. paniculata could be contrib- uted by at least these two diterpenoids.

V. EFFECTS OF DA AND DDA ON ISOLATED RAT THORACIC AORTA

Further studies were done with DA and DDA to investigate their effects on phenylephrine- and high-K + -induced contractions of the rat thoracic aorta.

The results showed that both DA and DDA had a vasorelaxant prop- erty as they inhibited contractions induced by phenylephrine and high K + in a concentration-dependent manner in endothelium-intact aorta. They also antagonized the concentration-response curve of phenylephrine in a non- competitive manner. The effect was attenuated in endothelium-denuded aorta without modifying the maximal response. This suggested that the vasorelax- ant effect of DA and DDA was partly dependent on the endothelium.

The vascular endothelium plays an important role in controlling the vascular tone via secretion of both relaxant and contractile factors (21). The most potent known are the vasodilators NO and PGI 2 and the vasoconstric- tors angiotensin II and endothelin. NO is synthesized from the amino acid L -arginine,a family of nitric oxide synthetase (NOS) isoenzymes,including endothelial NOS (eNOS),neuronal NOS (nNOS),and inducible NOS

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(iNOS). NO stimulates cyclic GMP production by activating soluble guanyl- ate cyclase (21) and thus causes vasodilatation.

Like verapamil,both DA and DDA produced a much greater vaso- relaxant effect in aorta precontracted by KCl than by phenylephrine. In Ca 2+ -free medium,these diterpenoids antagonized Ca 2+ -induced vasocon- traction in a concentration-dependent manner and almost abolished both caffeine- and norepinephrine-induced transient contractions. Their vasore- laxant effects were partly antagonized by the competitive nitric oxide (NO)-

synthase inhibitor N G -nitro- L -arginine methyl ester (L-NAME),and also by methylene blue,a soluble guanylate cyclase inhibitor,but were unaffected by both indomethacin,a cyclo-oxygenase inhibitor and glibenclamide,an ATP- sensitive K + -channel blocker. These results suggest that the vasorelaxant activity of DA and DDA may be mediated via the activation of nitric oxide synthase and guanylate cyclase,as well as the blockade of Ca 2+ influx through both voltage- and receptor-operated Ca 2+ channels. Compared to DA,DDA had a stronger vasorelaxant activity.

F IGURE 7 The effects of ganglionic, a- and h-adrenergic, muscarinic cholinergic and histaminergic receptor blocking agents, and captopril on the hypotensive acatin of DDA. Columns represent the mean percent change in MAP of six animals; bars indicate the SEM. *Denotes that hypotensive responses of DDA were significantly reduced from those of control ( p < 0.05, t-test).

A. paniculata and Cardiovascular System 451

VI. EFFECTS OF DDA IN ANESTHETIZED SD RATS AND ISOLATED RAT RIGHT ATRIA

We found that DDA produced significant decreases in both MAP and heart rate in a dose-dependent manner (Fig. 6). The ED50 value for MAP was 3.4 mmol/kg. Pharmacological antagonist studies were subsequently done with this dose. We found that the hypotensive action of DDA was not mediated through effects on the a-adrenoceptor,muscarinic cholinergic,or histamin- ergic receptors,for it was not affected by phentolamine,atropine,pyrilamine, or cimetidine (Fig. 7). However,in the presence of propranolol,hexametho- nium,and captopril,the hypotensive effect was negated or attenuated, suggesting the involvement of h-adrenoceptors,autonomic ganglia receptor, and ACE.

In spontaneously beating isolated rat right atria,DDA caused a negative chronotropic effect (Fig. 8),indicating that it may have direct h 1 - adrenoceptor blocking action on the heart in addition to its a-adrenergic receptor inhibitory activity. The bradycardic effect of DDA may also contribute to the hypotensive action. This discordant finding,compared to

F IGURE 8 Effect of DDA ( . , n = 10) on the beating rate of isolated right atria from normotensive rats. Vehicle-treated control group (DMSO, o, n = 5). Points represent the mean F SEM of values. All points on the DDA curve were sig- nificantly different from the corresponding points on the control ( p < 0.05, t-test).

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the earlier finding of a lack of effect of FB on the heart of the anesthetized SD rat,may be explained by the fact that DDA is either absent or else present in FB in such small amounts as to have no significant effect on the heart rate.

VII. EFFECTS OF DA AND DDA ON ENDOTHELIAL CELL PRODUCTION OF NITRIC OXIDE

As our earlier studies suggested that the relaxation of the isolated rat aorta caused by DA and DDA may be mediated through the L -arginine-NO synthase pathway and NO activation of guanylate cyclase,it was decided to study the effects of DDA on NO production by human endothelial cell cultures. NO was quantified spectrometrically by the accumulation of nitrite produced by endothelial cells cultured in F12 medium for up to 48 hr. This method has been documented by previous workers (22,23).

We found that DA and DDA significantly stimulated NO production by endothelial cells in a concentration-dependent manner. This suggests another possible mechanism for their hypotensive effect.

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VIII. THIN-LAYER CHROMATOGRAPHIC (TLC) ANALYSIS OF FB FOR THE PRESENCE OF DA AND DDA

As our studies thus far suggested that DA and DDA were compounds that, like FB,had significant effects on the cardiovascular parameters of experi- mental rats,the FB fraction was analyzed by TLC to confirm the presence of these bioactive compounds. No detectable amounts of either DA or DDA were,however,found in FB (Fig. 9). This provided the explanation for a lack of effect of FB on heart rate (unlike DDA) and suggested that the bioactivity in FB could be attributed to compounds other than DA and DDA and that furthermore there were other compound(s) in the crude water extract of

A. paniculata that could produce a hypotensive effect.

IX. CONCLUSION This series of studies have shown that DA and DDA,having pharmacological

actions that lead to a lowering in blood pressure,could serve as potential lead molecules for the development of new antihypertensive compounds. Further purification of FB could yield new compound(s) with hypotensive properties. Last but not least,our studies indicate that the practice of drinking an infusion of leaves of this bitter herb for treating hypertension may indeed have some scientific merit.

ACKNOWLEDGMENTS The authors acknowledge the generosity of Professor Masanori Kuroyana-

gi,School of Pharmaceutical Sciences,University of Shizuoka,52-1 Yada, Shizuoka-shi 422,Japan in supplying andrographolide,DA,and DDA for our studies. We also thank the National University of Singapore for providing the research grant (RP960329) for the study and the research scholarship awarded to C.-Y. Zhang.

REFERENCES 1. Cava MP,Chan WR,Stein RP,Willis CR. Andrographolide,further trans-

formations and stereochemical evidence: the structure of andrographolide. Tetrahedron 1965; 21:2617–2632. 2. Balmain A,Connolly JD. Minor diterpenoid constituents of Andrographis paniculata Nees. J Chem Soc Perk Trans 1973; 1:1247–1251. 3. Chan WR,Taylor DR,Willis CR,Bodden RL. The structure on stereochemistry of neoandrographolide,a diterpene glucoside from Andrographis paniculata. Tetrahedron 1971; 27:5081–5091.

4. Chen WM,Liang XT. Deoxyandrographolide-19- D -glucoside from the leaves of

454 Tan and Zhang 5. Hu CQ,Zhao BN. Studies on the diterpenes of Chun Xin Lian (Andrographis

paniculata ). Chin Trad Herb Drugs 1981; 12:531. 6. Fujita T,Fujitani R,Takeda Y,Takaishi Y,Yamada T,Kido M,Muira I. On the diterpenoid of Andrographis paniculata: Xray crystallographic analysis of an- drographolide and structure determination of new minor diterpenoids. Chem Pharm Bull 1984; 32:2117–2225. 7. Hu CQ,Zhao BN,Chou PN. Isolation and structure of two new diterpenoid glucosides from Andrographis paniculata Nee. Acta Pharmacol Sin 1982; 17: 435–440. 8. Matsuda T,Kuroyanagi M,Sugiyama S,Umehara K,Ueno A,Nishi K. Cell differentiation-inducing diterpenes from Andrographis paniculata Nees. Chem Pharm Bull 1994; 42:1216–1225. 9. Kuroyanagi M,Sato M,Ueno A,Nishi K. Flavonoids from Andrographis paniculata . Chem Pharm Bull 1987; 35:4429–4435. 10. Tang W,Eisenbrand G. Chinese Drugs of Plant Origin: Chemistry,Pharmacol- ogy,Use in Traditional and Modern Medicine. Berlin: Springer-Verlag,1992: 97–103. 11. Deng WL,Nie RJ,Liu JY. Comparison of pharmacological effect of four andrographolides. Chin Pharm Bull 1982; 17:195–198. 12. Ahmad M,Asmawi MZ. Some pharmacological effects of aqueous extract of Andrographis paniculata Nees. In: Gan EK,ed. The International Conference on the Use of Traditional Medicine and other Natural Products in Health Care. School of Pharmaceutical Sciences,University of Science, Malaysia,June 8–11, 1993. [abstr] 13. Wang DW,Zhao HY. Promotion of atherosclerotic stenosis and restenosis after angioplasty with Andrographis paniculata Nees and fish oil: experimental studies of effects and mechanism. Chin Med J (Eng). 1994; 107:464–470. 14. Cushman DW,Cheung HS. Studies in vitro of angiotensin converting enzyme of lung and other tissues. In: Genest J,Koiw E,eds. Hypertension. Berlin: Springer, 1972:532–541. 15. Suryaprabha P,Das UN,Koratkar R,Sangeetha SP,Ramesh G. Free radical generation,lipid peroxidation and essential fatty acids in uncontrolled essential hypertension. Prostag Leuko Essent Fatty Acids 1990; 41:27–33. 16. Ikemoto F,Song GB,Tominaga M,Yamamoto K. Oxidation-induced increase in activity of angiotensin converting enzyme in the rat kidney. Biochem Biophys Res Commun 1988; 153:1017–1031. 17. Gryglewski RJ,Palmer RMG,Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 1986; 320:454–456. 18. Shen YC,Chen CF,Chiou WF. Suppression of rat neutrophil reactive oxygen species production and adhesion by the diterpenoid alctone andrographolide. Planta Med 2000; 6694:314–317. 19. Zhang XF,Tan BKH. Antihyperglycaemic and anti-oxidant properties of Andrographis paniculata in normal and diabetic rats. Clin Exp Pharmacol Physiol 2000; 27(5–6):358–363.

A. paniculata and Cardiovascular System 455 20. Bagchi D,Prasad R,Das DK. Direct scavenging effects of free radicals by

captopril,an angiotensin converting enzyme inhibitor. Biochem Biophys Res Commun 1989; 158:52–57. 21. McQuillan LP,Leung GK,Marsden PA,Kostyk SK,Keurembanas S. Hypoxia inhibits expression of eNOS via transcriptional and post-transcriptional mech- anisms. Am J Physiol 1994; 267:H1921–H1297. 22. Feder LS,Laskin DL. Regulation of hepatic endothelial cell and macrophage proliferation and nitric oxide production by GM-CSF,M-CSF and IL-1h following acute endotoxaemia. J Leucocyte Biol 1994; 55:507–513. 23. Li JM,Fenton RA,Cutler BS,Dobson JG,Jr. Adenosine enhances nitric oxide production by vascular endothelial cells. Am J Physiol 1995; 269(Cell Physiol 38):C519–C523.