**Andrographolide a New Potential Drug for the Long Term Treatment of Rheumatoid Arthritis Disease**

María A. Hidalgo, Juan L. Hancke, Juan C. Bertoglio and Rafael A. Burgos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55642

## **1. Introduction**

*Andrographis paniculata,* (Burm. f.) Wall. ex Nees, a herbaceous plant belonging to the Family Acanthaceae, is one of the most commonly used medicinal plants in the traditional systems of Unani and Ayurvedic medicines. It grows in hedge rows throughout the plains of India and is also cultivated in gardens. It also grows in many other Asian countries and is used as a tradition‐ al herbal medicine in China, Hong Kong, the Philippines, Malaysia, Indonesia, and Thailand. It is an annual plant of 1-3 ft high, also known as the "king of bitters", being the aerial parts most commonly used. *A. paniculata* have shown a broad range of pharmacological effects such as in‐ hibition of replication of the HIV virus, prevention of common cold, and antimalarial, antidiar‐ rheal, antibacterial, antihyperglycemic effects, suppression of various cancer cells, and principally anti-inflammatory properties. Andrographolide is the major labdane diterpenoid isolated from *A. paniculata* and exhibits anti-inflammatory and anticancer activities, either *in vi‐ tro* or *in vivo* experimental models of inflammation and cancer. Several immunomodulatory re‐ sponses of andrographolide have been observed in *in vitro* studies, such as reduction of iNOS, COX-2, NO, PGE2, TNF-alpha and IL-12 in macrophages and microglia. In neutrophils is able to reduce the radical oxygen species production, and Mac-1, IL-8 and COX-2 expression. In T cells, andrographolide inhibits the expression of IL-2, IFNγ and IL-6, reducing the humoral and cellu‐ lar adaptive immune response. Andrographolide was able to reduce the dendritic cells matura‐ tion and their ability to present antigens to T cells. Andrographolide administered in rodents reduced the Th2 cytokine IL-4, IL-5, IL-13 and serum immunoglobulin in an ovalbumin in‐ duced asthma model. A reduction of T cells response also has been observed in experimental au‐ toimmune encephalomyelitis and systemic lupus erythematosus mouse model. Several of immunomodulatory responses have been associated to the inhibition of Nuclear Factor-κB

© 2013 Hidalgo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

functions. It has been demonstrated that andrographolide inhibits the nuclear translocation of the p65 subunit of NF-κB and interferes with the NF-κB binding to the DNA. Also andrographo‐ lide can reduce NFAT function in T cells and reduce the phosphorylation of signal transducer and activator of transcription-3 (STAT3) in macrophages.

pholide is sparingly soluble in water; soluble in acetone, methanol, chloroform and ether. As a water soluble andrographolide derivative, the sodium bisulfite adduct has been synthesized

Andrographolide a New Potential Drug for the Long Term Treatment of Rheumatoid Arthritis Disease

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249

Preclinical properties include anti-retroviral [6, 7], antiproliferative and pro-apoptotic [8, 9], an‐ ti-diabetic [10, 11], anti-angiogenic [12], anti-thrombotic [13], anti-urothelial [14], anti-leishma‐ niasis [15], hepatoprotective [16, 17], protective activity against alcohol-induced hepatic and

O

HO <sup>O</sup>

renal toxicity [18], and cardioprotective [19] and anti-inflammatory [20-25] properties.

H CH2OH

The second diterpene isolated from *A. paniculata* was the minor non-bitter constituent neoan‐ drographolide, which was first described by Kleipool in 1952. The structure of neoandrogra‐ pholide (Figure 2) was described as a diterpene glucoside and its amount in the plant is around 0.5-1%. The main preclinical effects are anti-inflammatory [23, 26, 27], chemosensitizer [28],

Afterwards, more than 20 other diterpene lactones, both glycosylated and not, have been described. The most important among them, characterized by Balmain and Connolly in 1973, are: 14-deoxy-11,12- didehydroandrographolide, withan average content in the leaf of 0.1%, 14-deoxyandrographolide (0.02%), 14-deoxy-11-oxoandrographolide (0.12%) (Figure 2) [3]. In other hand has been described that 14-deoxy-11,12- didehydroandrographolide possess vasorelaxant and antihypertensive [30, 31], anti-herpes [7], antioxidant and hepatoprotective [32], antithrombotic [33], antiretroviral [6], and antidiabetic properties [34]. Meanwhile 14 deoxyandrographolide exert hepatoprotective [35], uterine smooth muscle relaxant [36],

H

HO

**Figure 1.** Chemical structure of andrographolide

anti-herpes-simplex virus [7] and antioxidant [29].

**2.1. Neoandrographolide**

**2.2. Minor labdane diterpenes**

for medical use as an antipyretic agent.

We propose the potential use of andrographolide in Rheumatoid Arthritis and other autoim‐ mune diseases. This is supported by the fact that andrographolide exerts anti-growth and proapoptotic effects on human rheumatoid arthritis fibroblast- like synoviocytes, the main cellular constituent of pannus, that combined with a massive infiltration of lymphocytes and macro‐ phages, invades and destroys the local articular structure. Recently, a prospective randomized placebo-controlled trial has suggested that *A. paniculata*, a standardized extract containing NLT 30% of andrographolide was effective for symptom relief in patients with Rheumatoid Arthritis. The use of andrographolide alone or a patented *A. paniculata* standardized extract in clinical trials shows mild and few side effects, and has the potential to be developed into a new alternative drug for Rheumatoid Arthritis treatment in the long term.

## **2.** *Andrographis paniculata* **and labdane diterpenoids**

The main and most interesting biological constituent of *A. paniculata* herb (aerial part) is a group of diterpene lactones belonging to the ent-labdane class, present in both free and glycosidic forms, and named andrographolides [1, 2].

Andrographolide is the bitter principle, a colourless, neutral crystalline substance, was first isolated by Boorsma from different parts of *Andrographis paniculata* [2]. In 1911 Gorter proved that it is structurally a lactone and named it andrographolide (in the Chinese literature it is sometimes cited as andrographis B). The bitter principle has been subjected to a number of chemical investigations. The properties of the compound and its diterpenoid lactone nature, as well as its stereochemistry, conformation and crystal structure were cleared by means of infrared, x, mass spectrometry and NMR analysis. Its chemical formula corresponds to the 3,14,15,18-tetrahydroxy-5,9 H,10-labda-8(20),12-dien-16-oic acid-lactone (Figure 1). Most recently, various epimers, geometric isomers, and rearrangement products of andrographolide have been isolated and structurally characterized [3, 4]. Andrographolide, as the other diterpene lactones of *A. paniculata*, are generally extracted with CHCl3/EtOH or acetone, and several methods are described in the literature to determinate its content in the plant, in commercial formulation, i.e. standardized extract and in biological samples: titration with alkalis, TLC/UV spectrophotometry and HPLC methods. The maximum content of androgra‐ pholide and related diterpenoids is in the mature leaves. It has been described that the stem contained 0.2±0.02%, seeds 0.13±0.01%; root 0.44±0.01%; and leaves 2.39±0.008% of androgra‐ pholide [5]. Regional variation in the andrographolide content was also observed. The content of andrographolide varies with the harvest season. The leaves contain more than 2% androg‐ rapholide before the plant blossoms; afterward the contents decreases to less than 0.5% [2]. The pH modifies the stability of andrographolide, and hydrolysis is extremely slow below pH 7, but considerably faster on the alkaline side, producing some structural changes. Androgra‐ pholide is sparingly soluble in water; soluble in acetone, methanol, chloroform and ether. As a water soluble andrographolide derivative, the sodium bisulfite adduct has been synthesized for medical use as an antipyretic agent.

Preclinical properties include anti-retroviral [6, 7], antiproliferative and pro-apoptotic [8, 9], an‐ ti-diabetic [10, 11], anti-angiogenic [12], anti-thrombotic [13], anti-urothelial [14], anti-leishma‐ niasis [15], hepatoprotective [16, 17], protective activity against alcohol-induced hepatic and renal toxicity [18], and cardioprotective [19] and anti-inflammatory [20-25] properties.

**Figure 1.** Chemical structure of andrographolide

#### **2.1. Neoandrographolide**

functions. It has been demonstrated that andrographolide inhibits the nuclear translocation of the p65 subunit of NF-κB and interferes with the NF-κB binding to the DNA. Also andrographo‐ lide can reduce NFAT function in T cells and reduce the phosphorylation of signal transducer

We propose the potential use of andrographolide in Rheumatoid Arthritis and other autoim‐ mune diseases. This is supported by the fact that andrographolide exerts anti-growth and proapoptotic effects on human rheumatoid arthritis fibroblast- like synoviocytes, the main cellular constituent of pannus, that combined with a massive infiltration of lymphocytes and macro‐ phages, invades and destroys the local articular structure. Recently, a prospective randomized placebo-controlled trial has suggested that *A. paniculata*, a standardized extract containing NLT 30% of andrographolide was effective for symptom relief in patients with Rheumatoid Arthritis. The use of andrographolide alone or a patented *A. paniculata* standardized extract in clinical trials shows mild and few side effects, and has the potential to be developed into a new

The main and most interesting biological constituent of *A. paniculata* herb (aerial part) is a group of diterpene lactones belonging to the ent-labdane class, present in both free and

Andrographolide is the bitter principle, a colourless, neutral crystalline substance, was first isolated by Boorsma from different parts of *Andrographis paniculata* [2]. In 1911 Gorter proved that it is structurally a lactone and named it andrographolide (in the Chinese literature it is sometimes cited as andrographis B). The bitter principle has been subjected to a number of chemical investigations. The properties of the compound and its diterpenoid lactone nature, as well as its stereochemistry, conformation and crystal structure were cleared by means of infrared, x, mass spectrometry and NMR analysis. Its chemical formula corresponds to the 3,14,15,18-tetrahydroxy-5,9 H,10-labda-8(20),12-dien-16-oic acid-lactone (Figure 1). Most recently, various epimers, geometric isomers, and rearrangement products of andrographolide have been isolated and structurally characterized [3, 4]. Andrographolide, as the other diterpene lactones of *A. paniculata*, are generally extracted with CHCl3/EtOH or acetone, and several methods are described in the literature to determinate its content in the plant, in commercial formulation, i.e. standardized extract and in biological samples: titration with alkalis, TLC/UV spectrophotometry and HPLC methods. The maximum content of androgra‐ pholide and related diterpenoids is in the mature leaves. It has been described that the stem contained 0.2±0.02%, seeds 0.13±0.01%; root 0.44±0.01%; and leaves 2.39±0.008% of androgra‐ pholide [5]. Regional variation in the andrographolide content was also observed. The content of andrographolide varies with the harvest season. The leaves contain more than 2% androg‐ rapholide before the plant blossoms; afterward the contents decreases to less than 0.5% [2]. The pH modifies the stability of andrographolide, and hydrolysis is extremely slow below pH 7, but considerably faster on the alkaline side, producing some structural changes. Androgra‐

and activator of transcription-3 (STAT3) in macrophages.

248 Innovative Rheumatology

alternative drug for Rheumatoid Arthritis treatment in the long term.

**2.** *Andrographis paniculata* **and labdane diterpenoids**

glycosidic forms, and named andrographolides [1, 2].

The second diterpene isolated from *A. paniculata* was the minor non-bitter constituent neoan‐ drographolide, which was first described by Kleipool in 1952. The structure of neoandrogra‐ pholide (Figure 2) was described as a diterpene glucoside and its amount in the plant is around 0.5-1%. The main preclinical effects are anti-inflammatory [23, 26, 27], chemosensitizer [28], anti-herpes-simplex virus [7] and antioxidant [29].

#### **2.2. Minor labdane diterpenes**

Afterwards, more than 20 other diterpene lactones, both glycosylated and not, have been described. The most important among them, characterized by Balmain and Connolly in 1973, are: 14-deoxy-11,12- didehydroandrographolide, withan average content in the leaf of 0.1%, 14-deoxyandrographolide (0.02%), 14-deoxy-11-oxoandrographolide (0.12%) (Figure 2) [3]. In other hand has been described that 14-deoxy-11,12- didehydroandrographolide possess vasorelaxant and antihypertensive [30, 31], anti-herpes [7], antioxidant and hepatoprotective [32], antithrombotic [33], antiretroviral [6], and antidiabetic properties [34]. Meanwhile 14 deoxyandrographolide exert hepatoprotective [35], uterine smooth muscle relaxant [36], immunomodulator [37], platelet activating factor antagonist [38], and vasorelaxant and antihypertensive [39] effects. In addition, 14-deoxy-11-oxoandrographolide only has been reported antileishmaniasis effect [40].

*ta* (20 mg/100 g b.w.) one hour before the injection of carrageenin, reduced the edema in 65.3%

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Andrographolide, shows anti-inflammatory and anticancer activities in both *in vitro* and *in vivo*. The effects of andrographolide on two cells types that play an important role in the inflammatory processes, e.g. leukocyte (neutrophils, macrophages and T-cells) and endothelial cells, demonstrates the ability of this compound to reduce the expression and production of

Several *in vitro* studies show that andrographolide reduces the production of the oxygen radical superoxide anion and hydrogen peroxide, as well as the adhesion induced by che‐ moattractant in isolated neutrophils [50, 51]. Other antecedents describe a reduction of the expression of cyclooxygenase-2 (COX-2), inducible enzyme producing prostaglandins, in a human model of neutrophils [21]. In mouse peritoneal macrophages, andrographolide re‐ duces the production stimulated by lipopolysaccharide (LPS) of two important cytokines that participate in the amplification and activation of the inflammatory process, the cyto‐ kines tumoral necrosis factor TNFα and granulocyte macrophage colony-stimulating fac‐ tor (GM-CSF). The inhibition of the release of these cytokines by andrographolide was compared to the synthetic glucocorticoid dexamethasone, showing andrographolide to have a similar effect as dexamethasone, but with a lower potency [24, 52]. Also, the ef‐ fect of andrographolide on the cellular chemotaxis, a response that allows the movement of inflammatory cells to the injured tissue, show that it reduces the chemotactic migra‐ tion of macrophage induced by C5a, which may contribute to its anti-inflammatory activ‐ ity [53]. In local or systemic inflammatory disorders there is an enhanced formation of nitric oxide (NO) following the expression of inducible nitric oxide synthase (iNOS). The inhibition of NO formation may have therapeutic benefit in patients with inflammatory diseases as Rheumatoid Arthritis [54]. Thus, andrographolide reduces the LPS-induced iNOS and COX-2 expression in RAW264.7 macrophages [55, 56]. Additionally, androgra‐ pholide may have an effect on inflammation-mediated neurodegeneration, since it re‐ duces the production of reactive oxygen species (ROS), TNFα, NO and prostaglandin E2 in microglia, the counterpart of macrophages in the brain [25]. Andrographolide reduces the *in vitro* activation of human and murine T-cells, T-cells proliferation, interleukin-2

Interaction of leukocyte-endothelium plays a key role in the initiation and maintenance of inflammation, being the adhesion molecule ICAM-1 important in mediating leukocyte adhesion, arrest and transmigration to the inflammatory site. In this respect, certain antece‐ dents show that andrographolide reduces the adhesion of HL-60 cells onto human vein endothelial cells (HUVEC) and the expression of TNFα-induced ICAM-1[61, 62]. In addition, andrographolide reduces the endothelial cell proliferation, migration and invasion, suggesting a role in angiogenesis [63]. Moreover, andrographolide reduces the growth factor deprivation-

in rats. The effect was comparable to oxyphenilbutazone 76.5% [2]

**3.1.** *In vitro* **studies**

pro-inflammatory mediators.

(IL-2) and IFNγ production [57-60].

induced apoptosis in endothelial cells [64].

Andrographiside, the 19-glucoside of andrographolide, was isolated in 1981, and only a hepatoprotective effect has been described [41].

*A. paniculata* contains also minor andrographolide-like compounds such as andropanoside (19 glucoside of 14-deoxy-andrographolide), or andrograpanin (3,14-dideoxy-andrographolide), which are mostly all 14-deoxy- and/or 3-deoxy-derivatives. These compounds show antiinflammatory properties in preclinical studies [42, 43].

Isoandrographolide is present in the whole plants and has been described as a cellular differentiation inducer [3], antiproliferative [44], and cytotoxic [45] effects.

Also three salts of labdanic acids, named as magnesium andrographate, disodium androgra‐ phate and dipotassium andrographate 19-*O*-D-glucoside have been isolated hydrophylic extract from the leaf.

Since the total synthesis of andrographolide and analogues, many libraries of new derivatives have been created using andrographolide as a template with the purpose to obtain compounds with improved pharmacological profiles. Andrographolide is also a starting point for the semisynthesis of other labdane diterpenes [46-48].

**Figure 2.** Chemical structure of minor labdane diterpenes isolated from *Andrographis paniculata*.

## **3. Anti-inflammatory and immunomodulatory effects of andrographolide** *in vitro* **and** *in vivo*

Different preparations of *A. paniculata* administered orally reduced the pyrexia within or after 5 hrs of administration of yeast in rats [49]. On the other hand, administration of *A. panicula‐* *ta* (20 mg/100 g b.w.) one hour before the injection of carrageenin, reduced the edema in 65.3% in rats. The effect was comparable to oxyphenilbutazone 76.5% [2]

#### **3.1.** *In vitro* **studies**

immunomodulator [37], platelet activating factor antagonist [38], and vasorelaxant and antihypertensive [39] effects. In addition, 14-deoxy-11-oxoandrographolide only has been

Andrographiside, the 19-glucoside of andrographolide, was isolated in 1981, and only a

*A. paniculata* contains also minor andrographolide-like compounds such as andropanoside (19 glucoside of 14-deoxy-andrographolide), or andrograpanin (3,14-dideoxy-andrographolide), which are mostly all 14-deoxy- and/or 3-deoxy-derivatives. These compounds show anti-

Isoandrographolide is present in the whole plants and has been described as a cellular

Also three salts of labdanic acids, named as magnesium andrographate, disodium androgra‐ phate and dipotassium andrographate 19-*O*-D-glucoside have been isolated hydrophylic

Since the total synthesis of andrographolide and analogues, many libraries of new derivatives have been created using andrographolide as a template with the purpose to obtain compounds with improved pharmacological profiles. Andrographolide is also a starting point for the

O

O

O

H CH2OH H

HO

O

differentiation inducer [3], antiproliferative [44], and cytotoxic [45] effects.

**Neoandrographolide 14-deoxy-11,12- didehydroandrographolide 14-deoxyandrographolide**

**Figure 2.** Chemical structure of minor labdane diterpenes isolated from *Andrographis paniculata*.

HO

H CH2OH H

**3. Anti-inflammatory and immunomodulatory effects of andrographolide**

Different preparations of *A. paniculata* administered orally reduced the pyrexia within or after 5 hrs of administration of yeast in rats [49]. On the other hand, administration of *A. panicula‐*

reported antileishmaniasis effect [40].

extract from the leaf.

250 Innovative Rheumatology

H CH2 O Glu

H

*in vitro* **and** *in vivo*

hepatoprotective effect has been described [41].

inflammatory properties in preclinical studies [42, 43].

semisynthesis of other labdane diterpenes [46-48].

O

O

Andrographolide, shows anti-inflammatory and anticancer activities in both *in vitro* and *in vivo*. The effects of andrographolide on two cells types that play an important role in the inflammatory processes, e.g. leukocyte (neutrophils, macrophages and T-cells) and endothelial cells, demonstrates the ability of this compound to reduce the expression and production of pro-inflammatory mediators.

Several *in vitro* studies show that andrographolide reduces the production of the oxygen radical superoxide anion and hydrogen peroxide, as well as the adhesion induced by che‐ moattractant in isolated neutrophils [50, 51]. Other antecedents describe a reduction of the expression of cyclooxygenase-2 (COX-2), inducible enzyme producing prostaglandins, in a human model of neutrophils [21]. In mouse peritoneal macrophages, andrographolide re‐ duces the production stimulated by lipopolysaccharide (LPS) of two important cytokines that participate in the amplification and activation of the inflammatory process, the cyto‐ kines tumoral necrosis factor TNFα and granulocyte macrophage colony-stimulating fac‐ tor (GM-CSF). The inhibition of the release of these cytokines by andrographolide was compared to the synthetic glucocorticoid dexamethasone, showing andrographolide to have a similar effect as dexamethasone, but with a lower potency [24, 52]. Also, the ef‐ fect of andrographolide on the cellular chemotaxis, a response that allows the movement of inflammatory cells to the injured tissue, show that it reduces the chemotactic migra‐ tion of macrophage induced by C5a, which may contribute to its anti-inflammatory activ‐ ity [53]. In local or systemic inflammatory disorders there is an enhanced formation of nitric oxide (NO) following the expression of inducible nitric oxide synthase (iNOS). The inhibition of NO formation may have therapeutic benefit in patients with inflammatory diseases as Rheumatoid Arthritis [54]. Thus, andrographolide reduces the LPS-induced iNOS and COX-2 expression in RAW264.7 macrophages [55, 56]. Additionally, androgra‐ pholide may have an effect on inflammation-mediated neurodegeneration, since it re‐ duces the production of reactive oxygen species (ROS), TNFα, NO and prostaglandin E2 in microglia, the counterpart of macrophages in the brain [25]. Andrographolide reduces the *in vitro* activation of human and murine T-cells, T-cells proliferation, interleukin-2 (IL-2) and IFNγ production [57-60].

Interaction of leukocyte-endothelium plays a key role in the initiation and maintenance of inflammation, being the adhesion molecule ICAM-1 important in mediating leukocyte adhesion, arrest and transmigration to the inflammatory site. In this respect, certain antece‐ dents show that andrographolide reduces the adhesion of HL-60 cells onto human vein endothelial cells (HUVEC) and the expression of TNFα-induced ICAM-1[61, 62]. In addition, andrographolide reduces the endothelial cell proliferation, migration and invasion, suggesting a role in angiogenesis [63]. Moreover, andrographolide reduces the growth factor deprivationinduced apoptosis in endothelial cells [64].

The therapeutic potential of andrographolide for the treatment of rheumatoid arthritis has been suggested by using of human rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) as a cellular model. Andrographolide exerts anti-proliferative and pro-apoptotic effects in RAFLSs, with G0/G1 cell cycle arrest, increases the expression of cell-cycle inhibitors p21 and p27 and reduces cyclin-dependent kinase 4 [65].

**4. Anti-inflammatory molecular mechanisms of andrographolide**

release of the p52 NF-κB bound to RelB [71].

arthritis via AP-1 and/or STAT3 modulation.

[73], bronchial epithelial cells [20], and dendritic cells [58].

All immunomodulatory effects of andrographolide have been attributed to modulation of different intracellular mediators, however three main mechanisms are commonly descri‐ bed. A first anti-inflammatory mechanism involved in the reduction of COX-2 expression by andrographolide in neutrophils comprises the modulation of the NF-κB pathway. The NF-κB is a family of transcription factors that regulate the expression of a large number of pro-inflammatory genes, such as COX-2, iNOS, TNF-alpha, IL-8 or IL-1, that are in‐ volved in the pathogenesis of Rheumatoid Arthritis. The activation of NF-κB compromis‐ es two main routes: the canonical and alternative pathways. The canonical NF-κB signaling pathway is the most important one. Inflammatory receptor activation results in IκB kinase (IKK) activation, and the IKK complex phosphorylate the IκB protein, leading to its polyubiquitination. The ubiquitinated IκB is degraded via 26S proteasome, thereby exposing the nuclear localization signal on NF-κB dimer and inducing nuclear transloca‐ tion. The alternative NF-κB pathway has been implicated in lymphoid organogenesis and B cell development, and is based in the processing of p100 NF-κB by IKKα, resulting in

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Andrographolide reduces the luciferase activity controlled by NF-κB and inhibits the DNA binding of NF-κB induced by chemoattractants, however not affecting IκB degradation [21]. The detailed mechanism of DNA binding inhibition indicates that andrographolide form a covalent adduct with reduced cysteine 62 of p50 subunit NF-κB, which block the binding of NF-κB to DNA [72]. The NF-κB pathway inhibition by andrographolide has been described in different cells involving in inflammatory processes such as endothelial cells [62], monocytes

A second mechanism describes an inhibitory effect of andrographolide on iNOS and COX-2 expression in macrophages, attributable to the modulation of transcription factors AP-1 and STAT3. AP-1 and STAT3, which are important for the production of pro-inflammatory cytokines such as IL-1β, IL-6 and IL-10, plays a major role in Rheumatoid Arthritis. It has been reported an overexpression of activated STAT3 and high DNA binding activity of AP-1 in synovial tissue from patients with Rheumatoid Arthritis [74, 75]. In fact, andrographolide reduced the LPS-induced AP-1 DNA-binding activities, and also decreased the STAT3 phosphorylation, which is crucial for nuclear translocation and DNA binding [56]. Thus, andrographolide may also be contributing to reduce the inflammatory process in rheumatoid

A third mechanism involves the interference of the transcription factor Nuclear Factor of Activated T cells (NFAT) induced by andrographolide in T-cells. The interference of NFAT activation by andrographolide is related to the increase of andrographolide-induced JNK

In addition to the immunomodulatory andrographolide mechanism described above, there are several cellular pathways, such as PI3K/Akt and ERK1/2 pathways, involved in the antiinflammatory effect of andrographolide and in the pathogenesis of the Rheumatoid Arthritis

phosphorylation, which controls the export of NFAT from nucleus [57].

#### **3.2.** *In vivo* **studies**

The anti-inflammatory activity of andrographolide has been studied in diverse *in vivo* inflammatory diseases models.

Earlier studies with andrographolide show that it inhibited carrageenin, kaolin and nistatininduced paw oedema. Moreover, andrographolide p.o. significantly inhibited the weight of granuloma induced by cotton pellets, and decreased the edema in adjuvant-induced arthritis (0.1-0.4% dead *Mycobacterium tuberculosis* suspension). Andrographolide (300 mg/kg) also inhibited dye leakage in acetic acid-induced vascular permeability. It was devoided of any ulcerogenic effect on the stomach in acute and chronic studies in rats. These effects were dose dependent, but inferior to phenylbutazone. Other diterpenic lactones, have shown to possess antipyretic effect in rabbits and rats with fever induced by 2-4-dinitrophenol. The potency was: 14-deoxy-11,12-didehydroandrographolide > deoxyandrographolide, and neoandrographo‐ lide > andrographolide [66].

In a model of ovalbumin-induced asthma in mice the intra-peritoneal administration of 30 mg/ kg andrographolide reduces the levels of TNFα and GM-CSF (92 and 65 %, respectively) in bronchoalveolar fluid, and the accumulation of lymphocytes and eosinophils, supporting a potential use in asthma. Andrographolide also reduced the Th2 cytokine IL-4, IL-5, IL-13 and serum immunoglobulin [20, 52].

Andrographolide also is helpfulness in the reduction of the symptoms of a mice experimental autoimmune encephalomyelitis (EAE), an animal model of human Multiple Sclerosis, by inhibiting T-cell and antibody responses directed to myelin antigens [59]. Similarly, in another model of autoimmune disease, the administration of andrographlide reduces the susceptibil‐ ity, prevents the symptoms and reduces anti-nuclear antibodies and kidney damage of systemic lupus erythematous [67, 68].

The potential effect of andrographolide on rheumatoid arthritis could involve angiogenesis inhibition. In fact, the development of new vessels, is important process that might facilitate the incoming of inflammatory cells into the synovium and, therefore, stimulate the pannus formation. [69]. In a model of induction of angiogenesis in C57BL/6 mice, andrographolide reduced the serum levels of cytokines of IL-1β, IL-6, TNFα and GM-CSF, the angiogenic factor VEGF and the NO production. Additionally, it is observable an increase of the levels of antiangiogenic factors TIMP-1 and IL-2 [12]. Andrographolide also suppresses breast tumor growth, which correlates with the inhibition of the pro-angiogenic molecules OPN and VEGF, in the NOD/SCID mice model [70].

## **4. Anti-inflammatory molecular mechanisms of andrographolide**

The therapeutic potential of andrographolide for the treatment of rheumatoid arthritis has been suggested by using of human rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) as a cellular model. Andrographolide exerts anti-proliferative and pro-apoptotic effects in RAFLSs, with G0/G1 cell cycle arrest, increases the expression of cell-cycle inhibitors p21 and

The anti-inflammatory activity of andrographolide has been studied in diverse *in vivo*

Earlier studies with andrographolide show that it inhibited carrageenin, kaolin and nistatininduced paw oedema. Moreover, andrographolide p.o. significantly inhibited the weight of granuloma induced by cotton pellets, and decreased the edema in adjuvant-induced arthritis (0.1-0.4% dead *Mycobacterium tuberculosis* suspension). Andrographolide (300 mg/kg) also inhibited dye leakage in acetic acid-induced vascular permeability. It was devoided of any ulcerogenic effect on the stomach in acute and chronic studies in rats. These effects were dose dependent, but inferior to phenylbutazone. Other diterpenic lactones, have shown to possess antipyretic effect in rabbits and rats with fever induced by 2-4-dinitrophenol. The potency was: 14-deoxy-11,12-didehydroandrographolide > deoxyandrographolide, and neoandrographo‐

In a model of ovalbumin-induced asthma in mice the intra-peritoneal administration of 30 mg/ kg andrographolide reduces the levels of TNFα and GM-CSF (92 and 65 %, respectively) in bronchoalveolar fluid, and the accumulation of lymphocytes and eosinophils, supporting a potential use in asthma. Andrographolide also reduced the Th2 cytokine IL-4, IL-5, IL-13 and

Andrographolide also is helpfulness in the reduction of the symptoms of a mice experimental autoimmune encephalomyelitis (EAE), an animal model of human Multiple Sclerosis, by inhibiting T-cell and antibody responses directed to myelin antigens [59]. Similarly, in another model of autoimmune disease, the administration of andrographlide reduces the susceptibil‐ ity, prevents the symptoms and reduces anti-nuclear antibodies and kidney damage of

The potential effect of andrographolide on rheumatoid arthritis could involve angiogenesis inhibition. In fact, the development of new vessels, is important process that might facilitate the incoming of inflammatory cells into the synovium and, therefore, stimulate the pannus formation. [69]. In a model of induction of angiogenesis in C57BL/6 mice, andrographolide reduced the serum levels of cytokines of IL-1β, IL-6, TNFα and GM-CSF, the angiogenic factor VEGF and the NO production. Additionally, it is observable an increase of the levels of antiangiogenic factors TIMP-1 and IL-2 [12]. Andrographolide also suppresses breast tumor growth, which correlates with the inhibition of the pro-angiogenic molecules OPN and VEGF,

p27 and reduces cyclin-dependent kinase 4 [65].

**3.2.** *In vivo* **studies**

252 Innovative Rheumatology

inflammatory diseases models.

lide > andrographolide [66].

serum immunoglobulin [20, 52].

systemic lupus erythematous [67, 68].

in the NOD/SCID mice model [70].

All immunomodulatory effects of andrographolide have been attributed to modulation of different intracellular mediators, however three main mechanisms are commonly descri‐ bed. A first anti-inflammatory mechanism involved in the reduction of COX-2 expression by andrographolide in neutrophils comprises the modulation of the NF-κB pathway. The NF-κB is a family of transcription factors that regulate the expression of a large number of pro-inflammatory genes, such as COX-2, iNOS, TNF-alpha, IL-8 or IL-1, that are in‐ volved in the pathogenesis of Rheumatoid Arthritis. The activation of NF-κB compromis‐ es two main routes: the canonical and alternative pathways. The canonical NF-κB signaling pathway is the most important one. Inflammatory receptor activation results in IκB kinase (IKK) activation, and the IKK complex phosphorylate the IκB protein, leading to its polyubiquitination. The ubiquitinated IκB is degraded via 26S proteasome, thereby exposing the nuclear localization signal on NF-κB dimer and inducing nuclear transloca‐ tion. The alternative NF-κB pathway has been implicated in lymphoid organogenesis and B cell development, and is based in the processing of p100 NF-κB by IKKα, resulting in release of the p52 NF-κB bound to RelB [71].

Andrographolide reduces the luciferase activity controlled by NF-κB and inhibits the DNA binding of NF-κB induced by chemoattractants, however not affecting IκB degradation [21]. The detailed mechanism of DNA binding inhibition indicates that andrographolide form a covalent adduct with reduced cysteine 62 of p50 subunit NF-κB, which block the binding of NF-κB to DNA [72]. The NF-κB pathway inhibition by andrographolide has been described in different cells involving in inflammatory processes such as endothelial cells [62], monocytes [73], bronchial epithelial cells [20], and dendritic cells [58].

A second mechanism describes an inhibitory effect of andrographolide on iNOS and COX-2 expression in macrophages, attributable to the modulation of transcription factors AP-1 and STAT3. AP-1 and STAT3, which are important for the production of pro-inflammatory cytokines such as IL-1β, IL-6 and IL-10, plays a major role in Rheumatoid Arthritis. It has been reported an overexpression of activated STAT3 and high DNA binding activity of AP-1 in synovial tissue from patients with Rheumatoid Arthritis [74, 75]. In fact, andrographolide reduced the LPS-induced AP-1 DNA-binding activities, and also decreased the STAT3 phosphorylation, which is crucial for nuclear translocation and DNA binding [56]. Thus, andrographolide may also be contributing to reduce the inflammatory process in rheumatoid arthritis via AP-1 and/or STAT3 modulation.

A third mechanism involves the interference of the transcription factor Nuclear Factor of Activated T cells (NFAT) induced by andrographolide in T-cells. The interference of NFAT activation by andrographolide is related to the increase of andrographolide-induced JNK phosphorylation, which controls the export of NFAT from nucleus [57].

In addition to the immunomodulatory andrographolide mechanism described above, there are several cellular pathways, such as PI3K/Akt and ERK1/2 pathways, involved in the antiinflammatory effect of andrographolide and in the pathogenesis of the Rheumatoid Arthritis [76]. The PI3 kinase pathway, is activated by TNF-α and IL-1, within fibroblastic synovial cells, and can activate the transcription factors NF-κB and AP-1 [77]. Also, the participation of the ERK1/2 MAPK in the initiation and progression of rheumatoid arthritis suggest that ERK inhibitors may emerge as a new therapeutic tool. The use of an ERK inhibitor in the animal model of collagen-induced arthritis suppressed the antigen-specific activation of T cells [78]. *In vitro*, andrographolide reduced the Akt phosphorylation in macrophages, HUVEC and microglia, and decreased the ERK1/2 phosphorylation in macrophages, suggesting that the signaling pathways PI3K/Akt and ERk1/2 may be associated to its anti-inflammatory effect [24, 61, 79]. Additionally, andrographolide also have the ability to reduce ERK1 and ERK5 phosphorylation [57].

**5. Effect of andrographolide on rheumatoid arthritis**

Number of patients 28 30

paniculata standardized extract (modified from Burgos et al., 2009).

Rheumatoid Arthritis [80].

Age (mean years) (min-max) 44.82 (13-63) 47.1 (20-70) Years with diagnosed (min-max) 6.5 (0.7-22.3) 6.7 (0.7-44.5) BMI (Kg/m2) (min-max) 30.0 (19.7-41.4) 29.2 (18.3-44.5) Height (m) (min-max) 1.52 (1.30-1.75) 1.51 (1.38-1.69) Weight (kg) (min-max) 69.9 (43.0-106.0) 67.2 (39.5-100.0) Intake of NSAIDs, n (%) 17 (60.7%) 18 (60.0%)

**5.1. Efficacy of an Andrographis paniculata composition (Paractin®) for the relief of rheumatoid arthritis symptoms: A prospective randomized placebo-controlled trial**

3 times a day. The demographic characteristic of the patients is shown in table 2.

In a prospective, double blind against placebo controlled clinical trial with chronic active Rheumatoid arthritis, the effect of a standardized patented *A. paniculata* extract (Paractin®) administration to 60 patients during 14 weeks in the reduction of symptoms and signs was studied. Each patient received either a tablet containing 30 mg of andrographolide or a placebo

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**Treatment group**s

**Table 1.** Demographic characteristics of Rheumatoid Arthritis patients included in the double blind study of A.

The results of the study show a significant reduction at the end of the treatment in tender joint, number of swollen joints, total grade of swollen joint, number of tender joints, total grade of swollen joints, total grade of tender joints HAQ 0.52 and SF36 (two health questionnaires) within the group treated with the active drug when comparing day 0 against week 14 (figure 4). The effect was associated to a reduction of rheumatoid factor, IgA, and C4. The study concludes that the drug was significantly effective in reducing symptoms and serological parameters of the disease and therefore useful as natural complement in the treatment of

The clinical efficacy of *A. paniculata* could be explained by the anti-inflammatory properties of andrographolide. Andrographolide present in the extract is a potent inhibitors of NF-κB [21], a transcription factor linked to pro-inflammatory expression of several proteins such as COX-2, iNOS, and TNF-α, IL-6. Since NF-κB is involved in the pathogenesis of Rheumatoid Arthritis and other rheumatoid conditions [81], we hypothesized that *A. paniculata* extract tablets (Paractin®) can reduce inflammatory symptoms, signs, serological parameters in these patients. In fact, the clinical findings suggest that the *A. paniculata* formulation may have an additional therapeutical effect over Prednisone and MTX in reducing pain and inflammatory clinical symptoms during treatment period. The beneficial effect in reducing pain and other

**Placebo Active drug**

In the following figure we propose the main anti-inflammatory effects of andrographolide that include the inhibition of several intracellular signaling pathways (Figure 3).

**Figure 3.** Proposed molecular mechanism of andrographolide in inflammation. Andrographolide shows inhibitory effect (x) on the PI3K/Akt pathway, ERK1/2 MAPK, NF-κB, NFAT, AP-1 and STAT3, and increases the JNK phosphorylation.

## **5. Effect of andrographolide on rheumatoid arthritis**

[76]. The PI3 kinase pathway, is activated by TNF-α and IL-1, within fibroblastic synovial cells, and can activate the transcription factors NF-κB and AP-1 [77]. Also, the participation of the ERK1/2 MAPK in the initiation and progression of rheumatoid arthritis suggest that ERK inhibitors may emerge as a new therapeutic tool. The use of an ERK inhibitor in the animal model of collagen-induced arthritis suppressed the antigen-specific activation of T cells [78]. *In vitro*, andrographolide reduced the Akt phosphorylation in macrophages, HUVEC and microglia, and decreased the ERK1/2 phosphorylation in macrophages, suggesting that the signaling pathways PI3K/Akt and ERk1/2 may be associated to its anti-inflammatory effect [24, 61, 79]. Additionally, andrographolide also have the ability to reduce ERK1 and ERK5

In the following figure we propose the main anti-inflammatory effects of andrographolide that

**Figure 3.** Proposed molecular mechanism of andrographolide in inflammation. Andrographolide shows inhibitory effect (x) on the PI3K/Akt pathway, ERK1/2 MAPK, NF-κB, NFAT, AP-1 and STAT3, and increases the JNK phosphorylation.

include the inhibition of several intracellular signaling pathways (Figure 3).

phosphorylation [57].

254 Innovative Rheumatology

#### **5.1. Efficacy of an Andrographis paniculata composition (Paractin®) for the relief of rheumatoid arthritis symptoms: A prospective randomized placebo-controlled trial**

In a prospective, double blind against placebo controlled clinical trial with chronic active Rheumatoid arthritis, the effect of a standardized patented *A. paniculata* extract (Paractin®) administration to 60 patients during 14 weeks in the reduction of symptoms and signs was studied. Each patient received either a tablet containing 30 mg of andrographolide or a placebo 3 times a day. The demographic characteristic of the patients is shown in table 2.


**Table 1.** Demographic characteristics of Rheumatoid Arthritis patients included in the double blind study of A. paniculata standardized extract (modified from Burgos et al., 2009).

The results of the study show a significant reduction at the end of the treatment in tender joint, number of swollen joints, total grade of swollen joint, number of tender joints, total grade of swollen joints, total grade of tender joints HAQ 0.52 and SF36 (two health questionnaires) within the group treated with the active drug when comparing day 0 against week 14 (figure 4). The effect was associated to a reduction of rheumatoid factor, IgA, and C4. The study concludes that the drug was significantly effective in reducing symptoms and serological parameters of the disease and therefore useful as natural complement in the treatment of Rheumatoid Arthritis [80].

The clinical efficacy of *A. paniculata* could be explained by the anti-inflammatory properties of andrographolide. Andrographolide present in the extract is a potent inhibitors of NF-κB [21], a transcription factor linked to pro-inflammatory expression of several proteins such as COX-2, iNOS, and TNF-α, IL-6. Since NF-κB is involved in the pathogenesis of Rheumatoid Arthritis and other rheumatoid conditions [81], we hypothesized that *A. paniculata* extract tablets (Paractin®) can reduce inflammatory symptoms, signs, serological parameters in these patients. In fact, the clinical findings suggest that the *A. paniculata* formulation may have an additional therapeutical effect over Prednisone and MTX in reducing pain and inflammatory clinical symptoms during treatment period. The beneficial effect in reducing pain and other

in patients affected with Rheumatoid Arthritis [85]. We propose that *A. paniculata* could be useful in decreasing the radiological progression in long-term treatments of Rheumatoid Arthritis patients. In support of this, andrographolide reduces NFAT activity, a transcription factor linked with bone erosion [88]. In MC3T3, a murine osteoblast cell line, we observed that andrographolide is able to induce differentiation and calcium mineralization, via expression

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257

On the other hand, no side effects were observed, indicating that *A. paniculata* treatment was safe, non-toxic, and well tolerated. In the literature, side effects associated with *A. paniculata* or andrographolide, administered in higher doses (4-6mg/kg), have caused isolated cases of allergic reactions, tiredness, headache, pruritus/rash, diarrhea, nausea, metallic taste, bitter taste, dry tongue, eyes sensitive to light, decreased short- term memory, dizziness, heartburn, tender lymph nodes, and lymphadenopathy [89]. None of these effects were observed in

Despite, the fact that was no difference between *A. paniculata* and placebo treatment after 14 weeks, the intragroup analysis showed a significant decrease of clinical symptoms and serological parameters in the *A. paniculata* group. This effect could become more evident in a long term administration of the drug and follow up Rheumatoid Arthritis patients for several

**5.2. Monotherapy with an** *Andrographis paniculata* **standardized extract (Paractin®) for the symptomatic relief of different chronic rheumatoid conditions: A prospective case report**

Presently, there is no specific or etiological cure for Rheumatoid Arthritis and these other rheumatoid conditions as well, and treatment aims to limit joint damage, prevent loss of function, and decrease pain. Therapies used for these purposes include nonsteroidal antiinflammatory drugs, disease-modifying anti-rheumatic drugs (DMARDs), and corticoste‐ roids. The American College of Rheumatology (ACR) Guidelines recommends the administration of DMARD within 3 months of diagnosis and methotrexate (MTX) as the standard treatment in monotherapy or in combination with other DMARDs [90]. MTX, as a standard therapy, induces significant improvement in the number of tender and swollen joints, pain, and functional status, in addition to physician and patient global assessment. The onset of MTX- induced improvement is generally within 3 months in the majority of patients who will eventually respond, and a plateau in the response is often reached after 6 to 12 months. However, as an anti-metabolic agent, MTX may cause adverse events such as cytopenia, serious infections, liver damage and muco-cutaneous problems. The long term use of MTX, is associated with prevalence of significant liver enzymes in aprox. 13% of the patients and 3.7%

Considering that in the clinical study in patients with Rheumatoid arthritis there was a significant decrease in the group with *A. paniculata* in the symptoms over time (after 14 weeks) on the progression of the diseases, it was proposed that long term treatment could

of COX2 (Burgos et al., data unpublished).

years.

**and long term follow up**

*5.2.1. Background*

Rheumatoid Arthritis patients after 14 weeks of treatment [80].

of the patients discontinue MTX permanently for liver toxicity [91]

**Figure 4.** Effect of *A. paniculata* extract (Paractin®) on tender joints, total grade of tender joints and rheumatoid factor [80].

inflammatory symptoms with the *A. paniculata* formulation could be associated to the high standardization of total andrographolides (NLT 30%) in the extract considering. This is closely associated with the inhibition of COX-2 [21] and the reduction of PGE2 production [25], one of the main mechanisms for the control of inflammation and pain in Rheumatoid Arthritis by NSAIDs [82]. The dose of Andrographolide used in the present study was around 1.2 mg per kg. It has been reported that 1mg/kg reaches a steady state plasma concentration of 1.9 μM [83], a concentration able to reduce the PGE2 production [25]. Moreover, in patients treated with *A. paniculata* extract a decrease of rheumatoid factor (RF), creatine kinase, hemoglobin, IgA and IgM were observed. A correlation between RF titers and clinical disease activity has been reported widely [84]. RF titers decrease with methotrexate, suggesting an indirect link with disease activity [85]. Andrographolide can reduce the TNFα production in macrophages, an effect that could be associated with the reduction of auto-antibodies. It is known that a reduction of TNFα can diminish significantly the RF levels [86]. The ability of andrographolide to reduce antibody titer has also been demonstrated in other autoimmune diseases such as experimental autoimmune encephalomyelitis and lupus (see above). A reduction of immuno‐ globulin, such as IgM and IgA, could also be beneficial in long-term treatment because there is a positive correlation between the grade of cartilage damage in active Rheumatoid Arthritis [87] and decrease of RF. Moreover, treatment with DMARDs reduces the level of IgM and IgA in patients affected with Rheumatoid Arthritis [85]. We propose that *A. paniculata* could be useful in decreasing the radiological progression in long-term treatments of Rheumatoid Arthritis patients. In support of this, andrographolide reduces NFAT activity, a transcription factor linked with bone erosion [88]. In MC3T3, a murine osteoblast cell line, we observed that andrographolide is able to induce differentiation and calcium mineralization, via expression of COX2 (Burgos et al., data unpublished).

On the other hand, no side effects were observed, indicating that *A. paniculata* treatment was safe, non-toxic, and well tolerated. In the literature, side effects associated with *A. paniculata* or andrographolide, administered in higher doses (4-6mg/kg), have caused isolated cases of allergic reactions, tiredness, headache, pruritus/rash, diarrhea, nausea, metallic taste, bitter taste, dry tongue, eyes sensitive to light, decreased short- term memory, dizziness, heartburn, tender lymph nodes, and lymphadenopathy [89]. None of these effects were observed in Rheumatoid Arthritis patients after 14 weeks of treatment [80].

Despite, the fact that was no difference between *A. paniculata* and placebo treatment after 14 weeks, the intragroup analysis showed a significant decrease of clinical symptoms and serological parameters in the *A. paniculata* group. This effect could become more evident in a long term administration of the drug and follow up Rheumatoid Arthritis patients for several years.

#### **5.2. Monotherapy with an** *Andrographis paniculata* **standardized extract (Paractin®) for the symptomatic relief of different chronic rheumatoid conditions: A prospective case report and long term follow up**

#### *5.2.1. Background*

inflammatory symptoms with the *A. paniculata* formulation could be associated to the high standardization of total andrographolides (NLT 30%) in the extract considering. This is closely associated with the inhibition of COX-2 [21] and the reduction of PGE2 production [25], one of the main mechanisms for the control of inflammation and pain in Rheumatoid Arthritis by NSAIDs [82]. The dose of Andrographolide used in the present study was around 1.2 mg per kg. It has been reported that 1mg/kg reaches a steady state plasma concentration of 1.9 μM [83], a concentration able to reduce the PGE2 production [25]. Moreover, in patients treated with *A. paniculata* extract a decrease of rheumatoid factor (RF), creatine kinase, hemoglobin, IgA and IgM were observed. A correlation between RF titers and clinical disease activity has been reported widely [84]. RF titers decrease with methotrexate, suggesting an indirect link with disease activity [85]. Andrographolide can reduce the TNFα production in macrophages, an effect that could be associated with the reduction of auto-antibodies. It is known that a reduction of TNFα can diminish significantly the RF levels [86]. The ability of andrographolide to reduce antibody titer has also been demonstrated in other autoimmune diseases such as experimental autoimmune encephalomyelitis and lupus (see above). A reduction of immuno‐ globulin, such as IgM and IgA, could also be beneficial in long-term treatment because there is a positive correlation between the grade of cartilage damage in active Rheumatoid Arthritis [87] and decrease of RF. Moreover, treatment with DMARDs reduces the level of IgM and IgA

**Figure 4.** Effect of *A. paniculata* extract (Paractin®) on tender joints, total grade of tender joints and rheumatoid factor [80].

256 Innovative Rheumatology

Presently, there is no specific or etiological cure for Rheumatoid Arthritis and these other rheumatoid conditions as well, and treatment aims to limit joint damage, prevent loss of function, and decrease pain. Therapies used for these purposes include nonsteroidal antiinflammatory drugs, disease-modifying anti-rheumatic drugs (DMARDs), and corticoste‐ roids. The American College of Rheumatology (ACR) Guidelines recommends the administration of DMARD within 3 months of diagnosis and methotrexate (MTX) as the standard treatment in monotherapy or in combination with other DMARDs [90]. MTX, as a standard therapy, induces significant improvement in the number of tender and swollen joints, pain, and functional status, in addition to physician and patient global assessment. The onset of MTX- induced improvement is generally within 3 months in the majority of patients who will eventually respond, and a plateau in the response is often reached after 6 to 12 months. However, as an anti-metabolic agent, MTX may cause adverse events such as cytopenia, serious infections, liver damage and muco-cutaneous problems. The long term use of MTX, is associated with prevalence of significant liver enzymes in aprox. 13% of the patients and 3.7% of the patients discontinue MTX permanently for liver toxicity [91]

Considering that in the clinical study in patients with Rheumatoid arthritis there was a significant decrease in the group with *A. paniculata* in the symptoms over time (after 14 weeks) on the progression of the diseases, it was proposed that long term treatment could demonstrate a mayor therapeutic response similar to other DMARs treatment. We report six case reports, with different rheumatoid arthritis conditions, that support the fact that *A. paniculata* standardized extract reduces symptoms of chronic joint pain, stiffness and serological inflammatory parameters in a prospective individual case controlled follow up study over a period of 42 months.

#### *5.2.2. Intervention*

The drug of botanical origin used for the treatment of these cases is a patented (US patent *8084495*) standardized extract of *A. paniculata* known as Paractin®, manufactured and distributed by Herbal Powers (USA). Paractin® contains andrograpolide NLT 30%, neoan‐ drographolide NLT 0.2% and deoxyandrographolide NLT 3%. Paractin® was supplied directly for this study and stored according to the instructions of the manufacturer. The batch number for the *A. paniculata* extract used in this study was PAR-070801-2. A secondary and identical batch was retained (No 20050520) and kept at Herbal Powers. Each tablet contained 150 mg of the extract. During all duration of this treatment, two tablets were given before meals three times a day. This dosage regimen was determined in previous preclinical and clinical trials with the pure compound and other commercially available *A. paniculata* extracts [80, 83]. The content of these compounds was evaluated by HPLC using reference standards as described elsewhere [92].

#### *5.2.3. Patients and method*

The group consisted of 6 (five adults and one pediatric) patients, 3 male and 3 female, all with a long history of active diseases as shown in Table 2.

> consent by their parents. Advice and indications to test Paractin® was done by the rheuma‐ tologist, who requested the approval of each individual pharmacological protocol and supply of the product. The rationale and main objective was that Paractin® could reduce long term clinical symptoms and serological parameters of inflammation in these patients. Inclusion criteria were confirmed by clinical and laboratory diagnosis, that included active clinical and serological parameters of inflammation, no underlying standard treatment, poor or no response to standard treatment, or important side effects of Methotrexate and Prednisone, like in the female pediatric patient. From day 0, two tablets of Paractin® orally containing 150 mg of standardized *A. paniculata* extract (90 mg andrographolide per day) was administered during 48 month. Total withdrawal of the standard therapy was commonly decided by the treating physician and patient upon improvement observed with Paractin® treatment and informed to the investigators. All patients were controlled monthly during the first six months, then every three months thereafter at their respective place of residence and coordinated by their rheumatologist. After 24 months the treatment with Paractin® tablets, administered orally to patients with Rheumatoid Arthritis, Psoriatic Arthritis and Ankylosing spondylitis, reduced symptoms. In a similar fashion the serum immunological parameters of inflammation

> **Figure 5.** Erythrocyte sedimentation rate (ESR) in patients with chronic Rheumatoid Arthritis compared with ESR value at beginning of treatment. Continuous observation during 48 month. Each point represents the mean and range

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When Paractin® was given alone; no side effects and good tolerability were observed during the complete period of administration. Only two cases reported a temporary and early and

were reduced progressively during 48 month of Paractin® treatment.

(maximum-minimum value). In dashed line the normal value.


**Table 2.** Antecedents of patients treated with *Andrographis paniculata* standardized extract (Paractin®)

All patients were individually recruited and controlled by their treating physician from the Hospital Regional de Valdivia, Unit of Rheumatology in the city of Valdivia, Chile and complying confirmed diagnosis of Rheumatoid Arthritis conditions before they were enrolled. They all signed a written informed consent, including the one pediatric case that was given

Andrographolide a New Potential Drug for the Long Term Treatment of Rheumatoid Arthritis Disease http://dx.doi.org/10.5772/55642

demonstrate a mayor therapeutic response similar to other DMARs treatment. We report six case reports, with different rheumatoid arthritis conditions, that support the fact that *A. paniculata* standardized extract reduces symptoms of chronic joint pain, stiffness and serological inflammatory parameters in a prospective individual case controlled follow up

The drug of botanical origin used for the treatment of these cases is a patented (US patent *8084495*) standardized extract of *A. paniculata* known as Paractin®, manufactured and distributed by Herbal Powers (USA). Paractin® contains andrograpolide NLT 30%, neoan‐ drographolide NLT 0.2% and deoxyandrographolide NLT 3%. Paractin® was supplied directly for this study and stored according to the instructions of the manufacturer. The batch number for the *A. paniculata* extract used in this study was PAR-070801-2. A secondary and identical batch was retained (No 20050520) and kept at Herbal Powers. Each tablet contained 150 mg of the extract. During all duration of this treatment, two tablets were given before meals three times a day. This dosage regimen was determined in previous preclinical and clinical trials with the pure compound and other commercially available *A. paniculata* extracts [80, 83]. The content of these compounds was evaluated by HPLC using reference standards as

The group consisted of 6 (five adults and one pediatric) patients, 3 male and 3 female, all with

 Female 51 Rheumatoid Arthritis 6 50 Male 36 Rheumatoid Spondylitis 7 50 Female 15 Rheumatoid Arthritis/Vasculitis 3 48 Female 39 Psoriatic Arthritis 15 60 Male 67 Rheumatoid Arthritis/ Serositis 8 38 Male 34 Psoriatic Arthritis/ Erythroderma 4 40

**Table 2.** Antecedents of patients treated with *Andrographis paniculata* standardized extract (Paractin®)

All patients were individually recruited and controlled by their treating physician from the Hospital Regional de Valdivia, Unit of Rheumatology in the city of Valdivia, Chile and complying confirmed diagnosis of Rheumatoid Arthritis conditions before they were enrolled. They all signed a written informed consent, including the one pediatric case that was given

**Diagnosis Prevalence of**

**Disease (Years)**

**Duration of Treatment (Months)**

study over a period of 42 months.

*5.2.2. Intervention*

258 Innovative Rheumatology

described elsewhere [92].

*5.2.3. Patients and method*

**Patient Sex Age at**

a long history of active diseases as shown in Table 2.

**Diagnosis Year. "0"**

**Figure 5.** Erythrocyte sedimentation rate (ESR) in patients with chronic Rheumatoid Arthritis compared with ESR value at beginning of treatment. Continuous observation during 48 month. Each point represents the mean and range (maximum-minimum value). In dashed line the normal value.

consent by their parents. Advice and indications to test Paractin® was done by the rheuma‐ tologist, who requested the approval of each individual pharmacological protocol and supply of the product. The rationale and main objective was that Paractin® could reduce long term clinical symptoms and serological parameters of inflammation in these patients. Inclusion criteria were confirmed by clinical and laboratory diagnosis, that included active clinical and serological parameters of inflammation, no underlying standard treatment, poor or no response to standard treatment, or important side effects of Methotrexate and Prednisone, like in the female pediatric patient. From day 0, two tablets of Paractin® orally containing 150 mg of standardized *A. paniculata* extract (90 mg andrographolide per day) was administered during 48 month. Total withdrawal of the standard therapy was commonly decided by the treating physician and patient upon improvement observed with Paractin® treatment and informed to the investigators. All patients were controlled monthly during the first six months, then every three months thereafter at their respective place of residence and coordinated by their rheumatologist. After 24 months the treatment with Paractin® tablets, administered orally to patients with Rheumatoid Arthritis, Psoriatic Arthritis and Ankylosing spondylitis, reduced symptoms. In a similar fashion the serum immunological parameters of inflammation were reduced progressively during 48 month of Paractin® treatment.

When Paractin® was given alone; no side effects and good tolerability were observed during the complete period of administration. Only two cases reported a temporary and early and

259

mild gastric discomfort with the tablets. Plasma biochemical parameters showed normal hematological, liver, kidney and metabolic functions. Interestingly, a moderate reactivation of joint pain and stiffness in two of the Rheumatoid arthritis patients and the one Ankylosing spondylitis patient was observed, due to an interruption of the treatment during 15, 11 and 22 days, respectively. Interestingly, these withdrawal and continuity incidents suggest that after peak and steady efficacy is reached and according to clinical and serological parameters follow up, a residual activity of the product is maintained between two and three weeks, disappearing at week four, and then recovered back again to previous status after four weeks. Also, we have so far not observed any loss of efficacy, or the need to increase dosages of the product, proving that no adaptation or refractoriness has yet been developed in this treated group. After one to five years follow up of these six rheumatologic patients, given a daily monotherapy of three Paractin® – tablets per day, we can conclude this product is well tolerated, safe and efficacious for the symptomatic relief and serological control of underlying inflammation related to their disease activity.

> **Figure 7.** Rheumatoid Factor (RF) in patients with chronic rheumatoid disease treated with Paractin® during 48 month. Each point represents the mean and range (maximum-minimum value). In dashed line the normal value.

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**Figure 8.** Variation on Rheumatologic stiffness in patients with chronic Rheumatoid Arthritis, treated with Paractin®

during 24 month. Each point represents the mean and range (maximum-minimum value).

**Figure 6.** C Reactive protein (CRP) in patients with chronic rheumatoid disease compared with the CRP value at the beginning of treatment with Paractin®. Continuous observation during 48 months. Each point represents the mean and range (maximum-minimum value). In dashed line the normal value.

Andrographolide a New Potential Drug for the Long Term Treatment of Rheumatoid Arthritis Disease http://dx.doi.org/10.5772/55642 261

mild gastric discomfort with the tablets. Plasma biochemical parameters showed normal hematological, liver, kidney and metabolic functions. Interestingly, a moderate reactivation of joint pain and stiffness in two of the Rheumatoid arthritis patients and the one Ankylosing spondylitis patient was observed, due to an interruption of the treatment during 15, 11 and 22 days, respectively. Interestingly, these withdrawal and continuity incidents suggest that after peak and steady efficacy is reached and according to clinical and serological parameters follow up, a residual activity of the product is maintained between two and three weeks, disappearing at week four, and then recovered back again to previous status after four weeks. Also, we have so far not observed any loss of efficacy, or the need to increase dosages of the product, proving that no adaptation or refractoriness has yet been developed in this treated group. After one to five years follow up of these six rheumatologic patients, given a daily monotherapy of three Paractin® – tablets per day, we can conclude this product is well tolerated, safe and efficacious for the symptomatic relief and serological control of underlying inflammation related to their

**Figure 6.** C Reactive protein (CRP) in patients with chronic rheumatoid disease compared with the CRP value at the beginning of treatment with Paractin®. Continuous observation during 48 months. Each point represents the mean

and range (maximum-minimum value). In dashed line the normal value.

disease activity.

260 Innovative Rheumatology

**Figure 7.** Rheumatoid Factor (RF) in patients with chronic rheumatoid disease treated with Paractin® during 48 month. Each point represents the mean and range (maximum-minimum value). In dashed line the normal value.

**Figure 8.** Variation on Rheumatologic stiffness in patients with chronic Rheumatoid Arthritis, treated with Paractin® during 24 month. Each point represents the mean and range (maximum-minimum value).

**6. Conclusion**

treatment similar to other DMARDs.

FONDEF Grant DO9I1085 and DO4I1240

sidad Austral de Chile, Valdivia, Chile

Bull (Tokyo). (1994). , 42(6), 1216-25.

**Acknowledgements**

**Author details**

**References**

(1992).

403-5.

Several studies describe a potent anti-inflammatory action of *Andrographis paniculata* and andrographolide. Andrographolide shows a reduction of the production of pro-inflammatory mediators, such as COX-2, iNOS and cytokines. The molecular mechanism of andrographolide implies the reduction of the activation of transcription factors as NF-κB, AP-1, STAT3 and NFAT and the inhibition of intracellular signaling pathways. *A. paniculata* standardized extract (30% andrographolide) in clinical trials showed effectiveness for symptom relief and reduce serological parameters in patients with Rheumatoid Arthritis, and the data support a long term

Andrographolide a New Potential Drug for the Long Term Treatment of Rheumatoid Arthritis Disease

http://dx.doi.org/10.5772/55642

263

María A. Hidalgo1,2, Juan L. Hancke1,2, Juan C. Bertoglio1,2 and Rafael A. Burgos1,2

and cancer. Clin Exp Pharmacol Physiol. (2012). , 39(3), 300-10.

1 Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Science, Univer‐

2 Institute of Medicine, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile

[1] Lim, J. C, Chan, T. K, Ng, D. S, Sagineedu, S. R, Stanslas, J, & Wong, W. S. Androgra‐ pholide and its analogues: versatile bioactive molecules for combating inflammation

[2] Tang, W, & Eisenbrand, G. Chinese Drugs of Plant Origen. Berlin: Springer Verlag;

[3] Matsuda, T, Kuroyanagi, M, Sugiyama, S, Umehara, K, Ueno, A, & Nishi, K. Cell differentiation-inducing diterpenes from Andrographis paniculata Nees. Chem Pharm

[4] Pramanick, S, Banerjee, S, Achari, B, Das, B, & Sen, A. K. Sr., Mukhopadhyay S, et al. Andropanolide and isoandrographolide, minor diterpenoids from Andrographis paniculata: structure and X-ray crystallographic analysis. J Nat Prod. (2006). , 69(3),

**Figure 9.** Effect of Paractin® on Fatigue in patients with chronic Rheumatoid Arthritis, treated during 24 month. Each point represents the mean and range (maximum-minimum value).

**Figure 10.** Effect of Paractin® on pain in patients with chronic Rheumatoid Arthritis, treated during 24 month. Each point represents the mean and range (maximum-minimum value).

## **6. Conclusion**

Several studies describe a potent anti-inflammatory action of *Andrographis paniculata* and andrographolide. Andrographolide shows a reduction of the production of pro-inflammatory mediators, such as COX-2, iNOS and cytokines. The molecular mechanism of andrographolide implies the reduction of the activation of transcription factors as NF-κB, AP-1, STAT3 and NFAT and the inhibition of intracellular signaling pathways. *A. paniculata* standardized extract (30% andrographolide) in clinical trials showed effectiveness for symptom relief and reduce serological parameters in patients with Rheumatoid Arthritis, and the data support a long term treatment similar to other DMARDs.

## **Acknowledgements**

FONDEF Grant DO9I1085 and DO4I1240

## **Author details**

**Figure 9.** Effect of Paractin® on Fatigue in patients with chronic Rheumatoid Arthritis, treated during 24 month. Each

**Figure 10.** Effect of Paractin® on pain in patients with chronic Rheumatoid Arthritis, treated during 24 month. Each

point represents the mean and range (maximum-minimum value).

262 Innovative Rheumatology

point represents the mean and range (maximum-minimum value).

María A. Hidalgo1,2, Juan L. Hancke1,2, Juan C. Bertoglio1,2 and Rafael A. Burgos1,2

1 Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Science, Univer‐ sidad Austral de Chile, Valdivia, Chile

2 Institute of Medicine, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile

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**Chapter 12**

**Efficacy of Nandrolone Decanoate in Treating**

Rheumatoid cachexia, a consequence of chronic inflammation, is a common feature of rheu‐ matoid arthritis (RA) [1]. It is characterised by reduced muscle mass and increased, predom‐ inantly truncal, adiposity, which in turn both contribute to physical weakness and disability [2,3]. Additionally, as in other catabolic conditions, these adverse changes in body compo‐ sition exacerbate risk of falls and fracturing, contribute to impaired physical disability and reduced quality of life, and increase morbidity and mortality [e.g. 4-6]. In RA, again as in other chronic conditions, these outcomes are most marked in severe forms of cachexia where there is frank weight loss with reductions in both lean and fat mass. This overt wasting has been estimated to occur in up to 10% of RA patients and is associated with a three-fold

There is evidence that rheumatoid cachexia is established early in the course of the disease [8], and that it is resistant to antirheumatic drug treatment. This non-responsiveness to standard treatment is highlighted by the high prevalence of significant muscle wasting (ap‐ proximately 67%) and the even higher incidence of obesity (approximately 80%) observed in patients with stable, controlled disease [9-13]. Despite the fact that TNF-α is considered to be a major factor driving rheumatoid cachexia, even anti-TNF treatment fails to reverse or at‐ tenuate these perturbations to body composition [8,14,15]. In fact, evidence is emerging that anti-TNF therapy increases fat mass, particularly trunk adiposity, relative to standard DMARDs [14,15]. Consequently, specific potential anabolic interventions need to be as‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Lemmey et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Rheumatoid Cachexia in Male Rheumatoid**

Andrew B. Lemmey, Srinivasa Rao Elamanchi, Samuele M. Marcora, Francesco Casanova and

Additional information is available at the end of the chapter

**Arthritis Patients**

Peter J. Maddison

**1. Introduction**

higher mortality [7].

http://dx.doi.org/10.5772/53236

