1. Introduction
We propose the potential use of andrographolide in Rheumatoid Arthritis and other autoimmune diseases. This is supported by the fact that andrographolide exerts anti-growth and pro-apoptotic effects on human rheumatoid arthritis fibroblast- like synoviocytes, the main cellular constituent of pannus, that combined with a massive infiltration of lymphocytes and macrophages, invades and destroys the local articular structure. Recently, a prospective randomized placebo-controlled trial has suggested that
2. Andrographis paniculata and labdane diterpenoids
The main and most interesting biological constituent of
Andrographolide is the bitter principle, a colourless, neutral crystalline substance, was first isolated by Boorsma from different parts of
Preclinical properties include anti-retroviral [6, 7], antiproliferative and pro-apoptotic [8, 9], anti-diabetic [10, 11], anti-angiogenic [12], anti-thrombotic [13], anti-urothelial [14], anti-leishmaniasis [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
The second diterpene isolated from
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].
Andrographiside, the 19-glucoside of andrographolide, was isolated in 1981, and only a hepatoprotective effect has been described [41].
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 andrographate and dipotassium andrographate 19-
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
3. Anti-inflammatory and immunomodulatory effects of andrographolide in vitro and in vivo
Different preparations of
3.1. In vitro studies
Andrographolide, shows anti-inflammatory and anticancer activities in both
Several
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 antecedents 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-induced 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].
3.2. In vivo studies
The anti-inflammatory activity of andrographolide has been studied in diverse
Earlier studies with andrographolide show that it inhibited carrageenin, kaolin and nistatin-induced 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
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 susceptibility, 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 anti-angiogenic 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
All immunomodulatory effects of andrographolide have been attributed to modulation of different intracellular mediators, however three main mechanisms are commonly described. 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 involved in the pathogenesis of Rheumatoid Arthritis. The activation of NF-κB compromises 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 translocation. 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 anti-inflammatory 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 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
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
Number of patients | 28 | 30 |
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%) |
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].

Figure 4.
Effect of
The clinical efficacy of
On the other hand, no side effects were observed, indicating that
Despite, the fact that was no difference between
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
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 anti-inflammatory drugs, disease-modifying anti-rheumatic drugs (DMARDs), and corticosteroids. 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
5.2.2. Intervention
The drug of botanical origin used for the treatment of these cases is a patented (US patent
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.
1 | Female | 51 | Rheumatoid Arthritis | 6 | 50 |
2 | Male | 36 | Rheumatoid Spondylitis | 7 | 50 |
3 | Female | 15 | Rheumatoid Arthritis/Vasculitis | 3 | 48 |
4 | Female | 39 | Psoriatic Arthritis | 15 | 60 |
5 | Male | 67 | Rheumatoid Arthritis/ Serositis | 8 | 38 |
6 | Male | 34 | Psoriatic Arthritis/ Erythroderma | 4 | 40 |
Table 2.
Antecedents of patients treated with

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.
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 consent by their parents. Advice and indications to test Paractin® was done by the rheumatologist, 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
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 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 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.

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).

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