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

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

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 andrographolide can reduce NFAT function in T cells and reduce the phosphorylation of signal transducer and activator of transcription-3 (STAT3) in macrophages.
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 proapoptotic 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 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.

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 CHCl 3 /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 andrographolide 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 andrographolide [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% andrographolide 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.

Neoandrographolide
The second diterpene isolated from A. paniculata was the minor non-bitter constituent neoandrographolide, which was first described by Kleipool in 1952. The structure of neoandrographolide ( 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].
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-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][47][48].

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]

In vitro studies
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 chemoattractant 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 reduces the production stimulated by lipopolysaccharide (LPS) of two important cytokines that participate in the amplification and activation of the inflammatory process, the cytokines tumoral necrosis factor TNFα and granulocyte macrophage colony-stimulating factor (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 effect 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 migration of macrophage induced by C5a, which may contribute to its anti-inflammatory activity [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, andrographolide may have an effect on inflammation-mediated neurodegeneration, since it reduces 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][58][59][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 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 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].

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 neoandrographolide > 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 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 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].

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

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. 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 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 immunoglobulin, 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.

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 antiinflammatory 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 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.

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%, neoandrographolide 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 (N o 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].

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. 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 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  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.

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.