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2 ParActin Research Summary Address: th Ave W A randomized, double blind, and placebo-controlled study in patients with rheumatoid arthritis (RA) was performed. 100mg of Paractin were administered three times a day for 14 weeks, after a 2-week washout period to 60 patients with active RA. Patients were allowed to take prednisone or chloroquine (stable doses). MTX was administered to all patients as standard treatment. On day 1, and at the end of week 2, week 6, week 10, and week 14 the following exams were performed: 1. Evaluation of the number of inflammatory and painful joints. 2. Evaluation of pain by the patient utilizing the Visual Analog Scale (VAS). 3. Improvement in the evaluation of health quality of life questionnaires 4. Duration of morning rigidity. 5. Global evaluation of symptoms by the patient and researchers. 6. Global evaluation of the tolerance by the patient and researchers. Paractin Group showed significant diminishing in: 1. number of swollen joints (Paractin 9 vs Placebo 13) 2. total grade of swollen joint (Paractin 11 vs Placebo 16) 3. total grade of tender joints (Paractin 14 vs Placebo 17) 4. HAQ (Paractin 19 vs Placebo 24) 5. reduction of rheumatoid factor (Paractin 119 vs Placebo 130) 6. reduction in IgA (Paractin vs placebo 335)

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4 In this study, we showed that ParActin significantly reduced the RF value from 110 to 70 mg/dl. The placebo group, even though taking NSAIDs most of the study period, did not experience any significant change in RF value. Rheumatoid Factor (mg/dl) Placebo Paractin * * Weeks RCP (mg/dl) Weeks Placebo Paractin * Patients treated with ParActin are showing improvement in CRP level from a above 1mg/dl to 0.5mg/dl at the end of the study. The result suggest the capacity of ParActin to reduce C- reactive protein, which is one the most sensitive mediator reactants of acute and chronic inflammatory activity in RA. The placebo group, which is on NSAIDs most of the study period, only showed slight reduction in CRP protein level. This research showed that ParActin is effective in normalizing CRP level. Mechanism of Action Paractin works by inhibiting NF-κB and NFAT, important factors in regulating the inflammatory processes and progression of RA. Paractin inhibits COX-2 and reduce PGE2, one of the main mechanisms for the control of inflammation and pain in RA. Paractin decreases of Rheumatoid Factor via reduction in TNF-α production Paractin reduces IgA and IgM, which is beneficial as there is positive correlation between the grade of cartilage damage in active RA Paractin inhibits NFAT activity, a transcription factor linked with bone erosion

5 Andrographolide induces Osteoblast Mineralization Via COX-2 Expression (Publication: 2012) Andrographolide strongly increase the COX-2 mrna expression MC3T3-E1 cells were treated for 9 days with different concentrations of andrographolide (AP) (a) morphological changes dose dependent by andrographolide; the cells more rounded and the cytoplasm is more constricted with respect to the control (O. M. 40x (b) plaques stained with red alizarin, for the detection of mineralized matrix. Andrographolide has a mineralizing effect on the bones with the deposit of calcium. Therefore, it could be of use in osteoporosis.

6 Monotherapy with an Andrographis paniculata composition (Paractin capsules) for the symptomatic relief in different chronic rheumatoid conditions and Multiple Sclerosis: Long term follow up of 17 cases. (1)C.F. Zárate, MD;; (1)L.C. Cárcamo, MD and (2)J.C. Bertoglio, MD. Publication: 2012 Paractin were administered orally twice a day for an average of more than three years to this group of 17 independently treated patients, all with a long history of active disease: 3 with Adult Rheumatoid Arthritis, 1 with Juvenile Rheumatoid Arthritis, 2 with Psoriatic Arthritis, 1 with Sjögren's syndrome, 1 with Ankylosing spondilitis and 9 with Multiple Sclerosis. Dosage: 150mg Paractin, twice daily. Principal Findings: The intensity of joint pain and stiffness rapidly decreased at three to four weeks in all patients with active rheumatoid diseases and was thereafter maintained significantly low without recurrence with this therapy. These clinical findings correlated with a corresponding reduction in RF, HSR and CRP. The fatigue, number of relapses and progression rate of disease in the Multiple sclerosis group also decreased significantly, which correlated with overall signs of clinical activity and also with MR imaging. Result: After 42 weeks of treatment, long term treatment of Paractin improved symptom, serum immunological parameters of inflammation or MR images of lesions in this group. No side effects Normal liver, kidney and metabolic functions. Very significant improvement of fatigue and functional capacity Potent control of clinical, serological and MR inflammatory parameters Significant less consumption of NSAIDs Presently (three and a half years later), 8 rheumatologic patients still in continuous regular treatment only Paractin (monotherapy), all in full tolerability and remission of clinical and serological inflammatory parameters. Magnetic Resonance Imaging (MRI) Comparative imaging study of lesions by MRI before treatment and 4 month and 26 months No change in total number and no increase in size of demyelinating lesions in the brain white matter Highly significant reduction to no inflammatory activity of demyelinating lesions Paractin is a potent inhibitor of NF-kB, a transcription factor linked to COX-2, inos, and TNF-a. NF-kB is involved in the pathogenesis of RA and other rheumatoid conditions.

7 COX-2 and PGE2 production are one of the main mechanisms for the control of inflammation and pain in RA and rheumatoid conditions by NSAIDs. Paractin is able to reduce the PGE2 production, reduce Rheumatoid factor (RF), creatine kinase, hemoglobin, IgA, and IgM. Andrographolide reduce TNF-a; It is known that a reduction of TNF-a canreduce significantly the RF levels. The ability of Andrographolide to reduce antibody titer also has been demonstrated in experimental autoimmune encephalomyelitis as an inhibition of antibodies directed to myelin antigens. A reduction of immunoglobin, 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 RA and decrease of RF.

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9 The Journal of Pharmacology and Experimental Therapeutics Vol. 312 No.1 Paractin interferes with T cell activation and reduces Experimental Autoimmune Encephalomyelitis in the Mouse*. Mirentxu I. Iruretagoyena*, Jaime A. Tobar*, Pablo A. González*, Sofía E. Sepúlveda*, Claudio A. Figueroa*, Rafael A. Burgos, Juan L. Hancke and Alexis M. Kalergis* Multiple Sclerosis (MS) is a chronic neuron-inflammatory demyelinating disorder of the central nervous system (CNS) that predominantly affects young adults. Empirical evidence points toward an autoimmune pathogenesis, where myelin-specific CD4+ and CD8+ T cells are thought to play a central role by reacting against and destroying the myelin sheath. Previous research had shown ParActin (14- NEO-ANDRO) to effectively inhibit NFkappaB, reduce Interleukin-2 and Interferon gamma cytokines. Here we provide evidence for ParActin (14-NEO-ANDRO) as an inhibitor for T cell-mediated immune responses in vitro and in vivo.

10 6 to 8 weeks old female mice were injected with 50 mg of MOG35-55 peptide. Half the mice were treated daily with 13 mg/kg of ParActin (14-NEO-ANDRO) one week before MOG sensitization and continued through all the experiment. Clinical signs of disease were observed between day 15 and 18.. Interleukin-2 and Interferon gamma were measured with ELISA, Anti-MOG antibody response were analyzed, surface markers I-Ab, CD86 and CD40 were measured, T cell proliferation was assessed, and nonspecific IgG antibodies were measured. In vitro T cell activation is inhibited by ParActin (14-NEO-ANDRO) Lymph node cells incubated with ParActin (14-NEO-ANDRO) suppressed B and C T cell activation. ParActin (14-NEO-ANDRO) prevented DCs from activating both CD4+ and CD8+ OVA-specific T cell by inhibiting the ability of DCs to process OVA and generate the peptide-mhc complexes required for T cell activation. ParActin (14-NEO-ANDRO) also inhibited up-regulation of the maturation of DCs markers. Compared to untreated controls, mice treated with ParActin significantly reduced anti-np IgG titers compared to untreated mice. ParActin (14-NEO-ANDRO) treatment significantly reduces severity of EAE in mice. Symptoms of EAE were present on day 14 after sensitization. Clinical score was evaluated on a daily basis. Treatment with ParActin (14-NEO-ANDRO) not only delayed the onset of EAE, but also significantly reduced the severity and incidence of MS. ParActin (14-NEO-ANDRO) reduces anti-myelin T cell and antibody response 3 weeks after EAE induction, lymph nodes were evaluated. Interferon gamma, Interleukin 2, and anti- MOG IgG were observed only in lymph node obtained from untreated mice suffering from EAE. In contrast, Interferon gamma, Interleukin 2, and anti-mog IgG were not detected in lymph node from ParActin treated mice.

11 Result & Conclusion: Previous studies provide evidence suggesting that ParActin (14-NEO-ANDRO) interfered with the function of inflammatory cells and showed anti apoptotic activity which could contribute to reduce severity of EAE symptoms and inflammatory damage. In mice induced with EAE, ParActin (14-NEO-ANDRO) significantly reduced both the incidence and clinical severity of EAE, significantly reduced Interferon gamma and Interleukin 2 release, and inhibited T cell activation. The data presented here suggest that ParActin (14-NEO-ANDRO) act as T cell inhibitor both in vitro and in vivo. ParActin (14-NEO-ANDRO) prevent DCs from activating either CD4+ or CD8+ T cell, reduce the efficiency of DC maturation, significantly reduce antibody (IgG) secretion, and diminish DTH response. These results support the notion that T cell-mediated immune responses can be effectively reduced by ParActin (14-NEO-ANDRO). We conclude that PARACTIN (14-Neo-Andro) is a new T cell inhibitor exhibiting strong potential anti-inflammatory properties. Andrographolide prevents cognitive impairment, tau pathology and stimulates LTP in a model of Alzheimer s disease Felipe Serrano 1, Cheril Tapia-Rojas 1, Francisco J. Carvajal 1, Juan Hancke 2, Waldo Cerpa 3 and Nibaldo C. Inestrosa 1,* Publication: Journal Brain 2012 Abstract Although, the mechanisms involved in the pathogenic changes triggered by the amyloid-βpeptide are not clearly understood, the neuronal dysfunction which includes oxidative stress, astrogliosis, cytoskeletal alterations and synaptic failure are all features that contributes to Alzheimer s disease. The use of natural compounds it is an interesting conceptual strategy in the search of drugs with therapeutic potential for the treatment of neurodegenerative diseases. We report here our studies on a drug, called Andrographolide, a diterpenoid and major component of Andrographis paniculata, on the prevention of some cognitive and synaptic plasticity deficits associated to Alzheimer s disease. Our results indicated that Andrographolide acts on brain neuropathology, learning and memory in the amyloid precursor protein/presenilin-1 transgenic mouse model of Alzheimer s disease. In particular, Andrographolide alleviates memory decline induced by amyloid-β-peptide deposits as indicated by the Morris Water Maze paradigm, probably by inducing postsynaptic proteins. Andrographolide also prevents the inflammatory oxidative stress; the astrocyte reaction triggered by the high amyloid-β-peptide deposit levels, as well as, tau phosphorylation, but did not affect the amyloid-β-peptide burden in the hippocampus. Andrographolide generates in vitro an increase in field excitatory postsynaptic potentials in hippocampal CA1 slices, and prevents the amyloid-β-peptide oligomer effects allowing the induction of long-term potentiation. Our results suggest that Andrographolide has therapeutic potential for Alzheimer disease. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

12 Address: th Ave W Paractin prevents the spatial memory loss in APP-PS-1 double transgenic mice APP-PS-1 mice, which represent a transgenic mouse model of Alzheimer's disease, were used in this experiment. APP-PS-1 mice exhibit onset of cognitive impairment and increasing amyloid-ß plaques in their brains. Mice were divided into 3 groups. Group 1 as untreated control, Group 2 were treated with ParActin. Amyloid plaques in both the hippocampus and the cerebral cortex were tested at 12 weeks. Beta Amyloid plague deposits Ratón Tg (APPswe+PS-1) Untreated Control 4X, 10X, 10X, Ratón Tg (APPswe+PS-1) Paractin 10X, hipocampo 10X, corteza Inflamation (oxidative status) of the brain Naive control Paractin 4X Paractin significantly reduce the level of nitrotyrosynation (oxidative status of the brain) in transgenic mice. Study 2: In this experiment, the spatial memory in 25 to 30-week-old APP-PS-1 mice was analyzed in Morris water maze (MWM) tests where the mice were trained to escape to a hidden platform. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

13 Mice were divided into 4 groups: Group 1 is control; Group 2 is untreated APP-PS-1 mice; Group 3 is APP-PS-1 mice treated with Paractin. Group Day 1-2 Day 3-5 Week 2 Day sec 20 sec 20 sec 20 sec 2 60 sec 60 sec 60 sec 40 sec 3 20 sec 20 sec 20 sec 20 sec No of time it takes the mice to find the escape platform. This experiment showed that APP-PS-1 mice treated with 14-Neo-Andro compound were able to learn the escape hidden pathway as fast as regular control mice and confirm the ability of ParActin (14-Neo-Andro) to improve learning ability and may reverse memory loss in AD. In PSAPP mice treated with 14-Neo-Andro, there are no apparent changes in the levels of amyloid plaques in both the hippocampus and the cerebral cortex. However, a decrease in the levels of nitrotyrosynation (oxidative status of the brain) was observed in PSAPP transgenic animals treated with 14-Neo-Andro compound. Similar results were observed at the level of astrocyte activation (inflammation). These observations correlate with the observations at the behavioral level. Although ParActin (14-Neo-Andro) do not reduce the levels of amyloid plaques, it showed the ability to stop memory and deterioration better than hyperforin, and decrease in the inflammation and oxidative status of the brain. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

14 Findings: 1. Paractin prevents the spatial memory loss in APP-PS-1 double transgenic mice 2. Paractin protects from astrocytic proliferation and protein nitrotyrosination induced by Aβ in the double transgenic AD model. 3. Paractin did not affect Aβ burden in a double APP-PS-1 transgenic mice. 4. Paractin increases hippocampal synaptic proteins 5. Tau hyperphosphorylation decreases after Paractin treatment in APP-PS1 mice. 6. Paractin induces an increase in fepsp amplitude without affecting paired pulse facilitation 7. Paractin reverses the Aβ-induced damage allowing the induction of LTP in old APP-PS1 mice ParActin Human Clinical on Alzheimer 20 patients who manifested early stage of Alzheimer's disease (AD) were recruited. Two weeks before the clinical trial, the patients undergo appropriate psycho neurological tests such as the Mini Mental Status Exam (MMSE), the Alzheimer Disease Assessment Scale (ADAS), the Boston Naming Test (BNT), and the Token Test (TT). Neuropsychological tests are repeated on Day 0, 6 weeks and 3 months of the clinical trial. The tests are performed by neurophysiologists who are not aware of the patient's treatment regimen. Patients are randomly assigned either 250mg of ParActin (14-NEO-ANDRO) or placebo at the start of the study, to be taken orally twice daily. The ParActin (14-NEO-ANDRO) and placebo results are statistically compared for all the study periods. Patients using placebo show a significant cognitive deterioration, which is the natural course of AD. Patients treated with ParActin (14-NEO- ANDRO) ameliorate in a considerable way the test scores. This research shows that ParActin (14- NEO-ANDRO), a PPAR-γ agonist may help in cognitive and memory deterioration and have application for maintaining healthy brain and cognitive function. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

15 Emerging research has shown Nuclear Factor kappa B (NF-kB) to hold key to many inflammatory related diseases as they regulate our immune system and inflammatory response by regulating proinflammatory cytokines (interleukin-1, -2, -6, -8) and proinflammatory mediators (ICAM-1, inos and COX-2). NSAIDs and glucocorticoids both inhibit NFkappaB and have application in many inflammatory related diseases. In previous study, we have shown ParActin (14- NEO-ANDRO) to possess anti-inflammatory activity by inhibiting the expression of several proinflammatory cytokines such as IL-2 and INF-γ. In the present study we analyzed the effect of ParActin on the NF-kB activation induced by PAF and fmlp in human cells. We showed that ParActin inhibited the NF-kB activity, reduced the DNA binding of NF-kB in vivo and in vitro, and reduced COX 2 expression. ParActin inhibits NF-κB activation by PAF Human cells (HL-60) were pre-incubated with ParActin (5 and 50 µm) or vehicle for 30 min, and then stimulated with PAF 100 nm for 1 hr, NF-kB activity was measured. ParActin inhibited significantly the PAF induced luciferase activity in the NF-κB reporter, indicating that ParActin interferes with the NF-κB activation. ParActin interferes with the DNA binding to NF-κB HL-60/Neutrophils were stimulated with 100nM PAF or fmlp, and incubated with ParActin (30,150, or 300 M) for 15 minutes. ParActin inhibit directly the DNA binding of NF-κB stimulated by PAF and fmlp. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

16 ParActin reduces COX-2 expression Research has shown that COX-2 expression is reduced by NF-κB inhibitors. In this study we demonstrated that ParActin (14-NEO-ANDRO) reduced the COX-2 expression in HL- 60/neutrophils induced by fmlp and PAF. Conclussion: NF-κB has been described to regulate the cellular process of apoptosis. The inhibition of NF-κB by ParActin (14-NEO-ANDRO) shown by this research could explain the anti-proliferate effect of ParActin. We conclude that ParActin (14-NEO-ANDRO) is a natural NF-κB inhibitor exhibiting strong potential anti-inflammatory properties. ParActin (14-NEO-ANDRO) inhibits IFNγ and IL-2 cytokines production and protects against cell apoptosis 1 Juan L. Hancke, & 1 Rafael A. Burgos, Karina Seguel,Mirna Perez, Ada Meneses, Marcele Ortega. Published: Planta 34/0704/416, and Biochem, Mol, Biol, /Summer Previous clinical trials have demonstrated the anti-inflammatory properties of ParActin by reducing the activity of macrophages and neutrophils, which is related to innate immune response. In this study we present evidence that ParActin (14-NEO-ANDRO) reduces the IFNγ and IL-2 production in murine T-cells and protects against cell apoptosis. Method: T-cells and thymocytes were isolated from 6 to 8-week old RK mice. The. T-cells were incubated with concanavaline A (CON-A) and various concentration of ParActin. IFNγ, IL-2 and IL-4 production was measured using ELISA. ERK1/2 phosphorylation was analyzed and apoptosis was quantified by DNA fragmentations. All doses of ParActin inhibited dramatically the CON-A rise of IFN-γ with maximum inhibitory response at doses of 7.5µM and IC ±0.07µM. The reduction in cytokine production shown in our study could explain the anti-inflammatory effect of ParActin ParActin inhibited the IL-2 production induced by CON- A. The IL-4 production was not modified either by ParActin in T-cells. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

17 The expression ICAM-1, inos, COX-2 and proinflammatory cytokine are controlled by mitogen-activated proteinkinase (MAPK). Our results show that ParActin reduced the ERK1/2 phosphorylation induced by CON-A. ERK 1/2 is a MAPK that requires phosphorylation to become active. NF-κB regulate the cellular process of apoptosis. The DNA fragmentation of thymocytes was incubated with hydrocortisone and PMA. ParActin alone did not induce an increase in apoptosis. 50µM of ParActin reduced the apoptosis induced by hydrocortisone up to 20%. 100µM of ParActin significantly reduced the apoptosis induced by PMA up to 35%. Apoptosis induced by hydrocortisone increases the production of caspase-3 activity. ParActin was able to reduce significantly the spontaneous caspase-3 like activity. We conclude that ParActin inhibit significantly the production of IFNγ in T-ells and ERK1/2 phosphyloration and protects against apoptosis. The antiinflammatory effect of ParActin opens a potential use in several health imbalances associated with an increase in IFNγ and IL- 2. Abstract The nuclear factor of activated T cells (NFAT) is a transcription factor essential for cytokine production during T-cell activation and is the target of several immunosuppressive drugs. Andrographolide is a diterpenic labdane that possesses anti-inflammatory and immunomodulatory effects. Several studies propose that andrographolide can reduce the immune response through inhibition of the nuclear factor kappa B (NF-kappaB) and mitogenactivated protein kinases (MAPK) such as extracellular signal regulated kinase 1/2 (ERK1/2) pathways. Moreover, andrographolide reduces IFN-gamma and IL-2 production induced by concanavalin A in murine T-cell. Nevertheless, the mechanisms involved in the decrease of cytokine production are unknown. In the present study, we determined that andrographolide reduced IL-2 production in Jurkat cells stimulated with phorbol myristate acetate and ionomycin (PMA/Ionomycin). We then showed that andrographolide reduced NFAT luciferase activity and interfered with its nuclear distribution, with these effects being linked to an increase in c-jun-n-terminal kinase (JNK) phosphorylation. Additionally, reduction of NF- ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.

18 kappab activity in Jurkat cells treated with andrographolide was observed. Using Western blotting, we demonstrated that andrographolide decreased ERK1 and ERK5 phosphorylation induced by anti-cd3 or PMA/Ionomycin. Andrographolide did not affect cell viability at concentration of 10 and 50 mum; however, our results suggest that andrographolide increase early apoptosis at 100 mum. We concluded that andrographolide can exert immunomodulatory effects by interfering with NFAT activation and ERK1 and ERK5 phosphorylation in T-cells. ParActin is a registered trademark of HP Ingredients. Copyright HP Ingredients. All Rights Reserved. All content on this document is the exclusive property of HP Ingredients and is protected by U.S. and international copyright laws. The content on this document may be used as a resource for the sale of ParActin only. Any other use, including the reproduction, modification, distribution, transmission, or republication, of the content on this document for other products is strictly prohibited.