Stimuno Mechanism of Action

Introduction: Stimuno is made from extract of Phyllanthus niruri L. In some region in Indonesia, this plant is known as meniran. Since 2000 years ago, Phyllanthus niruri L. has been widely used as herbs in various countries in the world, one of them is 'Ayurvedic Medicine/Ayurveda' in India. The use of this plant is very broad, eg, for treating hepatitis, gonorrhea, urinary tract infections, stomachache, toothache, kidney stones, irregular period (menstruation), diabetes, dysentery and also as an antipyretic and diuretic.
The genus of Phyllanthus comprises of some species, 2 of the most common species usually made into herbs are Phyllanthus niruri L. and Phyllanthus urinaria. Phyllanthus niruri L. has pale green stalks, whereas Phyllanthus urinaria has reddish stalks. In some countries, Phyllanthus niruri L. is known as Phyllanthus amarus, Phyllanthus debilis, Phyllanthus fraternus or Phyllanthus rotundifolius.
The activity of the plant on immune system has been tested in several preclinical studies. Further preclinical studies in rats and mice were conducted to determine the safety and immunomodulatory characteristics of Phyllanthus niruri L. The results of the studies showed that the extract of Phyllantus niruri L. can modulate the immune system through proliferation and activation of T and B lymphocytes, secretion of specific cytokines eg, interferon-γ, TNF-α (tumor necrosis factor-α), interleukin and activation of complement system, phagocytes eg, macrophages and monocytes. In addition, the cytotoxic activity of natural killer (NK) cells is increased. Furthermore, several clinical studies have been conducted to assess immunomodulatory effects in some cases with certain conditions, eg, pulmonary TB (tuberculosis), hepatitis B, upper respiratory tract infections, herpes zoster, acute exacerbation of chronic obstructive pulmonary diseases (COPD) and vaginal candidiasis. From preclinical and clinical studies similar results were found in immunological parameter, which is P. niruri L. extract in Stimuno acts as immunomodulator, to strengthen the body immune system and increase success rate in infection therapy.
Stimuno is the only immunomodulator with phytopharmaca certificate from Indonesian National Agency for Drug and Food Control. Phytopharmaca certificate is given only to herbal products, containing standardized extract, with efficacy proven by clinical trial, hence it shows comparable efficacy to synthetic drugs. Stimuno got the phytopharmaca certificate after going through 4 stages of trial, ie, 1st stage, preclinical trial to study the safety (ie, toxicity studies) and efficacy (ie, pharmacodynamic studies) in animals; 2nd stage, simple standardization of raw material; 3rd stage, pharmaceutical standardization to ensure the identity and standardization of finished goods; 4th stage, clinical trials in both healthy volunteers and patients. In conclusion, as phytopharmaca product, Stimuno is certified for its quality, safety and efficacy.
Stimuno has been marketed since 1999 as prescribed product in Indonesia. Stimuno received good responses from doctors for its quality and safety. Since August 1st, 2005, Stimuno was switched to over-the-counter (OTC) allowing customers to buy from outlets without prescriptions. This new approach is welcome by customers as they have been aware on the efficacy and safety of the product since the prescription period.
Pharmacology: Pharmacodynamics: Mechanism of Action: Stimuno activates both specific and non-specific immune system, and also both cellular and humoral immune system. (See Table 1.)

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Intracellular Bacterial Infection: Stimuno Acts On: Specific-Intracellular Bacteria: Stimuno increases the proliferation of lymphocytes, and convert it into T_h /CD₄₊, T_c /CD₈₊, T_s and T_dth, resulting in faster elimination of intracellular bacteria by T_h and T_c to prevent from any bacterial attachment to other cells and inhibit bacterial reproduction in the cells. T_h can activate macrophages that produce IFN-γ and cause bacterial lysis in the phagosomes. T_c plays a role in destroying the infected cells.
Nonspecific-Intracellular Bacteria: Phagocytes and NK cells are effectors which play a role in nonspecific im­mune system toward the intracellular bacterial infections. Stimuno increases the cytotoxici­ty of NK cells, hence, the infected cells are easier to destroy. In addition, Stimuno also plays a part in increasing monocytes/macrophages phagocytic activity, therefore, facilitates the pro­cess of bacterial lysis in phagosomes. Nevertheless, some of the intracellular bacteria are re­sistant to the degradation by the phagocytes. Macrophages will release IL-12 which produce and release IFN-γ from NK cells. IFN-γ will further activate macrophages to eliminate the phagocytized bacteria.
Extracellular Bacterial Infection: Stimuno Activates: Specific immune system: Increase B lymphocyte proliferation, hence, shortened the duration of antibody produc­tion, facilitate and accelerate the speed of neutralization and elimination of bacteria and its toxin by increasing antibody production, enhance T lymphocyte (T-cell) proliferation, thus, enhance T-cell differentiation into T_h , T_dth , T_c and T_s cells, increase production of the antibody IgE due to enhanced IL-4 secretion, decrease IL-10 secretion. Therefore, macrophage which works on non-specific im­mune system and produce IL-12 is not impeded.
Nonspecific Immune System: Facilitate and accelerate the speed of bacteria elimination by increasing macrophage/monocyte non-specific phagocytic ability, facilitate elimination of infectious bacteria by accelerating inflammation reaction through increment of chemotactic of neutrophil and macrophage activity. (See Figure 1.)
Virus: Stimuno Activates: Specific Immune System: Enhance T lymphocyte proliferation (T-cell), allowing T cell to differentiate into T_h , T_c , T_dth and T_s . T_h plays a role in activating B-cell proliferation and T_c has the ability to destroy virus infected cells, increased B lymphocyte proliferation will accelerate antibody production, prevent the occurrences of virus infection and destroy virus through opsonization, phagocytosis, and complement system activation by increasing antibody production.
Nonspecific Immune System: Increase NK cells cytotoxicity thus improve its ability to destroy infected cells, decrease the time needed to destroy virus by increasing complement system activity.

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Parasite: Stimuno Activates: Specific Immune System: Stimuno will increase IgE production by enhancing secretion of IL-4 from T_h2 subset. IgE will be bound to the parasites by eosinophyl (which is produced by IL-5), then secrete granulated enzyme that destroys the parasites.
Non-specific Immune System: Destroy parasites through enhancement of phagocytic activity of the immune system.
Tumor/Cancer: Stimuno Activates: Specific Immune System: Enhance T-cell proliferation, allowing T-cell to differentiate into T_h (T-cell, CD₄) and T_c (T-cell, CD₈) which are able to destroy tumor/cancer cells through activating various immune system components and direct destruction of tumor/cancer cells, increased B lymphocyte proliferation will accelerate antibody anticancer production that will act as a mediator for tumor/cancer cell lysis through 2 mechanisms: 1) complement-fixing antibodies complex will bind to tumor cell membrane, this will facilitate complement component to bind and create tiny holes on the tumor cell membrane. In addition, tumor/cancer cell will disrupt due to the loss of osmotic and biochemical integrity of the cell. 2) In the antibody-dependent cell-mediated cytotoxicity (ADCC), antibodies, especially IgG, creates an intercellular bridge through binding antibody with antigen specific determinant and through binding F_c with effector cell that express F_c receptor.
Non-specific Immune System: Increase macrophage activity. Active macrophages can bind to tumor cells and destroy tumor cells through an unknown mechanism. This unknown mechanism might resemble the mechanism of pathogen microorganism destruction by macrophages. This mechanism consists of lysosomal enzymes liberation, reactive oxygen intermediate, and nitrite-oxide. Activated macrophages also produce cytokine TNF which are able to kill tumor cells by inducing thrombosis in tumor cell blood capillaries. Increases complement activity that will accelerate cancer cell destruction through complement-mediated cytotoxicity. Increase NK cell cytotoxicity, facilitate cancer cells destruction. Besides, NK cell also bridged tumor cell lysis through ADCC mechanism, where NK cell is an effector cell that binds to F_c region of the antibody IgG. Tumoricidal capacity of NK cell is increased by various cytokines eg, interferon and interleukins (IL-2 and IL-12). Natural killer (NK) cells also release proteinase and perforin, creating pores on target cell membrane, which finally resulting in target cell lysis.
Strong Features of Stimuno: The only immunomodulator with phytopharmaca standard, ensuring the quality, efficacy and safety of Stimuno through standardization as well as preclinical and clinical studies, activating both specific and nonspecific, and both humoral and cellular immune system, reducing IL-10 in the specific immune system so that macrophage activity and lymphocyte T are not inhibited, safe for long-term usage (proven through clinical trial on tuberculosis patients for 6 months) and can also be consumed altogether with other medicines or vitamins without any clinically significant adverse effects, Stimuno has been prescribed regularly and widely used by physicians for the last 5 years in Indonesia.
Summary: Stimuno, containing Phyllanthus niruri L. extract, works as immunomodulator with the ability to enhance body immune system by activating all components of the immune system, both specific and nonspecific; cellular and humoral. (See Figure 2.)

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Stimuno is able to improve the production of immune markers so it could be used to prevent infection. Long-term usage of Stimuno has been proven to be safe (result from a clinical trial on patients with pulmonary TB for 6 months). It can also be used concomitantly with other drugs or vitamin without side effects. Stimuno works synergistically with antimicrobe, as it increased the successful infection-therapy rate so that we could expect shorter healing process, prevention from re-occurrence of the disease, and finally medical cost reduction.
Preclinical Trials: Preclinical trials were performed to test the effects of Phyllanthus niruri L. use. Through studies, some experts found that the extract of Phyllanthus niruri L. herb could reduce the titre of HBV in infected ducks. The immunological test was performed by using adult male and female BALB/c mice. Those mice were divided into 2 groups. Treatment group was given the extract of Phyllanthus niruri L. extract and control group that was given distilled water. The objective of this test is to study the difference of immunocompetent cells functions and activity of among those 2 groups of mice after oral administration of Phyllanthus niruri L. extract. Several variables were measured using unpaired t-test statistical method. (See Table 2.)

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The effect of Phyllanthus niruri L. extract administration toward phagocytic activity of monocytes/macrophages was analyzed using analysis of variance (ANOVA) of carbon clearance test result. The result showed that oral administration of Phyllanthus niruri L. extract in mice could increase the phagocytic activity of macrophages. Based on preclinical trials, it can be concluded that: The oral administration of Phyllanthus niruri L. extract could increase the activity of innate/nonspecific immune response by: Increasing the nonspecific phagocytic activity of macrophages/monocytes, increasing the inflammation reactions through enhancing chemotactic activity of neutrophils and macrophages, increasing the cytotoxicity of NK cells. The oral administration of Phyllanthus niruri L. extract could increase the activity of acquired/specific immune response by: Stimulating B cells proliferation, increasing IgG and IgM production, increasing T cells proliferation, increasing TNF-α by T_h1 subset secretion, decreasing IL-2 by T_h1 subset secretion, increasing IL-4 by T_h2 subset secretion, decreasing IL-10 by T_h2 subset secretion. Functions and activities of immune system components which are not affected by oral administration of Phyllanthus niruri L are: Cytotoxic T-cells (T_c /CD₈₊) activity against target cells TG-PEC, TNF-α secretion by monocytes that was stimulated by LPS. From statistical analysis of all the test results, it can be concluded that Phyllanthus niruri L. extract has immunomodulator activity.
Clinical Trial: Clinical studies aimed to examine the efficacy of Phyllanthus niruri L. in patients with certain conditions or diseases were conducted in accordance with the Good Clinical Practice (GCP) guidelines. (See Table 3.)

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Pulmonary Tuberculosis (TB): Several clinical trials have been conducted by experts to study the efficacy of Stimuno in pulmonary TB patients. Efficacy parameters measured were improvement in clinical condition (sputum-acid-fast-bacilli conversion), radiology and immunology (as measured by the plasma levels of TNF-α, IFN-γ IL-10, CD₄ and CD₈).
Sputum Acid-Fast-Bacilli (AFB) Conversion: A double-blind, randomized and placebo-controlled clinical trial with 6 months therapy was conducted involving 67 pulmonary TB patients (33 patients in control group received standard anti-TB drugs (rifampicin, isoniazid, ethambutol, pyrazinamide) and 34 patients in Stimuno group received standard anti-TB drugs plus Stimuno). Results from the study showed that after 1 week of treatment, the proportion of patients who had sputum conversion in Stimuno group (52%) was greater than that of placebo group (39%), p=0.172. Although the difference was not statistically significant, this indicated a better trend toward Stimuno and suggested an important clinical impact particularly at the community level, ie, patients with sputum conversion would not be the source of pulmonary TB infection to their environment. Hence, the more patients who have sputum conversion, less risk of TB transmission to the environment. This result is of great importance with regard to the improvement of community health. (See Figure 3.)

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A favorable trend toward Stimuno was also seen in another double-blind, randomized and placebo-controlled clinical trial. The trial enrolled 40 pulmonary TB patients, divided into 2 groups: Stimuno group who received standard anti-TB drugs plus Stimuno and control group who received the standard anti-TB drugs only (rifampicin 450 mg, isoniazid 300 mg, ethambutol 750 mg, pyrazinamide 1500 mg). After 2 months of therapy, patients in both groups had their sputum-acid-fast-bacilli converted (became negative). At baseline, the severity grade of sputum acid-fast-bacilli (AFB) test in Stimuno group was higher than the control group, however. This means that Stimuno administration benefited even the pulmonary TB patients with more severe grade of sputum-AFB, as their sputum conversion occurred as fast as those with less severe grade of sputum-AFB.
Radiological Improvement (Thorax X-ray): Beside beneficial to the sputum conversion, the study also showed that Stimuno supplementation in addition to standard anti-TB drugs provided a better improvement in radiology (thorax x-ray) than the standard drugs alone. (See Table 4.)

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Immunological Improvement: IFN-γ Level: In their study, Amin Z et al showed that after 6 months of therapy, IFN-γ levels in Stimuno group consistently increased, while in placebo group decreased. The result indicated the positive effect of Stimuno in TB therapy. Interferon-γ produced by NK cells and T lymphocytes will inhibit Mycobacterium growth through the increase of macrophage activity. The elevated level of IFN-γ is in line with the concept that a gradual improvement in immunological status in TB patients is required to fight against Mycobacterium tuberculosis. The success of TB therapy is determined largely by the increased level of IFN-γ. (See Figure 4.)

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The immunological effect of Stimuno on IFN-γ level was also corroborated in another study involving 40 pulmonary TB patients with positive sputum-AFB test. Patients were randomly and blindly allocated into 2 groups. Twenty (20) patients in Stimuno group received standard anti-TB drugs and Stimuno 50 mg thrice daily and the other 20 patients in control group received the standard anti-TB drugs and placebo. After 2 months of therapy, the IFN-γ levels in Stimuno group (7.65 pg/mL), p<0.01) increased significantly than those of placebo group (0.41 pg/mL, p=0.261), as compared to their respective baseline. (See Figure 5.)

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Based on the results of the studies, Stimuno was proven to have an immunomodulatory effect through elevating the IFN-γ level, a cytokine which biologically affect the activity of macrophage and natural immunity, promote the antigen-presenting process, phenotyping development of T-helper cells and humoral immunity. In order to optimize and increase successful rate of TB therapy, Stimuno may be used synergistically with anti-TB drugs. TNF-α Level: The effect of Stimuno on TNF-α in pulmonary TB patients was also observed in the Amin Z, et al study. The synergistic action between TNF-α and IFN-γ in Mycobacterium eradication and Nitric Oxide (NO) production is highly important in granuloma formation which limits the spread of bacterial infection throughout the body. On the other hand, TNF-α also has immuno-pathological effects which may be manifested as fever, lowered body weight, and tissue necrosis. In Stimuno group, the insignificant decrease of TNF-α level after 2 months of therapy which was then followed by its elevation at the 6th month, indicated the occurrence of immunological response which positively continued toward a repairing process of the infected tissues. (See Figure 6.)

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IL-10 Level: There was also a clinical study to observe the effect of Stimuno on IL-10 level. In the double-blind, randomized and placebo-controlled study, 40 pulmonary TB patients with positive sputum-AFB test were divided into 2 group: 20 patients in Stimuno group received standard TB drug (rifampicin, isoniazid, ethambutol, pyrazinamide) and Stimuno, and the other 20 patients in control group received only the standard anti-TB drugs. After 6 months of therapy, the increment of IL-10 secretion in Stimuno group was insignificant as compared to baseline level, while a significant increase was seen in control group. This suggested that the administration of Stimuno might suppress IL-10 secretion by T_h2 subset. The immunological effects of IL-10 include: Inhibition of macrophage activity in producing cytokines eg, TNF-α, IL-1, chemokines and IL-12, inhibition of accessorial function of macrophages in T-cell activation induced by the suppressed expression of class II-MHC and several co-stimulators eg, B7.
When the secretion of IL-10 was suppressed by Stimuno, attenuation of humoral immune response would not occur, as there was no inhibition on the expression of class II-MHC and its co-stimulators, thus optimizing the action of T_h lymphocytes (CD₄). Furthermore, the inflammatory reaction necessary for the microbial eradication could still proceed. Decreased IL-10 level also means macrophage activity in producing cytokine to eliminate foreign objects is not inhibited.
CD₄ and CD₈ Level: Another double-blind, randomized and placebo-controlled clinical study to examine Stimuno effect on CD₄ and CD₈ levels in TB patients has also been completed. Forty pulmonary TB patients enrolled in the study were divided into 2 groups, Stimuno group who received standard TB drug plus Stimuno (3 x 1 capsule daily) and control group who received only the standard anti-TB drugs. After 4 weeks of therapy, increment of CD4 T- lymphocytes as well as the ratio of CD₄/CD₈ in Stimuno group (10.7 cell/mm³ and 0.32, respectively) were significantly greater (p=0.001 and p=0.01, respectively) than those of control group (4.45 cell/mm³ and 0.05, respectively). (See Figures 7 and 8.)

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Upper Respiratory Infection in Children: A clinical study involving 40 pediatric patients (aged 2-15 years, had fever for no more than 2 days at screening) was conducted to investigate the effectiveness of Stimuno in accelerating the healing process and clinical symptoms improvement in treating upper respiratory tract infection. Eligible patients were allocated at random into 2 groups. The treatment group received Stimuno while the control received placebo. After 7 days of therapy, the result is presented on Figure 9 (see Figure 9).

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The data indicatively showed that the administration of Stimuno for pediatric patients with upper respiratory tract infections could accelerate patients' recovery from fever.
Chronic Hepatitis B Infection: A randomized, double-blind preliminary study was conducted to investigate the efficacy of Stimuno against chronic hepatitis B infection, particularly in the reduction of HBV DNA levels. Forty patients with elevated ALT were recruited in the study. Eligible patients were divided into 2 groups, treatment group received Stimuno and control group received placebo (vitamin B complex), which were given 2 capsules twice daily for 3 months. Of those 40 patients with elevated ALT, only 37 patients could be evaluated. At baseline, most patients had had elevated ALT <2 times upper normal limit. Means of HBV DNA baseline levels in Phyllanthus and placebo groups were 8.40±0.64 log₁₀ copies/mL and 7.69±1.30 log₁₀ copies/mL, respectively. Sixty five percent (65%) (11 of 17) patients in the Phyllanthi group showed that their serum HBV DNA decreased by as much as 1.22±1.86 (Log-order) while 65% (13 of 20) patients in the placebo group decreased by only 0.69±0.80 (log-order). Thus, the levels of reduction in the Phyllanthi group in serum HBV DNA were observed to be twice as much when compared to those in the placebo. The remaining 35% (6 of 17) of patients in the Phyllanthi group showed their serum HBV DNA slightly increased (by only 0.08±0.05 log 10 copies/mL), while 35% (7 of 20) of patients in placebo group increased by 0.41±0.50 log 10 copies/mL. In this study, Phyllanthi extract alone was found to suppress the levels of HBV DNA. This result suggests that a synergistic combination of an antiviral agent and Phyllanthus niruri L. extract can be implemented in the management of chronic hepatitis B infection).
Herpes Zoster: A randomized, double-blind and placebo-controlled clinical study was conducted to investigate the effectiveness and safety of Phyllanthi herb extract combined with standard therapy compared to standard therapy alone in patients with uncomplicated herpes zoster. Sixty (60) uncomplicated herpes zoster patients were allocated to receive treatment with either oral acyclovir combined with Stimuno (Stimuno group) or oral acyclovir alone (Control group). A daily dose of 5 x 800 mg acyclovir for 5 days combined with either Phyllanthi herb extract (3x50 mg daily) or placebo for 6 days were administered. Effectiveness and safety evaluation of the treatment was performed at day 4, 8 and 15. The study showed that at day 8 after therapy, patients' responses in Stimuno group, as measured by the presence of vesicles, area of lesions, and cicatrix severity, were better than those in Placebo group. Postherpetic neuralgia evaluation showed that of 10 patients aged ≥40 years (4 patients in Stimuno group and 6 patients in Placebo group), only 1 patient in each group experienced such complication. Through the study, Stimuno was proven to be effective and safe as combined with acyclovir in enhancing treatment response (accelerating the healing process, particularly in reducing the scar severity and the size of lesions) in varicella-zoster virus infection. If the size of lesions was reduced, it would also prevent the occurrence of postherpetic neuralgia.
Chronic Obstructive Pulmonary Disease: In order to investigate the effectiveness of Stimuno in accelerating clinical as well as laboratorial improvement in patients with acute exacerbation of COPD due to bacterial infection, a randomized, double-blind clinical study has been conducted. As much as 70 patients were recruited, divided into 2 groups, treatment group received ciprofloxacin with Stimuno and control group received ciprofloxacin and placebo. All 35 patients in each group were measured for the C₃, IgG, IgM levels and of them, 15 patients in each group were measured for superoxide dismutase (SOD) levels before and after a 2-week treatment. Means of SOD and C₃ levels in the treatment group were higher than those of the control group. The high SOD level might quell the free radicals produced by the inflammation process due to bacterial infection. The high C₃ level was needed to accelerate improvement of inflammation process. IgG and IgM levels in both groups were reduced. These unexpected results occurred since both IgG and IgM levels should have been measured when the process of improvement had been completed; in this case, it takes >90 days for COPD to become stable.
Vaginal Candidiasis: A randomized, double-blind, and controlled study was conducted to examine efficacy in candidiasis. Thirty (30) eligible patients (15 patients in each group), who were married women, infected with vaginal candidiasis, were enrolled in the study. They were allocated at random to either control group or Stimuno group. Both groups received ketoconazole 200 mg twice a day for 5 days, and either Stimuno or its matching placebo (2 caps 3 times a day) for 7 days. The efficacy parameters were clinical signs, IFN-γ and IL-12 levels in vaginal secretes. The results showed that there was a significant difference in IFN-γ levels between control and Stimuno groups. IFN-γ levels in vaginal secretes in control group before and after 7 day of treatment were not significantly different, but IFN-γ levels after month 1 and month 3 of treatment were significantly lower than baseline level. While in Stimuno group, the IFN-γ levels at day-7, month-1, and month-3 were all significantly higher as compared to baseline. Elevated IFN-γ level indicates an increased activity of cellular immune response (T_h1 subset) which is concurrently suppressing the secretion of IL-4 and IL-10 (by T_h2 subset) and activating the macrophages eradicate the candida from the vaginal area. Besides, the IL-12 levels in Phyllanthi group at day 7, month 1 and month 3 were also significantly higher than baseline level, though they were not significantly different as compared with the match values of the control group. In specific cellular immunity, IL-12 plays a role to stimulate IFN-γ production by NK- and T-cells as well as to promote the NK cell cytotoxicity. (See Figure 10.)

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The other efficacy parameters measured in the study were the recovery and recurrence rates. Recovery rate in Stimuno group was higher (73.33%) than in control group (26.67%). Furthermore, the recurrence rates in Stimuno group in the 1st and 3rd month after stopping treatment (18.2% and 45.5%, respectively) were significantly lower than those in control group (50% and 100%, respectively). (See Table 5.)

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Throughout this study, Stimuno was proven to be effective as an immunomodulator and in increasing successful rate of vaginal candidiasis therapy with antifungal agent, as well as accelerating the recovery.
Phyllanthus niruri L. Standardization: Phyllanthus niruri L. grows in tropical region at around 1-1000 m above sea level. It grows on open spaces, sandy porous lands, fields, river-sides, and beaches. The raw material of Stimuno is taken from organic cultivation of Phyllanthus niruri, standardized at every process from planting, harvesting, post-harvesting, to production. The cultivation is located in Banyuwangi region (312 m above sea level) in compliance to Good Agricultural & Collection Practices (GACP). Stimuno is made from all parts of the Phyllanthus niruri L. adhering to Good Laboratory Practice (GLP) standard, and Current Good Manufacturing Practice (CGMP).
Extract Standardization: The extract is made of Phyllanthus niruri L. herb through 56% alcohol maceration.
Description: Dark brown powder dry extract, tastes bitter with specific scent. Phytochemical Standard: Dry extract with ≤5% concentration contains flavonoid no <0.25% (Switzerland Pharmacopoeia Method).
Finished Product Standardization: Each capsule (equivalent to 50 mg Phyllanthus niruri extract) contains total flavonoid of no less than 0.02 mg. Each 5 mL syrup (equivalent to 25 mg Phyllanthus niruri extract) contains total flavonoid total of no <0.01 mg.
Biological Standardization: Lymphocyte T proliferation index increase using: BALB/c line mice with 20-25 g weight. Mitogen Concanavalin-A. Criteria: Lymphocyte T proliferation index increase is <1.2 times. IgM increase Agglutination method using: BALB/c strain mice with 20-25 g weight. Mitogen Concanavalin-A, sheep RBC antigen. Criteria: The IgM synthesis increase is no <5 times.
Toxicology: Preclinical Safety Trial: Acute Toxicity: Phyllanthus niruri L. extract is classified as PNT (Practically Non-Toxic), because organ disorders and death of 50% animal trial population only occurred after oral administration of Phyllanthus niruri L. extract with dose of 14 g/kg body weight for 24 hrs in BALB/c strain mice.
Subchronic Toxicity: Oral administration of Phyllanthus niruri L. extract with dose of 2, 3, 4, 5 g/kg body weight/day for 12 weeks in Wistar rats showed none had pathological and histopathological abnormality in pulmonary, liver, lymph, intestine and muscle.
Chronic Toxicity: Oral administration of Phyllanthus niruri L. extract on Sprague-Dawley strain rats with dose 66.95, 267.8, and 1071.2 mg/kg body weight/day for 6 months does not interfere behavior, motor activity, hepatic function, renal function, blood biochemical, body weight, organ index and also macroscopic and histopathology of some organs.
Teratogenicity: Teratogenicity test was done on Wistar rats at 3 dose-ranging regimens ie, lowest dose (equivalent to usual dose in human), mid-dose (equivalent to the dose of 4 times usual dose in human) and the highest dose (equivalent to the dose of 16 times usual dose in human). In control group, all fetuses were alive. In the lowest dose group, mean number of living fetuses per animal was comparable to that of the control group, but with 6.25% resorption observed. Increasing dose was not accompanied by increased resorption (at the mid-dose, there was 3.23% resorption observed on the fetuses, and at the highest dose, 1.35%). The result showed that at the lowest dose (66.95 g/kg BW in rats or equivalent to the usual dose in human), there was no tissue abnormality observed on the tested fetuses. However, at the highest dose, formation of aberrant tissues on the face (forehead section), hydrocephalus and no covering tissue formation at the abdomen were found, each on different fetuses of different forebears. Even though Phyllanthus niruri L. extract is safe at human usual dose, but in the light of the teratogenicity trial result, the extract should be used with caution in pregnant women considering that at the high dose, the extract risked the fetus.
Clinical Safety Trial: Based on clinical trial, long-term use (6 months) in pulmonary TB patients do not show clinically significant side effect.
In 1 study, after oral administration of Phyllanthus niruri L. with dose of 2 capsules twice daily for 12 weeks, showed that Phyllanthus niruri L. extract was well tolerated and safe for chronic hepatitis B patients, with an incident of adverse events similar to placebo. No serious adverse events occur during the study. The majority of adverse events were mild. The most common adverse events were headache, fatigue, cold, hyperurinaria and loss of appetite. Of them, only hyperurinaria and loss of appetite were possibly related to Phyllanthus treatment. At the end of the study, all the adverse events were already resolved.
After oral administration of Phyllanthus niruri L. extract with dose of 3x50 mg/day for 2 weeks, it showed that Phyllanthus niruri L. extract was well tolerated and safe for COPD patients.
A clinical trial was conducted to investigate the effectiveness and safety of Phyllanthus niruri L. extract in combination with standard therapy for patients with uncomplicated herpes zoster. After oral administration of Phyllanthus niruri L. extract with dose of 50 mg for 15 days, it showed that adverse event were not significantly different between 2 groups. The most common adverse events were diuresis and hypotension.
Based on clinical trial which conducted to investigate the role of Phyllanthus niruri L extract in the treatment of upper respiratory tract infection in pediatric patients, it showed that Phyllanthus niruri L. extract was well-tolerated and safe. This study was conducted for 7 days.
A clinical trial was completed to compare the effectiveness of ketoconazole alone with ketoconazole combined with Phyllanthus niruri L. extract in the treatment of vaginal candidiasis. After oral administration of Phyllanthus niruri L. extract with dose of 2 capsules (100 mg) 3 times a day for 7 days, it showed that Phyllanthus niruri L. extract was well-tolerated and safe for vaginal candidiasis patients.
The study was conducted to investigate the role of Phyllanthy herba as an adjuvant therapy to some tropical skin disease eg, varicella-zoster infection and leprosy. The study was conducted from May 2000 until March 2002 in Manado. After oral administration of Phyllanthus niruri L. extract with dose of 3x50 mg/day, no patients perform any adverse reactions.
Overview on Immune System: The cells and molecules responsible for immunity constitute the immune system, and their collective and coordinated response to the introduction of foreign substances is called the immune response. The physiologic function of the immune system is defense against infectious microbes. Immunity is a reaction to foreign substances, including microbes, as well as to macromolecules eg, proteins and polysaccharides, regardless of the physiologic or pathologic consequence of such a reaction.
Innate and Adaptive Immunity: Defense against microbes is mediated by the early reactions of innate immunity and the later response of adaptive immunity. Innate immunity (natural or native immunity) consists of cellular or biochemical defense mechanisms that are in place even before infection and poised to respond rapidly to infections. These mechanisms react only to microbes and not to noninfectious substances and they respond in essentially the same way to repeated infections. The principal components of innate immunity are: Physical and chemical barriers eg, epithelia and antimicrobial substances produced at epithelial surfaces, phagocytic cells (neutrophils, macrophages) and NK cells. Blood proteins, including members of the complement system and other mediators of inflammation, proteins called cytokines that regulate and coordinate many of the activities of the cells of innate immunity.
Innate immunity provides the early lines of defense against microbes. In contrast to innate immunity, there are other immune responses that are stimulated by exposure to infectious agents and increase in magnitude and defensive capabilities with each successive exposure to a particular microbe. Because this form of immunity develops as a response to infection and adapts to the infection, it is called adaptive immunity. The defining characteristics of adaptive immunity are exquisite specificity for distinct molecules and an ability to "remember" and respond more vigorously to repeated exposures to the same microbe. The adaptive immune system is able to recognize and react to a large number of microbial and nonmicrobial substances. In addition, it has an extraordinary capacity to distinguish among different, even closely related, microbes and molecules, and for this reason, it is also called specific immunity. It is also sometimes called acquired immunity, to emphasize that potent protective responses are "acquired" by experience. The components of adaptive immunity are lymphocytes and their products. Foreign substances that induce specific immune responses or are the targets of such responses are called antigens.
Innate and adaptive immune responses are components of an integrated system of host defense in which numerous cells and molecules function cooperatively. The mechanisms of innate immunity provide effective defense against infections. However, many pathogenic microbes have evolved to resist innate immunity, and their elimination requires the powerful mechanisms to adaptive immunity. There are 2 important links between innate immunity and adaptive immunity. First, the innate immune response to microbes stimulates adaptive immune responses and influences the nature of the adaptive responses. Second, adaptive immune responses use many of the effector mechanisms of innate immunity to eliminate microbes, and they often function by enhancing the antimicrobial activities of the defense mechanisms of innate immunity.
Type of Adaptive Immune Responses: There are 2 types of adaptive immune responses, called humoral immunity and cell-mediated immunity, that are mediated by different components of the immune system and function to eliminate different types of microbes. Humoral immunity is mediated by molecules in the blood and mucosal secretions, called antibodies, that are produced γ-cells called B lymphocytes (B-cells). Antibodies recognize microbial antigens, neutralize the infectivity of the microbes, and target microbes for elimination by various effector mechanism. Humoral immunity is the principal defense mechanism against extracellular microbes and their toxins because secreted antibodies can bind to these microbes and toxins and assist in their elimination.
Cell-mediated immunity, also called cellular immunity, is mediated by T lymphocytes (T-cells). Intracellular microbes eg, viruses and some bacteria, survive and proliferate inside phagocytes and other host cells, where they are inaccessible to circulating antibodies. Defense against such infections is a function of cell-mediated immunity, which promotes the destruction of microbes residing in phagocytes or the killing of infected cells to eliminate reservoirs of infections.