|Year : 2018 | Volume
| Issue : 1 | Page : 10-15
The Potency of Nanoparticle of Pinus merkusii as Immunostimulatory on Male Wistar Albino Rat
Sri Agus Sudjarwo1, Giftania Wardani2, Koerniasari Eraiko3, Koerniasari4
1 Department of Pharmacology, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia
2 Department of Pharmacy Biology, Faculty of Pharmacy, Hang Tuah University, Surabaya, Indonesia
3 Department of Conservative Dentistry, Faculty of Dentistry, Airlangga University, Surabaya, Indonesia
4 Study Program of Environmental Health, Polytechnic of Health, Surabaya, Indonesia
|Date of Web Publication||15-Jan-2018|
Sri Agus Sudjarwo
Department of Pharmacology, Faculty of Veterinary Medicine, Airlangga University, Surabaya - 60115
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Medicinal herbs are the commonly used worldwide immunomodulators in the management of various disease conditions. The aim of this study was to evaluate the immunostimulatory activity of the nanoparticle extract of Pinus merkusii in Wistar albino rats. Materials and Methods: It was an experimental study that was conducted on various groups of animals each with six healthy adult rats. Neutrophil adhesion test, hemagglutinating antibody (HA) titer, delayed-type hypersensitivity (DTH) response, phagocytic activity, and cyclophosphamide-induced myelosuppression were determined in various groups of animals. Results: The nanoparticle of extract P. merkusii at doses 500 mg/kg BW but not at doses 125 mg/kg BW and 250 mg/kg BW induced a significant increase in percent neutrophil adhesion fibers as well as a dose-dependent increase in antibody titer values and potentiated the DTH reaction induced by sheep red blood cells. Also, it prevented myelosuppression in cyclophosphamide drug-treated rats and good response toward phagocytosis in carbon clearance assay. Conclusion: From these findings, it can be concluded that nanoparticle extract of P. merkusii possesses immunostimulatory activity and has therapeutic potential for the prevention of immune-depressed conditions.
Keywords: Immunostimulatory, nanoparticle, Pinus merkusii
|How to cite this article:|
Sudjarwo SA, Wardani G, Eraiko K, Koerniasari. The Potency of Nanoparticle of Pinus merkusii as Immunostimulatory on Male Wistar Albino Rat. Int J Nutr Pharmacol Neurol Dis 2018;8:10-5
|How to cite this URL:|
Sudjarwo SA, Wardani G, Eraiko K, Koerniasari. The Potency of Nanoparticle of Pinus merkusii as Immunostimulatory on Male Wistar Albino Rat. Int J Nutr Pharmacol Neurol Dis [serial online] 2018 [cited 2020 Feb 28];8:10-5. Available from: http://www.ijnpnd.com/text.asp?2018/8/1/10/223265
| Introduction|| |
Immunomodulatory is a substance which suppresses or stimulates the components of immune system including both innate and adaptive the immune responses. It has been reported that immunomodulation of the immune response could provide an alternative to conventional chemotherapy for a variety of disease conditions, especially when impaired immune responsiveness of the host’s in under conditions or when a selective immunosuppressant has to be induced in a situation like autoimmune disorders and organ transplantation.,
The immunostimulation of the immune system of medicinal plant products has become subject to scientific investigations currently worldwide and their active components provide a potential alternative to conventional immunotherapy for a variety of immunologic diseases. In this context, the development of medicinal plant product-based drug candidates as immunostimulatory has gained momentum in research studies directed toward design and discovery of drugs., Medicinal plant products have long been used as immunostimulatory in traditional medicines, for the treatment of many immunological disorders. Andrographis paniculata, Allium sativum, Cajanus indicus, Gymnema sylvestre, Asparagus racemosus, Piper longum Linn., Curcuma longa, Phyllanthus emblica Linn., Ocimum sanctum Linn., Tinospora cordifolia, and Pinus radiata are among the medicinal plants claimed to possess potential immunostimulatory agent.,
It has been demonstrated that the medicinal properties of Pinus plant were due to the phytochemicals possessed, including alkaloids, polyphenols, flavonoids, lignans, triterpenes, sterols, glycosides, triterpenoids, and saponins., Recent research activities have shown that Pinus plant is an important source of pycnogenol that contains proanthocyanidins (procyanidins).,, Proanthocyanidins are potent, free radical scavengers, antibacterial agents, exhibit vasodilatory, antiallergic, antiinflammatory, cardioprotective, immune-stimulating, antiviral, and estrogenic activities.,
In recent years, synthesis of nanoparticles is an interesting issue of the nanoscience and nanobiotechnology. There is a growing attention to biosynthesis the nanoparticles using herbal. Among these organisms, plants seem to be the best candidate, and they are suitable for large-scale biosynthesis of nanoparticles. Nanoparticles produced by plants are more stable, and the rate of synthesis is faster than that in the case of other organisms. Moreover, the nanoparticles vary greatly in shape and size in comparison with those produced by other organisms., Pinus plant nanoparticles have drawn the attention of researchers because of their suitable applications in the fields of material science and medicine. The objective of the present study was to evaluate the immunostimulatory activity of the nanoparticle extract of Pinus merkusii in Wistar albino rats.
| Materials and Methods|| |
Male Wistar albino rat weighing approximately 200–250 g (2.5–3 months) were obtained from Gadjah Mada University, Yogyakarta, Indonesia for experimental purpose. They were housed in plastic cages in an air-conditioned room with a temperature maintained at 26 ± 2°C and 12 h alternates light and dark cycles. The rats were given ad libitum with tap water and fed with standard commercial rat chow. This study was reviewed by the Ethical Clearance Committee for preclinical research, Institute of Tropical Disease, Airlangga University and obtained ethical clearance under No. 93/ITD/5/2017.
Preparation of ethanol extract of P. merkusii
Plant material and extract preparation of P. merkusii leaf were collected from Surabaya, Indonesia. P. merkusii leaf materials were cleaned with running tap water and chopped into pieces. They were dried under shade at ambient temperature for 5 days, and the air-dried P. merkusii were then ground to powder for extraction. The powdered P. merkusii (1 kg) was macerated with ethanol (5 L) for a week at 37°C. The supernatant was then collected and filtered through Whatman No. 1 filter paper in a Buchner funnel under vacuum. The filtrate was concentrated by evaporation with a vacuum rotary evaporator at 45°C. The extract was dried at reduced pressure, stored at 0–4°C, and used for the experimentation.
Preparation of P. merkusii extract nanoparticle
P. merkusii extract nanoparticles were prepared by ionic gelation of chitosan and sodium tripolyphosphate (TPP) anions. One gram of P. merkusii extract was dissolved in 50 ml Tween 80 solutions and added 100 ml chitosan in glacial acetic acid (0.25% v/v). Three hundred and fifty milliliters of 0.84% w/v TPP was added under magnetic stirring at room temperature in drop wise using 10 ml syringe needle into P. merkusii extract and chitosan solution. The pH of the solution was adjusted by adding 0.1 M NaOH solution to the chitosan P. merkusii complex and stirred for 2 h on a magnetic stirrer. Chitosan P. merkusii complex was centrifuged at 12,000 rpm for another 30 min and decanted., The supernatant was kept and dried in an oven at 40°C overnight for onward usage.
Sheep red blood cells preparation
Fresh blood was collected from a sheep sacrificed in the local slaughter house. Sheep red blood cells (SRBCs) were washed three times in large volumes of pyrogen free 0.9% normal saline and adjusted to a concentration of 0.5 × 109 cells/ml for immunization and challenge.
Neutrophil adhesion test
Joshua et al.’s method was employed for neutrophil adhesion test. Rats of the control group were given 10 ml/kg normal saline, whereas treatment groups were pre-treated with different concentrations of nanoparticle extract of P. merkusii (125; 250 and 500 mg/kg), peroral for 14 days, respectively. On day 14 of nanoparticle extract of P. merkusii treatment, blood samples were collected by puncturing retro-orbital plexus into heparinized vials and analyzed for total leukocyte cell (TLC) and differential leukocyte cell (DLC) counts. After initial counts, blood samples were incubated with nylon fibers for 15 min at 37°C. The incubated blood samples were again analyzed by TLC and DLC, respectively, to give the neutrophil index of blood samples. The percent neutrophil adhesion was calculated by the following formula:
where NIu is the neutrophil index of untreated blood samples, and NIt is the neutrophil index of treated blood samples.
Hemagglutinating antibody titer
Joshua et al. described the procedure for hemagglutinating antibody (HA) titer. The rats of all groups were immunized by injecting 0.1 ml of an SRBCs suspension containing 0.5 × 109 cells intraperitoneally on day 0. The rats, which were divided into control group were given 10 ml/kg normal saline, whereas treatment groups were pre-treated with different concentrations of nanoparticle extract of P. merkusii (125; 250 and 500 mg/kg), peroral for 7 days, respectively. Blood samples were collected from the heart of each rat for serum preparation on the 7th day. The blood samples were centrifuged, and serum was obtained. Antibody levels were determined by the hemagglutination technique. Equal volumes of individual serum samples of each group were pooled. Twofold serial dilutions of pooled serum samples made in 25 μl volume of normal saline in microtitration plates were added to 25 μl of 1% suspension of SRBCs in saline. After mixing, the plates were incubated at 37°C for 1 h and the value of antibody titer was assigned to the highest serum dilution showing visible hemagglutination.
Delayed-type hypersensitivity response
The delayed-type hypersensitivity (DTH) response was determined using the method of Allen. The rats of all the groups were immunized by injecting 0.1 ml of a SRBCs suspension containing 0.5 × 109 cells intraperitoneally on day 0. The rats, which were divided into control group were given 10 ml/kg normal saline, whereas treatment groups were pre-treated with different concentrations of nanoparticle extract of P. merkusii (125; 250 and 500 mg/kg), peroral for 8 days, respectively. On the 7th day of immunization, all the rats were challenged with 0.5 × 109 cells in the left hind foot pad. The right footpad was injected with the same volume of normal saline, which served as the control for nonspecific swelling. Increase in footpad thickness was measured 24 h after the challenge.
Phagocytic response (carbon clearance method)
The method was described by Singh et al. The rats, which were divided into control group were given 10 ml/kg normal saline, whereas treatment groups were pre-treated with different concentrations of nanoparticle extract of P. merkusii (125; 250 and 500 mg/kg), peroral for 7 days, respectively. On the 7th day, immediate after the last dose administered to all the rats of each group control as well as treated received an intravenous injection of carbon suspension (1:50 dilution of Indian ink, Hi-Media Laboratories Pvt. Ltd., Mumbai, India) in a dose of 1 ml/200 g body weight. Blood was withdrawn from the retro orbital venous plexus before injection (0 min) and 15 min after injection of the carbon suspension, and 50 µl of blood was lysed with 4 ml of 0.1% sodium carbonate solution (Na2CO3). The optical density was measured spectrophotometrically at 650 nm wavelength.
The results were expressed as a phagocytic index:
where OD12 min and OD0 min are the optical densities at time t15 min and t0 min, respectively.
The method described by Bin-Hafeez et al. was employed for cyclophosphamide-induced myelosuppression. Albino rats were divided into five groups designated as a negative control; positive control and treatment groups, each group containing six rats. The negative control group received a saline solution. The positive control group was administered with only cyclophosphamide at the dose of 30 mg/kg, i.p. while treatment group rats received cyclophosphamide and varied concentrations of nanoparticle extract of P. merkusii (125; 250 and 500 mg/kg, p.o.). The nanoparticle extract of P. merkusii was given daily for 10 days and cyclophosphamide was injected with cyclophosphamide on the 8th, 9th and 10th day, 1 h after the administration of the respective treatment. Blood samples were collected on the 11th day of the experiment and analyzed for hematological parameters.
Data analyses were performed using the Statistical Package for the Social Sciences version 12.0 software for Windows (SPSS Inc., South Wacker Drive, Chicago, Illinois, USA). All data were expressed as means ± standard deviation (SD) values. The analysis of variance test was used to test for differences between the groups. Duncan’s multiple range test was used to analyze differences between the mean values and differences were considered statistically significant at P < 0.05.
| Results|| |
Effect of nanoparticle extract of P. merkusii on neutrophil adhesion
Incubation of neutrophils with nylon fibers produced a decrease in the neutrophil counts due to adhesion of neutrophils to the fibers. Effect of nanoparticle extract of P. merkusii on neutrophil activation by the neutrophil adhesion test is shown in [Table 1]. The neutrophil adhesion in control group rats was 5.74 ± 1.72. The nanoparticle extract of P. merkusii showed a significant increase in neutrophil adhesion at a dose of 500 mg/kg BW, but not at doses of 125 and 250 mg/kg, when the data were compared with control group rats, suggesting possible immunostimulant action of the nanoparticle extract of P. merkusii [Table 1].
|Table 1: Effect of administration of nanoparticle extract of Pinus merkusii on neutrophil adhesion|
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Effect of nanoparticle extract of P. merkusii on humoral immunity parameters
The hemagglutination antibody titer was used to assess humoral immune response. The humoral antibody titer value of control group was found to be 31.13 ± 3.85. Administration of nanoparticle extract of P. merkusii doses 500 mg/kg but not at doses of 125 and 250 mg/kg produced a significant increase in hemagglutination antibody titer, when the data were compared with control group rats, as an evidence from hemagglutination after incubation of serum with SRBCs [Table 2].
|Table 2: Effect of administration of nanoparticle extract of Pinus merkusii on hemagglutination titer|
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Effect of nanoparticle extract of P. merkusii on cell mediated immunity parameters
The cell-mediated immune response of nanoparticle extract of P. merkusii was assessed by DTH reaction, that is, foot pad reaction. As shown in [Table 3], the nanoparticle extract of P. merkusii produced a significant, dose-dependent manner increase in DTH reactivity in rats. The nanoparticle extract of P. merkusii at doses 500 mg/kg BW but not at dose 125 and 250 g/kg significantly increased the DTH reactivity as compared to the control. Increase in DTH reaction in rats in response to cell-dependent antigen revealed the stimulatory effect of nanoparticle extract of P. merkusii on T cells [Table 3].
|Table 3: Effect of administration of nanoparticle extract of Pinus merkusii on delayed-type hypersensitivity|
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Effect of nanoparticle extract of P. merkusii on phagocytic response
The faster removal of carbon particles has been correlated with the enhanced phagocytic activity. The phagocytic activity of the reticular-endothelial system was measured by the removal of carbon particles from the blood circulation. The phagocytic index of the control group was 4.18 ± 0.67 [Table 4]. Oral administration of nanoparticle extract of P. merkusii a dose-related increase in the clearance rate of carbon by the cells of the reticuloendothelial system. The nanoparticle extract of P. merkusii showed significant increase in the phagocytic index at doses of 500 mg/kg but not at doses of 125 and 250 mg/kg, when the data were compared with control group rats, suggesting phagocytic activity of the nanoparticle extract of P. merkusii.
|Table 4: Effect of administration of nanoparticle extract of Pinus merkusii on phagocytic index|
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Effect of nanoparticle extract of P. merkusii on cyclophosphamide-induced myelosuppression
Cyclophosphamide at the dose of 30 mg/kg, i.p. caused a significant reduction in the hemoglobin, red blood cells (RBCs); white blood cells (WBCs), and platelets count. Combined treatment of cyclophosphamide and the nanoparticle extract of P. merkusii a dose-dependent manner result in a restoration of bone marrow activity as compared with cyclophosphamide treatment alone. Significant reduction in WBC count was observed in rats treated with cyclophosphamide alone (positive control) as compared to the negative control. The nanoparticle extract of P. merkusii at doses 500 mg/kg BB significant increased the levels of hemoglobin, RBCs, WBCs and platelets count as compared to the positive control treated with cyclophosphamide, and it was observed that nanoparticle extract of P. merkusii at the doses of 125, 250 and 500 mg/kg, respectively, restored the levels of WBC back to normal [Table 5].
|Table 5: Effect of nanoparticle extract of Pinus merkusii on cyclophosphamide-induced myelosuppression|
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| Discussion|| |
The immune system is the vital defense against noninfectious and infectious diseases. A strong immune system comprises elements that are in balance with one another; if this balance is disturbed, our immune system will be incapable to protect the body against harmful substances., Immunomodulation using medicinal plants can provide a substitute to conventional immunotherapy for a range of diseases, especially when host defense mechanism has to be activated under the conditions of impaired immune response. There are several diseases where immunostimulant drugs are needed to overcome the immunosuppression induced by drugs or environmental factors. There is a strong necessity of the drugs that can enhance the immune system to combat the immunosuppressive consequences caused by stress, chronic diseases, and conditions of impaired immune responsiveness. Recently, medicinal plants and their products have been commonly used as immunomodulatory. Though medicinal plants have been investigated for diverse pharmacologic activities, the immunostimulatory potential of P. merkusii still remains unknown.
The results obtained in the present study indicate that nanoparticle extract of P. merkusii is a potent immunostimulant, stimulating both specific and nonspecific immune mechanisms. Neutrophils are an important component of the innate immune system, with the main role in the clearance of extracellular pathogens. Both localization and neutralization of microorganisms are functions of neutrophil that are regulated by specific inflammatory mediators released from the site of infection. The neutrophil, an end cell unable to divide and with limited capacity for protein synthesis is, nevertheless, capable of a wide range of responses, in particular chemotaxis, phagocytosis, exocytosis and both intracellular and extracellular killing. In the present study, nanoparticle extract of P. merkusii evoked a significant increase in percent neutrophils. This may potentially help in increasing immunity of body against microbial infections.
The augmentation of the humoral immune response to SRBCs by nanoparticle extract of P. merkusii, as evidenced by an increase in the antibody titer in rats indicated the enhanced responsiveness of T and B lymphocyte subsets, involved in the antibody synthesis. The high values of HA titer obtained in the case of nanoparticle extract of P. merkusii have indicated that immunostimulation was achieved through humoral immunity. B lymphocytes and plasma cells function in the humoral immunity component of the adaptive immune system by secreting antibodies such as IgG and IgM are the major immunoglobulins which are involved in the complement activation, opsonization, neutralization of foreign bodies.
Cell-mediated immunity (CMI) involves effectors mechanisms performed by T lymphocytes and their products (lymphokines). CMI responses are critical to defense against infectious microorganisms, infection of foreign grafts, tumor immunity, and DTH reactions., Therefore, an increase in DTH reaction in rats in response to T cell-dependent antigen revealed the stimulatory effect of nanoparticle extract of P. merkusii on T cells. In the present study, nanoparticle extract of P. merkusii showed an overall stimulatory effect on the immune functions in rats. Stimulatory effects were observed on both humoral and cellular immunity. In DHT test, the nanoparticle extract of P. merkusii showed an increase response in all doses, but this increase was significant only in dose 500 mg/kg. This activity could be due to the presence of pycnogenol that contains proanthocyanidins which augment the humoral response, by stimulating the macrophages and B lymphocytes subsets involved in antibody synthesis. The mechanism behind this elevated DTH during the CMI responses could be due to sensitized T-lymphocytes. When challenged by the antigen, they are converted to lymphoblast and secrete a variety of molecules including proinflammatory lymphokines, affecting more scavengers cells to the site of reaction. An increase in DTH response indicates a stimulatory effect of the plant which has occurred on the lymphocytes and accessory cell types required for the expression of this reaction.
Phagocytosis is the process by which certain body cells, collectively known as phagocytes, ingests and removes microorganisms, malignant cells, inorganic particles and tissue debris. Nanoparticle extract of P. merkusii appeared to enhance the phagocytic function by exhibiting a dose-related increase in the clearance rate of carbon by the cells of the reticulo endothelium system.
The administration of nanoparticle extract of P. merkusii significantly controls the total WBC count, RBCs count, and hemoglobin and platelets count and also restored the myelosuppressive effects induced by cyclophosphamide. The results of the present study indicate that the nanoparticle extract of P. merkusii can stimulate the bone marrow activity. The bone marrow a sensitive target particularly to cytotoxic drugs such as cyclophosphamide. In fact, bone marrow is the organ most affected during any immunosuppression therapy with this cyclophosphamide. Loss of stem cells and the inability of the bone marrow to regenerate new blood cells results in thrombocytopenia and leucopenia. Administration of the nanoparticle extract of P. merkusii was found to increase the total WBC count, which was lowered by cyclophosphamide, a cytotoxic drug, indicating that the nanoparticle extract of P. merkusii can stimulate the bone marrow activity.
It could be concluded that nanoparticle extract of P. merkusii may stimulate both cellular and humoral immune responses. The nanoparticle extract of P. merkusii not only potentiate nonspecific immune response but also improve humoral as well as CMI effectively. The effectiveness of nanoparticle extract of P. merkusii treated animals in overcoming the side effects of drug-induced myelosuppression provides sufficient evidence for balancing and adaptogenic efficacy of the nanoparticle extract of P. merkusii. Thus, from the results obtained, it can be concluded that nanoparticle extract of P. merkusii has therapeutic potential and could be served as an effective immunomodulatory candidate. Further studies on the mechanism of action of nanoparticle extract of P. merkusii, to establish its therapeutic potential for the prevention of autoimmune diseases are planned in the laboratory.
This study was supported by Ministry of Research, Technology and Higher Education of the Republic of Indonesia. Grant No.: 004/Sp2H/LT/DRPM/IV/2017, April 3, 2017.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]