Table of Contents    
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 1  |  Page : 84-92  

Immune-enhancing effect of bengkoang (Pachyrhizus erosus (l.) Urban) fiber fractions on mouse peritoneal macrophages, lymphocytes, and cytokines


1 Department of Pharmacology and Clinical Pharmacy, Universitas Gadjah Mada, Yogyakarta; Department of Pharmacy, Faculty of Health Sciences, Jenderal Soedirman University, Purwokerto, Central Java, Indonesia
2 Department of Pharmacology and Clinical Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia
3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia

Date of Submission06-Mar-2020
Date of Decision04-Apr-2020
Date of Acceptance20-May-2020
Date of Web Publication27-Jan-2021

Correspondence Address:
Arief Nurrochmad
Department of Pharmacology and Clinical Pharmacy, Universitas Gadjah Mada, Sekip Utara Yogyakarta
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_53_20

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   Abstract 


Background: This study was conducted to evaluate the immunomodulatory effects of bengkoang (Pachyrhizus erosus [L.] Urban) fiber extract (BFE) fraction on mouse peritoneal macrophages, lymphocytes, and cytokines. Materials and Methods: BFE was prepared by heat-extraction from the bengkoang fiber in distilled water at 121°C for 20 min. Fraction of BFE including BEF-A, BEF-B, BEF-D, and BEF-E were prepared by precipitation method with cold ethanol and potassium hydroxide. The phagocytic activity of macrophages was observed by a phagocytosis assay using mouse macrophages. The lymphocyte proliferation assay was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and measuring the absorbance at 550 nm. Also, the production of cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 was determined. Results: The BFE enhanced the phagocytic activity by increasing the phagocytic index and capacity of mouse peritoneal macrophages. The phagocytic capacity of mouse peritoneal macrophages was significantly increased after the treatment of BFE, BFE-B, and BFE-E compared with control. The fractions BFE-A, BFE-B, BFE-D, BFE-E, and pectin could stimulate phagocytic activity by increasing the phagocytic index. There were no significant differences after treatment with fiber fractions in enhancing lymphocyte proliferation, but pectin could stimulate the lymphocyte proliferation. Also, the fraction of BFE-A could enhance TNF-α and IL-10 production. After treatment with BFE-B, there were increases in TNF-α and IL-6 production but decreases in IL-10 production. The fraction of BFE-D could also stimulate TNF-α production, and BFE-E could reduce IL-10 production. Conclusion: The fiber fractions of bengkoang showed an immune-enhancing effect, stimulated both TNF-α and IL-6 production, and suppressed IL-10.

Keywords: bengkoang (Pachyrizus erosus [L.] Urban), dietary fiber fraction, immunomodulatory effect, phagocytic macrophages activity, lymphocyte proliferation


How to cite this article:
Baroroh HN, Nugroho AE, Lukitaningsih E, Nurrochmad A. Immune-enhancing effect of bengkoang (Pachyrhizus erosus (l.) Urban) fiber fractions on mouse peritoneal macrophages, lymphocytes, and cytokines. J Nat Sc Biol Med 2021;12:84-92

How to cite this URL:
Baroroh HN, Nugroho AE, Lukitaningsih E, Nurrochmad A. Immune-enhancing effect of bengkoang (Pachyrhizus erosus (l.) Urban) fiber fractions on mouse peritoneal macrophages, lymphocytes, and cytokines. J Nat Sc Biol Med [serial online] 2021 [cited 2021 Apr 13];12:84-92. Available from: http://www.jnsbm.org/text.asp?2021/12/1/84/307856




   Introduction Top


Bengkoang (Pachyrhizus erosus (L.) Urban) grows naturally in many tropical and subtropical countries in Indonesia and belongs to the Fabaceae family. Bengkoang is a plant that has the potential to be developed as an immunomodulator. Bengkoang contains daidzein, daidzein-7-O-β-glucopyranose. Both 5-hydroxyldaidzein-7-O-β-glucopyranose) and (8,9)-furanyl-pterocarpan-3-ol have been proven as antioxidants. Bengkoang has crude fibers, and according to the previous study contains 14.9% carbohydrates and 1.4% fiber.[1] Polysaccharide compounds have been shown to have biological activity, especially in the immune system, suggesting their role as potential immunomodulatory agents. The previous study was conducted to evaluate the effect of bengkoang fiber extract (BFE) as an immunomodulator. A previous study reported that BFE enhanced phagocytic activity and stimulated the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6.[2] Bengkoang could simplify the production of IgM by human hybridoma HB4C5 cells and the production of IgM, IgG, and IgA by mouse primary splenocytes in a dose-dependent manner.[3] BFE activated J774.1 cells via the MAPK and NF-κB signaling pathways.[4]

Immunomodulatory agents stimulate and maintain the immune system and improve host defenses against pathogens.[5] The activation of the immune system plays a role in reducing the risk of chronic diseases. There are two categories of immune responses, namely, the innate immune response and the adaptive immune response. Although the adaptive immune response takes a long time after infection, it provides highly specific protection against pathogens. Macrophages are cells that play several crucial parts in the innate immune system. Lymphocytes play an essential role in the adaptive immune response. Immunomodulatory agents from polysaccharides compounds can modulate adaptive immunity by regulating the levels of lymphocytes, cytokines, chemokines, and antibodies.[6]

The present study aimed to determine the effect of fraction of BFE as immunomodulatory in innate immunity and adaptive immunity (phagocytic macrophage activity, lymphocyte proliferation, and cytokine production). The objective of this study was to investigate the immunomodulatory effect of the fractions of BFE on mouse peritoneal macrophages, lymphocytes, and cytokines. Furthermore, bengkoang can be developed into an immunomodulatory agent that can be used to maintain human health and prevent chronic diseases.


   Materials and Methods Top


Materials

Ethanol, ammonium oxalate, and potassium hydroxide were purchased from Sigma-Aldrich Pte Ltd., Singapore. Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin 2%, fungizone 0.5%, fetal bovine serum 10%, and phosphate-buffered saline (PBS) were purchased from Life Technologies Corporation (GIBCO). Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and pectin were purchased from Sigma-Aldrich Pte Ltd., Singapore. All other reagents and chemicals were of the purest commercial grade available.

Plant material collection

Bengkoang tubers were taken from Prembun, Kebumen, Central Java, Indonesia. This plant was identified and authenticated by Dr. Djoko Santoso, Assistant Professor in the Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia.

Animals

Two 8-week-old male BALB/c mice (Animal Center of Pharmacology and Toxicology Laboratory, Gadjah Mada Universitas) were housed with a standard pelleted basal diet and water ad libitum, in an animal room under 12 h light/dark cycles at a temperature of 22°C ± 3°C and a humidity of 60%. All animals were acclimatized for 1 week before experimentation. The experimental procedures were approved by the Institutional Animal Ethics Committee of the Integrated Research and Testing Laboratory, Universitas Gadjah Mada (number 00073/04/LPPT/VII/2018).

Preparation of the bengkoang tuber extract

Bengkoang was sorted, washed, peeled, mashed, and soaked in distilled water (1:2). The suspension was allowed to settle overnight and then was filtrated to separate the starch (precipitate) and fiber (supernatant). The supernatant was evaporated over a water bath for 30 min. Then it was immersed in 80% ethanol (1:1) filtrated, evaporated at 60°C for 20 min, and filtrated. The precipitate was dried using a freeze dryer to obtain a dry powder that was then called the BFE.

BFE was added 0.5% w/v of ammonium oxalate solution. The mixture was heated in a boiling water bath for 30 min and then centrifuged (1400 ×g for 5 min). Filtration produces a supernatant and solid phase. The supernatant was added to four volumes of cold ethanol and kept at 5°C ± 2°C for 12 h. The fraction BFE-A (supernatant) and BFE-B (solid phase) were recovered by centrifugation (1400 ×g for 5 min), purified by dialysis over 72 h using tubular membrane, and then freeze-dried.

The solid phase obtained after precipitation with ammonium oxalate was added to the 5% w/v potassium hydroxide solutions (23°C ± 3°C for 12 h) in a shaking water bath (826 g). The solid phase was separated from the supernatant by centrifugation and then freeze-dried to produce the fiber fraction BFE-D. The fraction (BFE-E) was precipitated from the respective supernatant by the addition of four volumes of cold ethanol at 5°C ± 2°C, over 12 h. The fraction BFE-E was recovered by centrifugation (at 1400 × g for 5 min), purified by dialysis over 72 h using tubular membrane, and freeze-dried.

Fourier transform infra-red analysis

The fiber fraction was identified with a spectrophotometer Fourier Transform Infra-Red (FTIR) in wavenumbers 4000–400 cm−1. The ground powder of the fiber fraction was mixed with potassium bromide to produce a pellet and then was analyzed with the spectrophotometer FTIR.

Immunomodulatory effect on mouse peritoneal macrophages

Mice were sacrificed by cervical dislocation. A suspension of mouse macrophages was made by injecting 10 mL RPMI 1640 medium (GIBCO) into the peritoneal cavity. The peritoneal fluid was extracted and then centrifuged at 2000 rpm at 4°C for 10 min. The supernatant was removed and the precipitant was collected that contained macrophages inoculated into the containing RPMI 1640 medium, penicillin-streptomycin 2%, fungizone 0.5%, and fetal bovine serum 10%. The cell density of mouse macrophages was 2.5 × 106 cell/mL. The phagocytic activity of macrophages was performed in 24-well culture plates that contained coverslips. The macrophages were cultured in 24-well culture plates at 1 mL/well and incubated for 24 h at 37°C. After washing with one mL of RPMI 1640 medium, various concentrations of BFE 10, 100, and 1000 μg/mL, BFE-A, BFE-B, BFE-D, BFE-E, and pectin (10, 30, and 90 μg/mL) were added to each well. Culture plates were then incubated for one h at 37°C. The latex beads were added in 24-well culture plates at 50 μL/well and incubated for one h at 37°C. After washing three times with phosphate buffer saline (PBS-GIBCO) using 1 mL/well, wells were fixed with methanol and colored by Giemsa 20%. The coverslips were taken and viewed under a binocular microscope to classify the phagocytic macrophage activity into phagocytic capacity and the index of macrophage activity.

Immunomodulatory effect on lymphocyte proliferation

Mice were sacrificed by cervical dislocation, and their spleens were excised. A suspension of lymphocytes was made by injecting 10 mL RPMI 1640 solution into the spleen. The suspension was centrifuged at 2000 rpm at 4°C for 10 min. The pellets were hemolyzed two times using hemolysis buffer Tris ammonium was added to the RPMI 1640 and centrifuged at 2000 rpm, at 4°C for 10 min. The supernatant was removed, and the precipitant was collected so that it contained the lymphocytes inoculated into the RPMI 1640 medium, penicillin-streptomycin 2%, fungizone/amphotericin B (GIBCO) 0.5%, and fetal bovine serum (GIBCO) 10%. The cell density of mouse macrophages was 1.5 × 106 cell/mL. Cells were cultured in 96-well culture plates at 100 μL/well and incubated for 24 h at 37°C. After incubation, wells were added with various concentration of BFE (10, 100, and 1000 μg/mL), various concentration of fiber fractions BFE-A, BFE-B, BFE-D, BFE-E, pectin (10, 30, 90, and 270 μg/mL), and incubated for 48 h at 37°C. Cell viability was measured by the tetrazolium salt (MTT) assay, and the absorbance was read at 550 nm.

Immunomodulatory effect on cytokine tumor necrosis factor-α, interleukin-6, and interleukin-10 production

After sacrifice, the mouse spleen was excised and then used to make a suspension. The suspension of lymphocyte was made by injecting 10 mL RPMI 1640 solution into the spleen. The suspension was centrifuged at 2000 rpm at 4°C for 10 min. The pellets were hemolyzed using hemolysis buffer Tris ammonium, added with RPMI, and centrifuged at 2000 rpm at 4°C for 10 min. The supernatant was removed; the remaining cell pellet was washed twice with RPMI and then diluted with complete medium. Lymphocyte cells were counted with a hemocytometer and resuspended in complete medium to obtain a cell suspension of 1 × 107 per mL. The cell suspension 100 μL and 2 μL phytohemagglutinin (PHA) used as a mitogen, were seeded into 96-well plates and incubated at 37°C in a 5% CO2 humidified atmosphere for 24 h. After incubation, 100 μL fiber fractions with variation concentration (10, 30, 90, and 270 μg/mL) were added into each well and incubated again for 48 h. Then, culture cell supernates were centrifuged at 1000 rpm for 20 min at 2-8°C to remove cell debris. The clear supernates were collected and underwent the assay immediately (storage in the freezer at −20°C) by the Sandwich mouse ELISA kit (Fine Test).

Statistical analysis

Each experiment was performed in triplicate. Data were presented as the mean ± Standard deviation (SD). The statistical significance between various groups was analyzed by a one-way analysis of variance followed by LSD multiple comparison tests (GraphPad InStat version 3.10; GraphPad Software Inc., La Jolla, CA 92037, USA). A (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 were considered statistically significant.


   Results Top


Immunomodulatory effect on mouse peritoneal macrophages

The immunomodulatory effect on the innate immune response was evaluated. In this study, the phagocytic activity of macrophages was observed based on the phagocytic capacity and phagocytic index. The phagocytic capacity describes macrophage activity, and the phagocytic index describes the capacity of macrophages that are active in overcoming the occurrence of attacks from foreign objects. The phagocytosis of macrophages occurs when latex attaches to macrophage cells. Macrophage cells were isolated from the peritoneal cavity of mice. [Figure 1]a shows that the cell control without treatment had no phagocytic activity of macrophages toward latex, while [Figure 1]b shows the phagocytic activity of macrophages against latex.
Figure 1: The phagocytic activity of macrophages in the control group (a) and treatment group (b). Macrophages stained by Giemsa 20%, ×400. An active macrophage (purple) is visible, as indicated by the black arrow. The latex beads are shown by the red arrow

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BFE at concentrations 10 μg/mL, 100 μg/mL, and pectin 100 μg/mL had the phagocytic activity of macrophages that was characterized by an increase in phagocytosis capacity, although there were no significant differences from controls [Table 1]. As indicated in [Table 2], the fiber actions of BFE-B with a concentration of 10, 30, and 90 μg/mL significantly enhanced the phagocytic capacity of mouse peritoneal macrophages compared with control cells (P < 0.05). The fiber fraction BFE-E with a concentration of 90 μg/mL could stimulate phagocytic activity (P < 0.05). There was increasing in phagocytic capacity after treatment with pectin, BFE-A, and BFE-D but no significant differences with control. For the phagocytic index, the results showed that the BFE and pectin 10 and 90 μg/mL significantly increased phagocytosis index, but not at high doses 100 and 1000 μg/mL for BFE. The fiber extract fractions (BFE A, B, D, and E) also increased the phagocytic index, and the fraction BFE-B (10, 30, and 90 g/mL) demonstrated the highest effect of the phagocytic index [Figure 2].
Figure 2: Effect of bengkoang fiber extract (a), pectin (b), and fractions of fiber extract (bengkoang fiber extract-A [c], bengkoang fiber extract-B [d], bengkoang fiber extract-D [e], and bengkoang fiber extract-E [f]) on the phagocytic index of mouse peritoneal macrophages. Cells were treated with bengkoang fiber extract 10, 100, and 1000 μg/mL, and pectin or fiber extract fractions 10, 30, and 90 μg/mL, and then cultured for one h. After culture, cultures were colored by Giemsa 20%, and macrophages were observed. Each result is represented as the mean ± Standard deviation of three independent measurements. Significant differences were compared with the control and are represented as *P < 0.05; **P < 0.01; ***P < 0.001

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Table 1: Effect of bengkoang fiber extract and pectin on the phagocytic capacity of mouse peritoneal macrophages

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Table 2: Effect of bengkoang fiber extract fractions (bengkoang fiber extract-A, bengkoang fiber extract-B, bengkoang fiber extract-D, and bengkoang fiber extract-E) and pectin on the phagocytic capacity of mouse peritoneal macrophages

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Immunomodulatory effect on lymphocyte proliferation

The immunomodulatory effect on the adaptive immune response was addressed on the proliferation activity of lymphocytes. The immunomodulatory effect is shown by the ability of lymphocytes to proliferate when given fiber as an antigen. As indicated in [Figure 3], the fiber BFE (10–1000 μg/mL) and pectin (10–90 μg/mL) had no significant stimulatory effect on lymphocyte proliferation. The administration of pectin 270 μg/mL enhanced lymphocyte proliferation (P < 0.05), as shown in [Figure 3]. After treatment with BFE-A at a concentration of 30, 90, and 270 μg per mL, lymphocyte proliferation was inhibited compared with control, and an increased concentration showed increased activity. In contrast, BFE-B, BFE-D, and BFE-E had no significant effect on lymphocyte proliferation [Figure 3].
Figure 3: Effect of bengkoang fiber extract (a), pectin (b), and fractions of fiber extract (bengkoang fiber extract-A [c], bengkoang fiber extract-B [d], bengkoang fiber extract-D [e], and bengkoang fiber extract-E [f]) on the proliferation of phytohemagglutinin-stimulated mouse lymphocytes. Cells were treated with bengkoang fiber extract 10, 100, 1000 μg/mL, pectin, or bengkoang fiber extract-(A, B, D, or E) 10, 30, 90, 270 μg/mL, and then cultured for 48 h. After culture, cell viability was measured by the tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay by an ELISA reader at 550 nm. Each result is represented as the mean ± Standard deviation of three independent measurements. Significant differences compared with the control are represented as *P < 0.05; ***P < 0.001

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Immunomodulatory effect on cytokine tumor necrosis factor-α, interleukin-6, and interleukin-10 production

The effects of BFE fractions on cytokine production of TNF-α, IL-6, and IL-10 were also evaluated from cultured lymphocytes. The results of TNF-α after treatment with fiber fractions are shown in [Figure 4] TNF-α production was significantly enhanced with BFE-B of 10, 30, 90, and 270 μg/mL. After treatment with BFE-A of 30, 90, and 270 μg per mL, there were increases in TNF-α production (P < 0.05) [Figure 4]. As shown in [Figure 4], the cytokine production of TNF-α decreased after treatment with BFE-D and BFE-E. As indicated in [Figure 5], treatment with BFE-B significantly enhanced IL-6 production (P < 0.001). There were no significant differences in IL-6 production with control after treatment with BFE-A, BFE-D, and BFE-E. A treatment with BFE-A of 10 μg per mL (low concentration) could significantly enhance IL-10 production, but treatments with BFE-A of 30, 90, and 270 μg/mL (high concentration) decreased IL-10 production as indicated in [Figure 6]. IL-10 production decreased after treatment with BFE-B and BFE-E (P < 0.001) but increased after treatment with BFE-D of 10 and 90 μg/mL. While after treatment with BFE-D 30 and 270 μg/mL decreased IL-10 production.
Figure 4: Effect of bengkoang fiber extract fractions (bengkoang fiber extract-A, bengkoang fiber extract-B, bengkoang fiber extract-D, and bengkoang fiber extract-E) on TNF-α secretion by phytohemagglutinin-stimulated mouse lymphocytes. Cells were treated with fiber extract fractions 10, 30, 90, and 270 μg/mL, and then cultured for 48 h. Each result is represented as the mean ± Standard deviation of three independent measurements. Significant differences compared with the control are represented as *P < 0.05; **P < 0.01; ***P < 0.001

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Figure 5: Effect of bengkoang fiber extract fractions (bengkoang fiber extract-A, bengkoang fiber extract-B, bengkoang fiber extract-D, and bengkoang fiber extract-E) on IL-6 production by phytohemagglutinin-stimulated mouse lymphocytes. Cells were treated with fiber extract fractions 10, 30, 90, and 270 μg/mL, and then cultured for 48 h. Each result is represented as the mean ± Standard deviation of three independent measurements. Significant differences compared with the control are represented as *P < 0.05; **P <0.01; ***P < 0.001

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Figure 6: Effect of bengkoang fiber extract fractions (bengkoang fiber extract-A, bengkoang fiber extract-B, bengkoang fiber extract-D, and bengkoang fiber extract-E) on IL-10 production by phytohemagglutinin-stimulated mouse lymphocytes. Cells were treated with fiber extract fractions 10, 30, 90, and 270 μg/mL, and then cultured for 48 h. Each result is represented as the mean ± Standard deviation of three independent measurements. Significant differences compared with the control are represented as *P < 0.05; **P < 0.01; ***P < 0.001

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Fourier transform infra-red analysis

The infrared (IR) spectra of the fiber fractions are shown in [Figure 7]. This Figure also shows the absorption area of various functional groups. The IR spectrum of BFE-A indicated the presence of vibration bands at 3434.42 cm−1 designating a hydroxyl group, a C-H stretching around at 2927.08 cm−1; a C-H in-plane around at 1400.20 cm−1; and a C-O-C in glycoside bonds around at 1164.38 cm−1 [Figure 7]a. The BFE-B IR spectra indicated the presence of OH stretching groups in the vibration band 3384.43 cm−1, a CH group at 2927.79 cm−1, C=O at 1638.90 cm−1, and a C-O-C at 1154.05 cm−1 [Figure 7]b. As shown in [Figure 7]c, the IR spectrum of BFE-D showed a band at 3447.96, indicating an OH at 2923.85 cm, indicating a CH at 1654, indicating the presence of a carbonyl group. The IR spectrum of BFE-E showed a broad stretching spectrum around 3419.88 cm−1 for the hydroxyl group; at 2966.67 cm−1 indicating C-H stretching; C=O stretching around 1650.85 cm−1; and a C-H in-plane around at 1401.72 cm−1.[7]
Figure 7: Fourier Transform Infra-Red spectra of bengkoang fiber extract fractions (bengkoang fiber extract-A (a); bengkoang fiber extract-B (b); bengkoang fiber extract-D (c); and bengkoang fiber extract-E (d)

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As shown in [Figure 8], the functional group between standard pectin and BFE-B is almost the same. The absorption area of various functional groups of BFE-B was similar to pectin [Figure 8]. The absorption band at 3384.43 cm−1 showed a very strong absorption peak intensity and width that indicated the presence of strain O-H uptake. The absorption band at 2927.79 cm−1 showed strain C-H uptake. The absorption band at 1638.90 cm−1 showed absorption of C=O strain, which was thought to originate from the carboxyl acid carboxylic group. The absorption around 1164.38 cm−1 indicates a C-O-C symmetric (in glycoside bonds).[7] The results demonstrate that the fiber fraction BFE-B contained a pectin-like compound.
Figure 8: Fourier Transform Infra-Red spectra of bengkoang fiber extract-B and pectin

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   Discussion Top


Bengkoang had antioxidant and immunomodulatory activity in a previous study.[4],[8] In the present study, BFE-E demonstrated a significantly enhanced immune response. Thus, BFE has the potential to be developed as an adjuvant of immunopreventive drugs. Many studies have examined how the immune response can be modulated and activated by foods that contain dietary fiber.[9] Polysaccharide compounds from natural sources can affect immune responses, suggesting their roles as potential immunomodulators.[10] According to a previous study, bengkoang contains pectin and hemicellulose polysaccharides that might have immunomodulatory activity.[11] In our study, we evaluated the immunomodulatory activity of the fiber fractions of bengkoang in innate and adaptive immune responses (phagocytic macrophage activity, lymphocyte proliferation, and cytokine production).

Macrophages have an essential role in the immune response, namely, phagocytosis of foreign particles, such as microorganisms, including macromolecules, antigens, even cells, and damaged or dead tissues.[12] In the present study, we used PHA as a mitogenic agent that could stimulate T cells and B cells to proliferate.[13] Lymphocyte proliferation is the response of lymphocytes to antigenic stimulation. Lymphocytes are specific cells that recognize and respond to foreign antigens so that they can become mediators of humoral and cellular immunity.[12]

Our results showed that the fiber fractions of bengkoang could modulate the innate immune response and adaptive immune response that developed potential immunomodulatory activity. The fiber fractions BFE-A, BFE-B, BFE-D, and BFE-E from bengkoang activated the phagocytic activity of macrophages at 10, 30, and 90 μg mL−1. These results indicated that bengkoang had an immune-enhancing effect on phagocytic activity. Furthermore, in the previous study, bengkoang proved to have antioxidant activity.[8] The fiber extract from bengkoang could activate J774.1 cells through MAPK and NFκB pathways.[3]

The fiber extract from bengkoang increased the production of TNF-α and IL-6. Both IL-6 and TNF-α are pro-inflammatory cytokines that enhance the inflammatory response to microbial infection. In addition, IL-6 has functions in both non-specific and specific immunity. In non-specific immunity, it could stimulate the liver to produce acute-phase proteins and increase neutrophil production. In specific immunity, IL-6 stimulates B-cell proliferation.[12],[14] IL-6 also affects metabolism, but metabolism affects the immune system.[15] The main function of IL-10 is to inhibit the production of several types of cytokines (TNF, IL-1, chemokines, and IL-12). IL-6 also inhibits the function of macrophages and dendritic cells by helping to activate T cells so that they are immunosuppressed.[16]

The fiber fraction of BFE-A could stimulate TNF-α and IL-10 production at low concentrations. PEF-B could enhance TNF-α, IL-6, and decrease IL-10 production at high concentrations. BFE-D could enhance TNF-α production, and BFE-E could decrease IL-10 production. Based on these results, bengkoang could strengthen the adaptive immune system by regulating TNF-α, IL-6, and IL-10. In a previous study, the fiber extract from bengkoang had immunomodulatory effects both in vitro and in vivo through the production of IL-5 and IL-10 cytokines.[4]

The present study showed that pectin at a low dose (10 μg/mL) could enhance the phagocytic activity, but at a high dose (270 μg/mL), it stimulated lymphocyte proliferation. A previous study reported that water-soluble polysaccharide-like pectin from Rose damacena could enhance IL-6 production from macrophages.[17] Dietary pectin had an inhibitory effect on IL-10 production that was mediated by the influence of cytokine TNF-α of the Th2 type, like IL-10, and could suppress the activation of Th2 cells, and decrease the release of IL-6 and TNF-α.[18] A similar phenomenon was also observed after treatment with the fiber fraction of BFE-B. Another study showed that pectin and its modification had the activity of modulating pro-inflammatory cytokine production. Pectin with different chemical structures has the activity of modulating the secretion of IL-10 and TNF-α in THP-1 macrophages, which are also different. Modified pectin with a low esterified methyl could reduce the production of TNF-α and IL-10.[19]

The fiber fraction of BFE-B contained a pectin-like compound based on the similarity of the functional group between pectin and BFE-B.[7] Pectin is one of the polysaccharide compounds in bengkoang that contains carboxyl groups. A previous study showed that carboxyl groups are important in activating macrophages. After treatment with acidic arabinogalactans isolated from Vigna radiata, the secretion of TNF-α and IL-6 increased, but the ability to have phagocytic activity decreased with the reduction of carboxyl groups.[20]

According to a previous study, polysaccharide compounds have the potential to be developed into immunomodulators.[21] The mechanism responsible for transforming polysaccharides into immunomodulators remains unclear. Dietary fiber has been reported to affect gut-associated lymphoid tissue, which plays a role in the immune system in the gastrointestinal tract.[22] The yield of fiber fermentation, short-chain fatty acids (SCFA) in the intestine can stimulate the immune system in the gastrointestinal tract.[23] Pectin, one compound found in dietary fiber, can increase IgG levels in mesenteric lymph nodes.[18] Schley and Field (2002) reported that SCFA could modulate the immune system.[23]

In our study, the fiber fractions of bengkoang proved to have phagocytic activity on mouse peritoneal macrophages when compared with control cells. The fiber extract fraction of bengkoang had no significant effect on lymphocyte proliferation, but lymphocyte proliferation increased by pectin. Low doses of pectin could enhance the immune system by increasing the phagocytic index, but at high doses, it could stimulate lymphocyte proliferation. BFE had functional groups that were similar to pectin and was reported to stimulate a phagocytic effect, enhance TNF-α and IL-6 production, and inhibit IL-10. Thus, the fiber extract fractions of bengkoang tubers may provide potential beneficial effects on when used as adjuvants of immunopreventive drugs. However, the fiber fractions need to be investigated further using in vivo studies.


   Conclusion Top


The fiber fractions of bengkoang (BFE-A, BFE-B, BFE-D, and BFE-E) had phagocytic activity and modulation of cytokine production. Low doses of pectin could stimulate phagocytic activity, but at high doses, increased lymphocyte proliferation. BFE-B contained a pectin-like compound that had phagocytic activity, stimulated both TNF-α and IL-6 production, and suppressed IL-10. These results suggest that bengkoang might have a potentially beneficial effect on human health and on preventing immune system-related diseases.

Financial support and sponsorship

Lecturer and Doctoral Student Collaborative Research Grant, (Grant number: 43.05.04/UN1/FFA1/SET.PIM/PT/2019) from the Faculty of Pharmacy, Universitas Gadjah Mada.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2]



 

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