Immunomodulatory Activity of Crude Bromelain of Pineapple (Ananas comosus (L.) Merr.) Crown from Subang District, Indonesia

 

Nyi Mekar Saptarini1*, Driyanti Rahayu1, Endah Kartikawati2

1Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy,

Universitas Padjadjaran, West Java, Indonesia – 45363.

2Department of Pharmacy, Faculty of Mathematic and Natural Sciences, Al Ghifari University,

West Java, Indonesia – 40293.

*Corresponding Author E-mail: nyi.mekar@unpad.ac.id

 

ABSTRACT:

Crude bromelain, which extracted from pineapple (Ananas comosus (L.) Merr) crown from Subang district, West Java, Indonesia, has been shown to have protease and antioxidant activity. Both of these activities can be applied as immunomodulator agent. The purpose of this study was to determine the immunomodulatory activity of crude bromelain of the pineapple crown through phagocytic response and specific humoral immune response. The phagocytic response was observed from carbon clearance in induced-mice with black ink, while the specific humoral immune response was observed from antibody haemagglutination of mice which challenged with sheep red blood cells. Data were analyzed with one-way ANOVA and compared to the standard. The results showed that the immunomodulatory activity of crude bromelain was dose-independent in 1.56, 3.12, and 4.68 mg/20 g BW of mice. It was concluded that crude bromelain 3.12 mg/20 g BW has immunomodolatory activity comparable to the standard.

 

KEYWORDS: Carbon Clearance, Induced-Mice, Haemaglutination, Dose-Independent.

 

 


INTRODUCTION:

Pineapple (Ananas comosus (L.) Merr) is the main sources of bromelain, which scattered throughout the pineapple with different characters1. Bromelain composition consists of acid phosphatases, peroxidases, and sulfur-containing cysteine endopeptidases2. Fruit and stem bromelain are used as anti-inflammatory agent for tendonitis3, enhancing T-cell responses in immune-suppressed patients with cancer4, and immunomodulatory agent5,6.

 

Subang district, West Java, is one of the pineapple production centers in Indonesia7. This study used pineapple crown as agricultural waste, which improperly usage. Previous studies showed that crude bromelain, which extracted from Subang pineapple crown has protease and antioxidant activity.

 

 

There is no systematic study has examined the immunomodulatory activity in crude bromelain, which extracted from pineapple crown. This study, based on the ethnomedical claims, was to determine the scientific validity for immunomodulatory activity of crude bromelain of pineapple crown. The novelty of this study was to determine the immunomodulatory activity of crude bromelain from the Subang pineapple crown. Immune function modulation with medicinal plants and their products become an accepted therapeutic approach.

 

MATERIALS AND METHODS:

Materials:

The pineapple crowns were collected for 5 months old fruits which planted in Subang district, West Java, Indonesia. The plant was identified by Plant Taxonomy Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia with No. 521/HB/08/2018.

 

Swiss Webster male mice with 25-35g of body weight and aged 2-3 months in a healthy condition (active and not disabled) were used as experimental animals. The mice were fasted overnight, but water were provided ad libitum before the experiments.

 

Sheep red blood cells (SRBC) were collected from a healthy sheep from the local slaughterhouse in West Java, Indonesia. This blood was mixed in Alsever’s solution in a ratio of 1:1. The Alsever’s solution consists of 0.055g of citric acid, 0.8g of sodium citrate, 2.05g of glucose, and 0.42g of sodium chloride in 100mL of distilled water.  The blood was centrifuged for 10min at 5000rpm, on the day of immunization, then washed to remove plasma with saline solution. The 20% v/v SRBC suspension was prepared saline solution8.

 

Barbital and sodium carboxymethyl cellulose (Na-CMC) with pharmaceutical grade were purchased from China. All chemical reagents with analytical grade were purchased from Merck, i.e. ethanol, trichloroacetic acid (TCA), casein, tyrosine, sodium chloride, sodium citrate, citric acid, acetic acid, sodium hydroxide, monosodium phosphate, disodium phosphate, and glucose.

 

Ethical Clearance:

In vivo experimental procedures and protocols in this study were reviewed and approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Padjadjaran with No. 1026/UN6.KEP/EC/2019.

 

Bromelain Extraction:

The pineapple crown and aquadest with ratio 1:2 was mashed, then filtered. Ethanol with ratio 1: 4 was added, then stored at 4°C for 8 h. The upper part was decanted, and the bottom part was centrifuged for 15 min at 15000 rpm. The sediment was dried at 30°C and calculated the yield9.

 

Determination of Protease Activity:

Five concentrations of tyrosine standard, as a product of protease, were measured at 275nm. Casein solution (1 mg/mL) and crude bromelain (1mg/mL) were incubated for 30 min. The TCA solution was added and boiled at 90°C for 5 min, then cooled to room temperature. The absorbance was measured at 275nm10. Protease activity was calculated by formula 1.

 

               μmol produced tyrosine

IU/mg = –––––––––––––––––––                                 (1)

                  mg extract x time

 

In vivo Immunomodulatory Assay:

Dose Calculation:

Immunomodulator activity of bromelain is achieved at a dose of 3000 GDU11. This dose was converted to mice dose12.

 

Phagocytic Response:

Swiss Webster mice were divided into 5 groups (n = 5). The group I was given 1.0% Na-CMC suspension as control, group II was given an imboost® suspension (0.65mg/20g BW) as standard, groups III-V were given different concentration of crude bromelain suspension (1.56, 3.12, and 4.68mg/20g) for 7 days. At the end of the 7th day, mice were injected with 0.2mL/20g BW of colloidal carbon particles via the tail vein. Mice blood was withdrawn in 5μL of EDTA solution from the retro-orbital plexus at 0, 5, 10, 15, and 20 min. A 25μL of blood was mixed with 2mL of 1% sodium carbonate solution, then its absorbance was determined at 650nm. The rate constant of carbon clearance (k), phagocytic index (a), and stimulation index were calculated with formula 2 - 4. After 12 h, mice were sacrificed then liver and lymph were isolated and weighed8.

 

                                                                  log OD5-logOD20

Rate constant of carbon clearance (k) = –––––––––––––––––––        (2)

                                                                              t20-t5

 

                                            k 1/3   body weight

Phagocytic index (a) = ––––––––––––––––––––––                           (3)

                                         weight of liver + lymph

 

                                  phagocytic index of sample

Stimulation index = ––––––––––––––––––––––––––                         (4)

                                   phagocytic index of control

 

SRBC Specific Humoral Immune Response:

Swiss Webster mice were divided into 5 groups (n = 5). The group I was given 1.0% Na-CMC suspension as control, group II was given an imboost® suspension (0.65mg/20g BW) as standard, groups III-V were given different concentration of crude bromelain suspension (1.56, 3.12, and 4.68mg/20g BW) for 14 days. On day 0, mice were immunized intraperitoneally with 0.1mL of 20% fresh SRBC suspension. On the 7th day, mice were challenged with 20mL of SRBC suspension. Blood samples were collected from the retro-orbital plexus on the 7th day for primary antibody titer and on the 15th day for secondary antibody titer. Mice blood were centrifuged for 10 min at 5000rpm, then 25mL of serum were added to 25mL of 1% SRBC suspension, mixed and incubated for 1 h at room temperature to examine haemagglutination titer8.

 

Statistical Analysis:

Data were presented as the mean±standard deviation (SD). One-way ANOVA was conducted to statistical analysis, followed by Tukey-Kramer post hoc test. Values were considered statistically significant at p < 0.05.

 

RESULTS AND DISCUSSION:

Bromelain Extraction:

Subang district has temperature is 21-31°C, and relative humidity is 78-84% with altitude is 0-1500m above sea level7. This condition is suitable for pineapple growth13, which produced 1,633 tons of pineapple in 20177. Bromelain was extracted using water, due to  water-soluble property, then precipitated with ethanol, due to ethanol-insoluble practically14. The purpose of precipitation is to separate the protein from the solution, easier to handle15. The sediment was dried in a refrigerator to prevent enzyme damage16. A total of 1200g of pineapple crown was produced 5.43g of crude bromelain, the yield was 0.45%. This result was better than previous study using pineapple crown aged 12 months, i.e. 0.26%. These results were accordancing to bromelain content is higher in raw fruit than ripe fruit13. Crude bromelain was brown with slightly pineapple odor (Fig. 1).

 

Fig. 1: Crude Bromelain of Pineapple Crown

 

Determination of Protease Activity:

The calibration curve of tyrosine as a product of casein degradation was used to determine protease activity of crude bromelain. The correlation coefficient was 0.9986 (Fig. 2) showed that the concentration and the instrument response are proportional17. Casein degradation by crude bromelain was observed from tyrosine, which can be measured by UV spectrophotometry due to aromatic residue18. The absorbance of tyrosine from casein degradation was 1.379±0.015 which equivalent to 227.97±2.16mg/mL of tyrosine, so protease activity was 30.40±0.24 IU/mg. This result was higher than crude bromelain from pineapple crown aged 12 months, i.e. 7.2 ± 0.45 IU/mg. These result was accorded with protease activity of raw pineapple is better than ripe fruit13.

 

Fig. 2: Calibration Curve of Tyrosine (n = 3)

 

In vivo Immunomodulatory Assay:

Phagocytic Response:

 

Fig. 3: The Mouse’s Body Weight During Observation (n = 5)

 

The mouse’s body weight at the beginning of the study was in the range of 30.2-31.8g. The mouse’s body weight was decreased in the first 3 days (29.8-31.5g), due to adaption, but it was not statistically significant (p = 0.02). The last 4 days, the mouse’s body weight was increased to range 31.0-32.0g (Fig. 3). The treatments were no effect on the mouse’s body weight during the observation (p = 2.58 x 10-4).

 

Fig. 4: The Carbon Absorbance During Observation

 

The decreased carbon absorbance was proportional to the increased rate of carbon clearance (Fig. 4). There was a significant difference in carbon absorbance in the treated groups (p = 1.58 x 10-5). The carbon absorbance reduction was dose-independent in the range 1.56 to 4.68 mg/20 g. Crude bromelain 4.68mg/20 g showed a decreased absorbance lower than 3.12mg/20 g. Absorbance of crude bromelain 3.12mg/20 g was similar to standard, but there was a significant difference (p = 2.53 x 10-3).

 

The purpose of in vivo immunomodulatory assay of the carbon clearance method was to evaluate the effect of crude bromelain on the reticuloendothelial system (RES)19. This system is a diffuse system consisting of phagocytic cells, i.e. the Kupffer cells in the liver, will clearance the particles from the bloodstream20. The carbon clearance method used colloidal carbon particles which injected into the systemic circulation directly, the rate of carbon clearance from the blood by macrophage is governed by an exponential equation21. The faster removal of carbon particles correlates with the enhanced phagocytic activity (Fig. 4). An increased phagocytic activity was observed in the treated groups as compared to control. An increased carbon clearance is an indicator of enhanced in vivo phagocytic activity and competency of granulopoietic systems in removal of foreign particles, which as an indicator of enhanced immunological response against antigens20.

 

Non-specific cellular immune responses involve the lymphoid system, that protect the body from damage due to foreign substances. The lymph function is contributing in cell production, phagocytosis, and immune development. Increased liver weight and lymph were indicated the increased the proliferation of immune cells contained in these organs22. On the 7th day, mice were sacrificed, the liver and lymph were isolated and weighed (Table 1). The ratio of liver and lymph of body weight in the treated groups (Table 1) showed no significant difference (p = 0.61).

 


Table 1. Effect of Crude Bromelain on Weight of Liver and Lymph

Group

Body weight on 7th day (g)

Liver (g)

Ratio liver to body weight

Lymph (g)

Ratio lymph to body weight

Control

32.0 ± 0.21

1.62 ± 0.02

0.051

0.13 ± 0.03

0.004

Standard

31.0 ± 0.13

1.87 ± 0.03

0.060

0.21 ± 0.02

0.007

Bromelain 1.56 mg/20 g

31.2 ± 0.18

1.38 ± 0.01

0.044

0.15 ± 0.03

0.005

Bromelain 3.12 mg/20 g

31.7 ± 0.19

1.77 ± 0.01

0.056

0.20 ± 0.03

0.006

Bromelain 4.68 mg/20 g

31.6 ± 0.12

1.61 ± 0.02

0.051

0.17 ± 0.03

0.005

 


Phagocytosis index is used to determine the ability of phagocytosis in eliminating carbon particles as antigens8. There was a significant difference in the phagocytosis index in the treated groups (p = 2.93 x 10-11) (Table 2). Crude bromelain 1.56mg/20g was showed the highest activity. Phagocytosis index of crude bromelain 3.12 mg/20g was similar to control (p = 0.15), which mean this activity was equal. Crude bromelain 4.68 mg/20g showed that pathological mice occur that affect the absorption, distribution, metabolism, and excretion, thereby reducing the ability of phagocytosis.

 

Table 2. Effect of Crude Bromelain on Phagocytosis and Stimulation Index

Group

Phagocytosis index

Stimulation index

Control

3.05 ± 0.07

0

Standard

5.28 ± 0.15

1.73

Bromelain 1.56 mg/20 g

5.70 ± 0.13

1.87

Bromelain 3.12 mg/20 g

5.23 ± 0.11

1.71

Bromelain 4.68 mg/20 g

4.93 ± 0.18

1.62

 

A compound is immunostimulant if the stimulation index is greater than 1 and immunosuppressant if the stimulation index is smaller than 123. All treated groups had a stimulation index value of greater than 1 (Table 2), so crude bromelain was immunostimulant. The stimulation index of crude bromelain 1.56mg/20g was the highest, while crude bromelain 3.12mg/20g was similar to control, and crude bromelain 4.68mg/20g was the lowest.

 

SRBC Specific Humoral Immune Response:

The purpose of haemagglutination assay was to confirm the effect of crude bromelain on the humoral immune system. This activity was composed of the interaction of antigens with the B cells, the proliferating and differentiating into antibody producing cells. The antibody will bind antigens, then neutralizing it or facilitating its elimination by cross linking to form latex, which readily ingested by phagocytic cells24.

 

Fig. 5: Effect of Crude Bromelain on SRBC Specific Humoral Immune Response

 

Induction of SRBC as an immunogen causes the signal delivery to cells, which responsible for antibody production. The major immunoglobulins, i.e. IgG and IgM, are involved in the complement activation, opsonization, and neutralization of toxins25. The observation was adjusted to immunoglobulin production, i.e. IgM on 7th day and IgG on 15th day26. Antibody titer on the 7th day was more than the 15th day (Fig. 5). Increased antibodies titer, which indicate the enhanced responsiveness of B lymphocyte subsets, were involved in antibody synthesis by the augmentation of the humoral immune response to SRBC by crude bromelain25.

In the control group, there was no immunoglobulin formed on the 7th day, but formed on the 15th day (18.8± 9.09). It happens because the natural antibody formation without immunomodulators occurs at a longer time, due to the immune system must recognize, and adjust the active side first to the induced antigen27. Increased primary and secondary antibody due to administration of crude bromelain was dose-independent. The results showed the increased circulating antibodies in mice which were pretreated with crude bromelain (Fig. 5). There was a significant difference in the treated groups for the primary antibody production (p = 3.85 x 10-12) and secondary antibody production (p = 2.26 x 10-10). The production of primary antibodies was more prominent as compared to the secondary antibodies. Crude bromelain 3.12mg/20g had the greatest activity and similar to the standard, but was significantly different (p = 1.26 x 10-3). In crude bromelain 4.68 mg/20g, pathological mice occur that affect the ADME process, thereby reducing the ability of the humoral immune response.

 

The enhancement of phagocytic index and specific humoral immune response provide the first line of evidence about the immunomodulatory activity of crude bromelain. The significantly increased in the immunomodulatory activity of crude bromelain was attributed to the presence of protease activity and antioxidant activity. Bromelain activates natural killer cells and increases tumor necrosis factor-a, interferon g, interleukin (IL)-1, IL-2, IL-6, and granulocyte-macrophage colony stimulating factor29-31. Bromelain also decreases CD4+ T cell activation and reduces CD25 expression32. While imboost® as a standard immunostimulant contains Echinacea purpurea. Its activity is caused by phagocytosis activation, fibroblast stimulation, and the enhancement of respiratory activity that results in augmentation of leukocyte mobility33.

 

CONCLUSION:

Crude bromelain 3.12mg/20g BW has immunomodolatory activity comparable to the standard.

 

FUNDING:

This research received Kemenristek Dikti Grants in 2019 with contract number 1123aq/UN6.O/LT/2061

 

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

 

ACKNOWLEDGEMENT:

The authors would like to thank Kiki Malinda and Lady Thalia Rosqin for technical assistance.

 

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Received on 04.11.2019           Modified on 28.02.2020

Accepted on 06.03.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(11):5177-5182.

DOI: 10.5958/0974-360X.2020.00905.1