Some medicinal plants as immunostimulant for fish

Journal of Ethnopharmacology 88 (2003) 99–106

Some medicinal plants as immunostimulant for fish Süheyla Karata¸s Dügenci a,∗ , Nazlı Arda b , Akın Candan a a

b

Department of Fish Diseases, Faculty of Fisheries, Istanbul University, Ordu Cad. No. 200, 34470 Laleli/Istanbul, Turkey Section of Molecular Biology, Department of Biology, Faculty of Science, Istanbul University, 34459 Vezneciler/Istanbul, Turkey Received 27 November 2002; received in revised form 12 May 2003; accepted 15 May 2003

Abstract Immunostimulant effects of the dietary intake of various medicinal plant extracts on fish, rainbow trout (Oncorhynchus mykiss), were investigated. For this purpose fish were fed with diets containing aqueous extracts of mistletoe (Viscum album), nettle (Urtica dioica), and ginger (Zingiber officinale). Food containing lyophilized extracts of these plants as 0.1 and 1% was used at a rate of 2% of body weight per day for three weeks. At the end of the experimental period, various parameters of non-specific defence mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes and total plasma protein level were examined. Specific growth rates (SGRs) and condition factors (CFs) of the fish were also measured. Plant materials tested for immunostimulatory food additives caused an enhanced extracellular respiratory burst activity (P < 0.001) compared to the control group. Especially the rainbow trout fed with a diet containing 1% aqueous extract of powdered ginger roots for three weeks exhibited a significant non-specific immune response. Phagocytosis and extracellular burst activity of blood leukocytes were significantly higher in this group than those in the control group. All plant extracts added to fish diet increased the total protein level in plasma except 0.1% ginger. The highest level of plasma proteins was observed in the group fed with 1% ginger extract containing feed. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Rainbow trout; Immunostimulation; Zingiber officinale (ginger); Urtica dioica (nettle); Viscum album (mistletoe)

1. Introduction The outbreak of diseases is a limiting factor in fish culture. At many fish farms and hatcheries several antibiotics, vaccines, and chemotherapeutic agents as well as some immunostimulants have been used to prevent viral, bacterial, parasitic, and fungal diseases. Like man, fish rely on both specific and non-specific mechanisms to protect themselves against invading pathogens. In fish, the primary lines of non-specific defences are the skin and mucus. When pathogens enter the body, cellular and humoral non-specific defence are mobilized. Phagocytosis is one of the main mediators of non-specific immunity to pathogens including bacteria, viruses, and parasites in fish. The most important cells involved in this defence are the phagocytes. These are supported by several soluble factors, such as complement and lysozyme (Dalmo et al., 1997; Verlhac and Gabaudan, 1999; Yano, 1996). It is also well known that the innate immune system in fish can

∗ Corresponding

author. Fax: +90-212-5140379. E-mail address: [email protected] (S.K. Dügenci).

be triggered by many immunostimulants such as levamisole (Siwicki, 1987, 1989; Siwicki et al., 1990; Jeney and Anderson, 1993), glucan (Engstad et al., 1992; Jorgensen and Robertsen, 1995; Chen and Ainsworth, 1992; Ainsworth, 1994; Jeney et al., 1997), glucan plus vitamin C (Verlhac et al., 1996), yeast RNA (Sakai et al., 2001), lipopolisaccharide (Dalmo and Seljelid, 1995; Solem et al., 1995), growth hormone (Sakai et al., 1995, 1996), zeranol (Kele¸s et al., 2002) and kitosan (Siwicki et al., 1994). However, some of the immunostimulants could not be used because of various disadvantages, such as high cost, limited effectiveness upon parenterally administration, etc. On the other hand, a large number of plants have been used in traditional medicine for the treatment and control of several diseases (Duke, 1987). Three of such plants are mistletoe (Viscum album), nettle (Urtica dioica), and ginger (Zingiber officinale). Some of the medicinal plants have been used as the phytogenic basis immunostimulatory preparations. Such preparations have been used, as such as adjuvant therapy, in cancer and AIDS (Mentle et al., 2000; Zarkovic et al., 2001; Verpoorte et al., 1999). Especially mistletoe and nettle have been stated to possess immunomodulatory activity. On the other hand, powdered ginger root is used as spice

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as well as an antiemetic (Phillips et al., 1993) or anticancer agent (Yusof et al., 2002). The aim of the present work was to examine if mistletoe, nettle, or ginger extracts would influence various parameters of non-specific defense in rainbow trout including induced respiratory burst activity (extracellular and intracellular), phagocytosis in blood leukocytes, and total plasma protein level.

2. Materials and methods The experimental design was based on the feeding program established to improve a disease resistance in rainbow trout (Oncorhynchus mykiss). After one week of adaptation to a control diet, fish were fed with experimental diets containing plant extracts for three weeks. At the end of the experimental feeding period, the immune response of the animals was studied. 2.1. Preparation of plant extracts and fish food Viscum album (mistletoe) growing on pine trees (Pinus nigra Arn. ssp. pallasiana) and Urtica dioica (nettle) were collected from their natural habitat in Hacıhalimler Village (Bolu, Northwest of Turkey) and from the Botanical Garden of Istanbul University in July 2000, respectively. A voucher specimen was deposited in the Herbarium of the Section of Botany, Istanbul University. The leaves of the plants were dried at room temperature and powdered. Zingiber officinale was purchased from Ayfer Kaum Plant Products and a Spices Marketing Company (Grand Bazaar, Istanbul) in June 2000 as powdered roots. Plant materials (30 g each) were macerated in distilled water of 90 ◦ C in a rotating waterbath for 2 h. After filtration, aqueous extracts were lyophilized and added to the basal diet obtained from Pınar Feed Factory. Afterwards, food was steam pelleted and allowed to cool and dry. The concentrations of the plant extracts were 0 (control), 0.1, and 1.0, respectively. 2.2. Animals and rearing conditions Rainbow trouts of 41 g mean initial body weight were obtained from the Sapanca Freshwater Production Unit, Faculty of Fisheries, Istanbul University (Turkey) and kept in 1500 l fiberglass tanks. Water temperature was 12.8 ◦ C during the experiment and recorded daily. Fish were divided into seven groups of 30 fishes and were fed with test diet at a rate of 2% of body weight per day throughout three weeks. 2.3. Growth The initial and final weights of fish in each group were measured individually. Specific growth rates (SGRs) and

condition factors (CFs) were calculated according to Laird and Needham (1988) as follows: ln[final mean body weight (g)] − ln[initial mean body weight (g)] × 100 SGR = time interval (days) CF =

weight (g) [lenght (cm)]3

2.4. Immune parameters Rainbow trout blood was collected by caudal vein puncture with heparinised syringes. First, leukocytes were isolated to study extracellular and intracellular respiratory burst activities and phagocytosis. Afterwards, total protein concentration was determined in plasma. 2.4.1. Isolation of leukocytes Leukocytes were isolated from blood according to the density-gradient centrifugation method (Rowley, 1990; Jeney et al., 1997). One milliliter of histopaque 1.119 (Sigma, St. Louis, MO) containing bacto hemagglutination buffer, pH 7.3 was dispensed into a siliconized centrifuge tube. One milliliter of histopaque 1.077 containing bacto hemagglutination buffer and 1 ml of blood were carefully layered on top. The gradient was centrifuged at 500 × g for 15 min at +4 ◦ C by using a swing out bucket rotor. Interface of leukocyte suspension was gently collected with a Pasteur pipette and dispensed into a siliconized tube. Cells were washed twice in phenol red-free Hank’s balanced salt solution (HBSS, Sigma) and adjusted to 2 × 106 viable cells/ml. 2.4.2. Determination of respiratory burst activity Bactericidal activity of phagocytic cells was measured by two methods (detection of extracellular and intracellular activities) as described by Chung and Scombes (1988). Briefly, 100 ␮l of cytochrome c solution (2 mg/ml, in phenol red-free HBSS) containing phorbol 12-myristate 13-acetate (PMA, Sigma, 1 ␮g/ml) was added to a 100 ␮l of leukocyte suspension. In order to test the specificity, a 100 ␮l of cytochrome c solution containing PMA and superoxide dismutase (SOD, Sigma, 300 U/ml) was added to the leukocyte suspension (100 ␮l). Samples were then mixed and incubated at room temperature for 15 min. Readings were taken from a multiscan spectrophotometer operating at 550 nm, using cytochrome c solution as blank. OD values were converted to nanomoles O2 − and final results were expressed as nanomoles O2 − produced per 105 blood leukocytes. The intracellular production of superoxide anion was estimated by the formation of formazan crystals. A 100 ␮l of blood leukocyte solution was mixed with 100 ␮l of nitro blue tetrazolium (NBT) (0.2% in PBS) containing PMA (1 ␮g/ml), and SOD (300 U/ml). After incubation at room

Groups

Extracellular respiratory burst activity (nmol O2 / 105 leukocyte)

Intracellular activity (NBT) (OD at 650 nm)

Phagocytosis (OD at 510 nm)

Plasma protein level (g/dl)

Growth parameters of rainbow trouts experimental period (day) 0

21

Weight (g) 0.1% ginger 1% ginger 0.1% nettle 1% nettle 0.1% mistletoe 1% mistletoe Control

1.05 1.26 1.04 1.03 1.03 1.07 1.02

± ± ± ± ± ± ±

0.05 0.23 0.04 0.03 0.03 0.05 0.05

0.09 0.17 0.12 0.11 0.13 0.13 0.10

± ± ± ± ± ± ±

0.2 0.05 0.04 0.02 0.04 0.06 0.02

2.21 2.37 1.93 2.03 2.06 2.03 1.88

± ± ± ± ± ± ±

0.08 0.26 0.35 0.56 0.27 0.18 0.21

2.26 3.84 3.40 3.58 3.06 3.10 2.48

± ± ± ± ± ± ±

0.25 0.13 0.12 0.10 0.20 0.18 0.04

41.00 41.63 38.50 43.60 36.72 42.06 41.63

± ± ± ± ± ± ±

8.33 6.74 3.52 9.06 8.49 8.79 6.72

Length (cm) 15.71 15.94 15.68 15.87 15.04 15.80 15.62

± ± ± ± ± ± ±

0.83 0.78 0.57 1.04 1.20 1.17 1.19

Weight (g) 52.61 51.72 52.42 53.48 51.00 52.86 52.85

± ± ± ± ± ± ±

4.63 4.57 6.07 8.22 8.20 7.64 8.81

Length (cm) 16.68 16.95 17.27 17.13 16.77 17.09 17.08

± ± ± ± ± ± ±

0.81 0.96 0.57 0.83 1.25 0.85 1.00

S.K. Dügenci et al. / Journal of Ethnopharmacology 88 (2003) 99–106

Table 1 Mean (±S.D.) of growth and non-specific immune parameters in rainbow trouts (n = 10)

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temperature for 60 min with regular mixing, plates were centrifuged at 500 × g for 3 min and the supernatants were discarded. Cells were washed twice with HBSS and fixed in 70% methanol. Formazan crystals were dissolved by adding a 120 ␮l of 2 M KOH and 140 ␮l DMSO. After the formation of the turquoise-blue-colored solutions, absorbance values were read in a multiscan spectrophotometer operating at 620 nm using KOH/DMSO (120 ␮l of 2 M KOH/140 ␮l DMSO) as blank.

S.G.R

2.4.3. Phagocytosis Phagocytotic activity of leukocytes was determined by the method of Seeley et al. (1990). Congo red stained yeast cells, which phagocytozed by the cells, used in this assay. First, yeast cells (Saccharomyces cerevisiae) were stained with Congo red. Three milliliters of a Congo red solution, 0.87% in PBS, was added to yeast cell suspension (1.5 g), and this mixture was allowed to stain for 15 min at room temperature. Afterwards, 7 ml of distilled water was added and the resulting solution was thoroughly mixed, then autoclaved for 15 min to kill and fix the yeast. These cells were then washed several times in HBSS and stored at +4 ◦ C until use. Prior to use, cells were resuspended at 4 × 107 cells/ml in HBSS. Two hundred and fifty microliters of the leukocyte solution was mixed with 500 ␮l of the Congo red stained and autoclaved yeast cell suspension (yeast cell/leukocyte, 40/1). The mixtures were incubated at room temperature for 60 min. Following incubation, 1 ml ice-cold HBSS was

Table 2 SGRs and CFs of fish in each group Groups

SGR

Condition factor Initial

Final

0.1% ginger 1.0% ginger 0.1% nettle 1.0% nettle 0.1% mistletoe 1.0% mistletoe Control

1.114 1.032 1.470 0.972 1.563 1.028 1.136

1.061 1.021 0.997 1.068 1.067 1.068 1.066

1.137 1.072 1.014 1.059 1.068 1.054 1.049

added and 1 ml of histopaque (1.077) was layered to the bottom of each sample tube by a syringe. The samples were centrifuged at 850 × g for 5 min to separate macrophages from free yeast cells. Macrophages were harvested and washed twice in HBSS. The cells were resuspended in 1 ml trypsin–EDTA solution (5 g/l trypsin and 2 g/l EDTA, Sigma) and incubated at 37 ◦ C overnight. The absorbance values were measured at 510 nm using trypsin–EDTA as blank. 2.5. Protein concentration in plasma The total plasma protein content was determined by the commercial protein-kit (Sigma, P 5656) based on the principle of the Lowry reaction. Bovine serum albumin was used as a standard and the data were expressed in g/dl.

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.1 % ginger

1% ginger

0.1 % nettle

1% nettle

0.1 % mistletoe

1% mistletoe

control

groups 1.15

Condition Factor

initial 1.1

final

1.05 1 0.95 0.9 0.1 % ginger

1% ginger

0.1 % nettle

1% nettle

0.1 % mistletoe

groups Fig. 1. SGRs and CFs.

1% mistletoe

control

S.K. Dügenci et al. / Journal of Ethnopharmacology 88 (2003) 99–106

2.6. Statistical analysis Analysis of variance and Duncan’s multiple range tests were run to compare the dietary treatment values using the SPSS 7.5 program. Statistical analysis was run in the same way for all parameters tested. The means and standard errors were calculated for each treatment. The accepted levels of significance were 0.05 and 0.001.

3. Results Mean (±S.D.) of growth and non-specific immune parameters in rainbow trout (n = 10) were summarized in Table 1. 3.1. Growth performance SGRs and CFs of fish in each group (Table 2) were calculated by using the growth parameters in Table 1 and as also shown in Fig. 1. As shown in Table 2 the results of the SGRs and CFs in this study were in optimum level. 3.2. Non-specific defense mechanisms

The results of this trial showed that feeding rainbow trout with 1% doses of ginger (Zingiber officinale) for three weeks stimulated non-specific parameters of neutrophils. Fish have several types of phagocytic leukocytes, which are present in blood, the peritoneal cavity, and a variety of tissue locations. Phagocytosis and the production of oxygen free radicals via the respiratory burst are important events in

5

1.2 1 0.8 0.6 0.4 0.2 0 1% ginger

0.1 % nettle

(A)

Intracellular activity 650 nm

4. Discussion

1.4

0.1 % ginger

1% nettle

0.1 % mistletoe

1% mistletoe

control

groups

0.3 0.25 0.2 0.15 0.1 0.05 0 0.1 % ginger

(B)

oxidative radical production was higher in the groups fed with different plant extracts compared to the control group, but statistical analysis showed that there was a significant stimulatory effect of 1% ginger (P < 0.001) (Fig. 2A). Intracellular production of superoxide anion was detected by formation of formazan crystals, revealed that there was an overproduction of superoxide anion in all groups compared to control animals. But this enhancement was not statistically significant (P < 0.05) (Fig. 2B). Phagocytosis of blood leukocytes increased in all groups, but this increase was only significant for one group (fed with 1% ginger containing food) (P < 0.05) (Fig. 3). Statistical analysis showed that total protein level in plasma was significantly enhanced in all experiments compared to control (P < 0.001), except the group fed with 0.1% ginger (Fig. 4).

1.6

O2 /10 leukocyte)

Extracellular activity (nmol

Respiratory burst activities (extracellular and intracellular) of blood leukocytes were shown in Fig. 2. Extracellular

103

1% ginger

0.1 % nettle

1% nettle

0.1 % 1% control mistletoe mistletoe

groups

Fig. 2. Respiratory burst activities of blood leukocytes: (A) extracellular activity; (B) intracellular activity.

∗∗ P

< 0.001; vertical lines are mean ± S.E.

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Phagocytosis (O.D. at 520 nm)

104

3 2.5 2 1.5 1 0.5 0 0.1 % ginger

1% ginger

0.1 % nettle

1% nettle

0.1 % mistletoe

1% mistletoe

control

groups Fig. 3. Changes in phagocytosis of blood leukocytes isolated from rainbow trout fed with foods added different doses of plant extracts. ∗ P < 0.05; vertical lines are mean ± S.E.

Total protein (g/dL)

bactericidal pathways in fish, but mechanisms are not well established (Sharp and Secombes, 1992, 1993). Phagocytes have a unique membraneous enzyme, NADPH oxidase, capable of one-electron reduction of molecular oxygen into superoxide anion (O2 − ) during a process known as the respiratory burst. Since O2 − is the first product to be released from the respiratory burst, the measurement of O2 − has been accepted as a direct and accurate way of measuring respiratory burst activity (Secombes, 1990; Secombes and Olivier, 1997; Roch, 1999). We used two main methods to measure O2 − : The reduction of ferricytochrome c to determine extracellular O2 − and the reduction of the redox dye NBT to determine intracellular O2 − . The increase of intracellular activity has been reported in Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) fed with different doses of glucan (Jorgensen and Robertsen, 1995; Engstad and Robertson, 1995; Dalmo and Seljelid, 1995; Dalmo et al., 1996; Siwicki, 1989). Kele¸s et al. (2002) reported that intracellular activity was specially high in fish fed with dietary zeranol (10 and 20 ppm). It was also reported that intracellular activity was specially high in fish (Oncorhynchus mykiss) fed with three dietary immunos-

timulats (MacroGard, Candida utilis, Saccharomyces cerevisiae) (Siwicki et al., 1994). But we could not detect such a high intracellular activity in our study. In spite of this, our study showed that fish fed with ginger (Zingiber officinale) had significantly greater extracellular activity than fish fed with other diets. It is well known that fish treated with immunostimulants show increased phagocytosis as well as respiratory burst activity (Sakai, 1999; Siwicki et al., 1994; Robertsen et al., 1994; Anderson, 1992; Verlhac et al., 1998; Blazer and Wolke, 1984; Blazer et al., 1989). Our finding pointing out that reactive oxygen species occur in the interstial space might be important in the respiratory burst. Secombes and Olivier (1997) also proposed that the release of superoxide anion, hydrogen peroxide, and hypochlorous acid (HOCl) into the phagosome and extracellular space during the respiratory burst is considered to be one of the most important mechanisms involved in the bactericidal activity of macrophages. Siwicki (1989) and Siwicki et al. (1990) reported that oral administration of levamisole increased the phagocytic index of phagocytic cells. Additionally, it was reported that feeding

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0.1 % ginger

1% ginger

0.1 % nettle

1% nettle

0.1 % 1% mistletoe mistletoe

control

groups Fig. 4. Plasma protein concentrations in rainbow trout fed with foods added different doses of plant extracts.

∗∗ P

< 0.001; vertical lines are mean ± S.E.

S.K. Dügenci et al. / Journal of Ethnopharmacology 88 (2003) 99–106

yeast products (MacroGard, Candida utilis, Saccharomyces cerevisiae) give rise to an enhanced phagocytic activity of blood leukocytes in rainbow trout. This all beside, a wide variety of standard dietary ingredients reported to affect immune responses. In this study, phagocytic activity of blood leukocytes increased in rainbow trout fed with plant extracts containing food, especially 1% ginger (Zingiber officinale). One of the indicators of humoral defence mechanisms, plasma protein levels were also found to increase in fish fed with plant extracts. Our results are in line with the observation that humoral factors may enhance phagocytosis in fish (Chung and Secombes, 1987). In conclusion, cellular and humoral defense mechanisms against fish pathogens as viruses, bacteria, and fungi that enter fishes by means of injury and the mucosa can be stimulated by using ginger (Zingiber officinale) extract as immunostimulant. Nettle (Viscum album) and mistletoe (Urtica dioica) with a known immunomodulatory effect in human was only moderately active in this study. May be this, can be related to either the plant species, the extraction procedure, the percentage of extract in the diet and the effect genetics of the experimental animal used in this assay (Kele¸s et al., 2001). This work provides a new perspective for the use of medicinal plants as adjuvant therapy added to fish food to prevent diseases. Thus, if immunostimulants will be applied before outbreaks of disease, high numbers of mortalities might be avoided. Further studies including determination of optimal doses, the active principle of the plant extract and feeding protocols for food additives are in progress. Acknowledgements The authors would like to thank Research Assistant Dr. Yelda Aktan, Didem Demircan and Deniz Tosun from Istanbul University, Faculty of Fisheries, for their technical assistance. This work was supported by the Research Fund of Istanbul University (Project no. 1339/280799).

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