Effects of Prunella vulgaris labiatae extract on specific and non-specific immune responses in tilapia (Oreochromis niloticus)
© Park and Choi; licensee BioMed Central Ltd. 2014
Received: 7 April 2014
Accepted: 7 April 2014
Published: 15 May 2014
We examined the effects of Prunella vulgaris Labiatae (P. vulgaris L.) on specific and non-specific immune responses of Nile tilapia, Oreochromis niloticus. The optimal concentration without toxicity of P. vulgaris was determined to 30-40 μg/ml in vitro and 120 μg/100 g of fish in vivo. P. vulgaris significantly elicited an antibody titer compared to FCA or β-glucan. β-glucan plus P. vulgaris group synergistically enhanced antibody production. No significant difference in antibody production was observed between P. vulgaris and P. vulgaris plus β-glucan group. A respiratory burst activity of head kidney (HK) leucocytes of tilapia administered with 300 or 500 μg P. vulgaris was significantly (p < 0.05) enhanced compared with the PBS-injected control group and FCA-treated group. Maximum increase in the NBT reduction value was observed in 500 μg P. vulgaris group but no significant difference was found between 300 and 500 μg P. vulgaris group. The level of serum lysozyme activity was significantly (p < 0.05) higher in the 300 and 500 μg P. vulgaris than 100 μg P. vulgaris and FCA group. The phagocytic activities of HK leucocytes from tilapia administered with 300 and 500 μg P. vulgaris were significantly (p < 0.05) higher than 100 μg P. vulgaris and the control group. P. vulgaris was revealed with a good immunoadjuvant evoking the specific and non-specific immune responses of tilapia.
KeywordsPrunella vulgaris Respiratory burst activity Lysozyme activity Tilapia
One of the most promising methods for controlling diseases in aquaculture is strengthening the defense mechanisms of fish through prophylactic administration of immunostimulants . Immunostimulants are naturally occurring compounds that modulate the immune system by increasing host resistance to infectious pathogens, and they have been widely used in aquaculture [2–4]. Traditional disease control strategies employing antibiotics and chemical disinfectants are no longer recommended due to the emergence of bacterial resistance as well as concerns over the environment and wildlife protection. Although vaccination has been shown to be an effective prophylactic method for disease control in fish , there are some methodological problems related to high costs and stress . Already, remarkable success has been achieved using immunostimulants as a more environmentally friendly approach to disease management [7–9].
Several compounds, including β-glucans, chitin, algal and mistletoe extracts, and bacterial polysaccharides, have been used to enhance immunity and disease resistance in a variety of fish species [7, 10–12]. β-glucan administration has been reported to augment antibody production, complement activity, lysozyme activity, phagocytic activity, and respiratory bursts in channel catfish Ictalurus punctatus, Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, gilthead rainbow trout Sparus auratus, and sea bass Dicentrarchus labrax.
Prunella vulgaris is a perennial herb that is used in traditional medicine for the clinical treatment of sore throat, fever, and accelerated wound healing [18, 19]. The organic fraction of P. vulgaris exhibits antioxidative and antimicrobial activities , whereas aqueous extracts of P. vulgaris inhibit HIV-1 infection . In aquaculture, P. vulgaris was reported to have efficacy as a dietary supplement, although only up-regulation of natural immunity was observed . To further study the availability of P. vulgaris as an immunoadjuvant to elicit a vaccination effect, we measured the specific antibody titer following intraperitoneal injection of P. vulgaris with an antigen in tilapia as a fish model.
To overcome the disease problem affecting fish culture systems, the present study applied indigenous P. vulgaris as an appropriate immnoadjuvant in order to augment specific and non-specific immunities as well as disease resistance in fish.
Materials and methods
Nile tilapia, Oreochromis niloticus, fish weighing about 100-150 g each were obtained from a fish farm in Kunsan National University, Korea and acclimated for 2 weeks to laboratory conditions in 70 L glass aquaria containing re-circulated and aerated water at 23-23°C. They were acclimated to this environment for at least 2 weeks prior to use and fed daily using a commercial diet during the adaptation and experimental periods. The health status of the animals was checked daily by observing fish behavior, and there were no clinical symptoms such as abnormal swimming patterns or body color changes.
Nitroblue tetrazolium (NBT), Percoll, hemocyanin (HC), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), phorbol myristate acetate (PMA), and Minimum essential medium (MEM) were purchased from Sigma Chemicals CO. Hanks balanced salt solution (HBSS), fetal bovine serum (FBS), and antibiotic-antimycotic were obtained from Gibco BRL, Grand Island, NY. Sodium nitrite, sulfanylamide, and phosphoric acid were purchased from ICN Biomedicals. Bakers’ yeast and Saccharomyces cerevisiae purchased from Oriental Yeast Co. Ltd., Tokyo, and thioglycollate broth was obtained from Difco Laboratories, Detroit, USA.
Extraction of P. vulgaris
P. vulgaris originating from South Korea was kindly donated by an herbal medicine company (Sanyacho-Nongwon, Namyangju, Korea). Extraction of P. vulgaris was performed according to Lee’s method . Briefly, the chopped flowers, stems, and leaves of P. vulgaris (100 g) were placed in distilled water (1,000 ml) and stirred at 4°C overnight. After centrifugation at 15,000 × g for 20 min, the supernatant was filtered through 0.2 μm pore-sized filters. Protein content of P. vulgaris extract was determined using a commercial protein assay kit (Bio-Rad Lab, USA) and stored at 4°C.
In vitro and in vivo toxicities of P. vulgaris
The in vitro toxicity of P. vulgaris was tested against EPC and CHSE-214 fish cell lines. Respective cell lines (1 × 106 cells) were dispensed into each well of a 24-well plate (Costar, USA), followed by administration of various concentrations of P. vulgaris extract ranging from 10 ng to 100 μg/ml. After incubation at 24°C for 3 days, MTT assay was performed according to the method of Daly et al. . Briefly, the tissue culture plates were centrifuged at 500 × g for 10 min, after which the supernatant fluids were carefully removed without disturbing the cell pellet or formazan precipitate. The formazan crystals were then dissolved by addition of 200 μl of dimethyl sulphoxide (DMSO) (Sigma) to each well, followed by 25 μl of glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). Contents of the wells were then thoroughly mixed with a multichannel pipette. After 10 min, formazan development was read at 595 nm using an ELISA reader (ASYS HITECH, Austria).
To determine whether or not P. vulgaris has serious toxicity in vivo, 100 and 1000 μg of P. vulgaris/100 g of fish were intraperitoneally (I.P.) injected into seven fish per group. Fish blood was collected 4 days after injection, and the concentrations of glutamic oxaloacetatic trams aminase (GOT), glutamic pyruvate transaminase (GPT), and c-creatin were determined on a Fuji Dry Chem System (Fuji Photo Film Co. Ltd, Japan).
Administration of P. vulgaris to elicit non-specific immune response in tilapia
Tilapia were divided into five groups of seven fishes per group. Fish in each group were I.P injected with 100, 300, and 500 μg of P. vulgaris/100 g of fish in 0.5 ml of phosphate-buffered saline (PBS). The remaining group of fish was injected with an equivalent volume of sterile PBS or 1:1 emulsified Freund's complete adjuvant (FCA) (Sigma) as a control. On day 4 post-injection, blood and head kidney leucocytes were obtained from each fish.
Antibody production upon administration of P. vulgaris plus other immunostimulants
The immunostimulating effect of P. vulgaris was compared with those of FCA and β-glucan based on elevated antibody production. HC was used as an antigen to evoke a specific antibody response. For I.P. injection, 300 μg of P. vulgaris and 100 μg of HC were mixed and administered in a volume of 200 μl. Tilapia were divided into six groups (five fish per group), after which HC was injected alone (control) or mixed and injected with 100 μl of other adjuvants. The total injection volume was adjusted to 200 μl in all experiments. P. vulgaris was I.P. injected at a concentration of 300 μg suspended in 200 μl of phosphate-buffered saline (PBS). In the FCA group, 100 μl of FCA was 1:1 emulsified with HC suspended in 100 μl of PBS. In the FCA plus P. vulgaris group, 100 μl of FCA was added to the P. vulgaris and HC mixture and then I.P. injected at a volume of 200 μl. The optimized concentration (50 μg) of β-glucan was injected with HC at a volume of 200 μl. In the β-glucan plus P. vulgaris group, 50 μg of β-glucan and 300 μg P. vulgaris were mixed together and I.P. injected at a volume of 200 μl. On day 30 post-injection, blood was harvested from the fishes in each group, followed by antibody titer assay using an ELISA reader.
Isolation of head kidney (HK) leucocytes
The method described by Santarem et al.  was followed with some modifications. The tilapia HK was dissected out by ventral incision, cut into small fragments, and then transferred into 5 ml of HBSS. Cell suspensions of the HK were obtained by teasing HK tissues with two slide glasses in HBSS in a Petri dish (Coring, USA). After sedimentation of tissue debris at 4°C for 1 min, the supernatants were removed. HK cell suspensions were then layered over a 34-51% Percoll gradient and centrifuged at 1000 × g for 40 min at 14°C. After centrifugation, leucocyte bands located above the 34-51% interfaces were collected using a Pasteur pipette and washed twice at 120 × g for 8 min in HBSS. The concentration of viable cells was determined by trypan blue exclusion.
Blood was collected from the dorsal aorta of tilapia. Blood was allowed to clot at 20°C for 30 min and then cooled at 0°C for 1 h. Serum was obtained by centrifugation at 1000 × g for 8 min. Sera were frozen at -20°C until used.
Reactive oxygen intermediates (ROI) production assay
ROI production by tilapia kidney cells after administration of P. vulgaris was assessed by monitoring reduction of NBT . Leucocytes (1 × 105 cells) were washed once with HBSS at 60 × g for 3 min at 4°C and then incubated in 100 μl of complete media in the presence of PMA and 1 μg/ml of NBT. After 1 h of incubation at 25°C, excess NBT was washed out with PBS, and the leucocytes were fixed with 70% methanol. After discarding the methanol, the leucocytes were washed twice with PBS. The reduced formazan was then solubilized with 120 μl of KOH and 140 μg of DMSO, after which optical density values were read at 620 nm on an ELISA reader.
Serum lysozyme activity was measured using a modified turbidimetric microtiter plate technique according to Ellis . Briefly, a standard suspension of 0.15 mg/ml of Micrococcus lysodeikticus (Sigma) was prepared in 66 mM phosphate buffer (pH 6.0). Tilapia serum (50 μl) was then added to 1 ml of the bacterial suspension, after which the absorbance reduction was recorded at 0.5 and 4.5 min intervals at 450 nm on a spectrophotometer (SHIMADZU UV-1600PC). One unit of lysozyme activity was defined as a reduction in absorbance of 0.001/min.
Tilapia HK leucocytes were adjusted to 1 × 106 cells/200 μl/well in 5% FBS-MEM and dispensed in an 8-well slide chamber (Nunc, Denmark), followed by overnight incubation at 25°C. Following incubation, 1 × 107 cells/ml of zymosan (Sigma) was added. The mixture was incubated at 25°C for 1 h with occasional shaking, after which 50 μl of the mixture was smeared onto a glass slide, air-dried, and stained with Wright’s solution. Phagocytic activity (PA)  was calculated by enumerating 500 leucocytes per fish under a microscope. PA = number of cells ingesting zymosans/number of cells observed × 100.
Statistical significance of the differences between the groups was calculated by applying Student’s 2-tailed t-test.
Results and discussion
Prunella vulgaris is a perennial plant known for its self-healing properties in Western herbal medicine [27, 28], and it traditionally has been used for treating various diseases such as an allergies and inflammation in East Asian countries . In addition, P. vulgaris has been reported to have immunomodulatory effects such as activation of macrophages [27, 28, 30]. The effect of P. vulgaris on fish immunity in aquaculture has only been reported by Harikrishnan et al. . Specifically, they investigated the dietary effects of P. vulgaris on the non-specific immune response as well as disease resistance against Uronema marinum. In the present study, we tested the availability of P. vulgaris as a potent immunoadjuvant to achieve an enhanced vaccination effect. Furthermore, the in vitro and in vivo toxicities of P. vulgaris were investigated in tilapia as a fish model. Lastly, to determine the optimal amount of P. vulgaris extract that evokes an immune response in tilapia, we administered P. vulgaris extract by intraperitoneal (I.P.) injection.
The levels of GOT, GPT and c-creatin in tilapia sera following injection of P. vulgaris
Standards for toxicity
Doses of P. vulgaris injected in fish
19 ± 3 mg/ML
23 ± 5 mg/ML
25 ± 3 mg/ML
40 ± 6 mg/ML
39 ± 5 mg/ML
42 ± 6 mg/ML
31 ± 4 mg/ML
28 ± 5 mg/ML
33 ± 7 mg/ML
Although the materials used in the study were different, Gopalakannan and Aurl  and Luo et al.  also reported that fish treated with a high dosage of chitosan display significantly inhibited phagocytosis compared to low dosage. This result suggests that the high level of P. vulgaris directly induced phagocytosis, thereby exhausting the cells.
P. vulgaris elevated almost all non-specific immune parameters as well as specific humoral immunity. Therefore, P. vulgaris could be a promising immunomodulatory material for inducing specific and non-specific immune responses in fish. Further studies on using P. vulgaris as a dietary supplement in aquaculture are currently underway.
This work was supported by a grant from the Fisheries Science Institute in Kunsan National University in 2013.
- Robertsen B: Modulation of the non-specific defense of fish by structurally conserved microbial polymers. Fish Shellfish Immunol. 1999, 9: 269-90. 10.1006/fsim.1998.0186.View ArticleGoogle Scholar
- Ai QH, Mai KS, Zhang I, Tan BP, Zhang WB, Xu W: Effects of dietary β-1, 3 glucan on innate immune response of large yellow croaker, Pseudosciaena crocea. Fish Shellfish Immunol. 2007, 22: 394-402.View ArticlePubMedGoogle Scholar
- Selvaraj V, Sampath K, Sekar V: Administration of lipopolysaccharide increaseds specific and non-specific immune parameters and survival in carp infected with Aeromonas hydrophila. Aquaculture. 2009, 286: 176-183. 10.1016/j.aquaculture.2008.09.017.View ArticleGoogle Scholar
- Ringø E, Olsen RE, Gifstad TØ, Dalmo RA, Amlund H, Hemre GI, Bakke AM: Prebiotics in aquaculture: a reviews. Aquacul Nutr. 2010, 16: 117-136. 10.1111/j.1365-2095.2009.00731.x.View ArticleGoogle Scholar
- Midtlyng PJ, Reitan LJ, Lillehaug A, Ramstad A: Protection, immune responses and side effects in Atlantic salmon (Salmo salar L.) vaccinated against furunculosis by different procedures. Fish Shellfish Immunol. 1996, 6: 599-613. 10.1006/fsim.1996.0055.View ArticleGoogle Scholar
- Ellis AE: Immunity to bacteria in fish. Fish Shellfish Immunol. 1999, 9: 291-308. 10.1006/fsim.1998.0192.View ArticleGoogle Scholar
- Raa J: The use of immunostimulatory substances in fish and shellfish farming. Rev Fish Sci. 1996, 4: 229-288. 10.1080/10641269609388587.View ArticleGoogle Scholar
- Sakai M: Current research status of fish immunostimulants. Aquaculture. 1999, 172: 63-92. 10.1016/S0044-8486(98)00436-0.View ArticleGoogle Scholar
- Peddie S, Zou J, Secombes J: Immunostimulation in the rainbow trout (Oncorhynchus mykiss) following intraperitoneal administration of Ergosan. Vet Immunol Immunopathol. 2002, 86: 101-113.View ArticlePubMedGoogle Scholar
- Anderson DP: Environmental factors on fish health: immunological aspects. The fish immune system: organism, pathogen, and environment. Edited by: Iwama G, Nakanishi T. 1996, 289-310. San Diego, CA: AcademicGoogle Scholar
- Yoon TJ, Park KH, Choi SH: Korean mistletoe (Viscum album Coloratum) extract induces eel (Anguilla japonica) non-specific immunity. Immune Network. 2008, 8 (4): 124-129.View ArticleGoogle Scholar
- Park KH, Choi SH: The effect of mistletoe, Viscum album coloratum, extract on innate immune response of Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol. 2012, 32: 1016-1021.View ArticlePubMedGoogle Scholar
- Chen D, Ainsworth AJ: Glucan administration potentiates immune defense mechanisms of channel catfish, Ictalurus punctauts Rafinesque. J Fish Dis. 1992, 15: 295-304. 10.1111/j.1365-2761.1992.tb00667.x.View ArticleGoogle Scholar
- Engstad R, Robertsen B, Frivold E: Yeast glucan induces increase in lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish Shellfish Immunol. 1992, 2: 287-297. 10.1016/S1050-4648(06)80033-1.View ArticleGoogle Scholar
- Verlhac V, Gabaudan J, Obach A, Schuep W, Hole R: Influence of dietary glucan and vitamin D on non-specifici responses of rainbow trout (Oncorhynchus mykiss). Aquaculture. 1996, 144: 123-133.View ArticleGoogle Scholar
- Castro R, Couso N, Obach A, Lamas J: Effect of different beta-glucans on the respiratory burst of turbot (Psetta maxima) and gilthead rainbow trout (Sparus aurata) phagocytes. Fish Shellfish Immunol. 1999, 9: 529-541. 10.1006/fsim.1999.0210.View ArticleGoogle Scholar
- Bagni M, Romano N, Finoia MG, Abelli L, Scapigliate G, Tiscar PG: Short- and long-term effects of a dietary yeast β-glucan (Macrogard) and alginic acid (Ergosan)preparation on immune response in sea bass (Dicentrarchus labrax). Fish Shellfish Immunol. 2005, 18: 311-325.View ArticlePubMedGoogle Scholar
- Psotova J, Kolar M, Sousek J, Svagera Z, Vicar J, Ulrichova J: Biological activities of Prunella vulgaris extract. Phytother Res. 2003, 17: 1082-1087.View ArticlePubMedGoogle Scholar
- Psotova J, Svobodova A, Kotarova H, Walterova D: Photoprotective properties of Prunella vulgaris and rosmariinic acid on human keratinocytes. J Photochem Photobiol B. 2006, 84: 167-174.View ArticlePubMedGoogle Scholar
- Oh C, Price J, Brindley MA, Widrlechner MP, Qu L, McCoy JA, Murphy P, Hauck D, Maury W: Inhibition of HIV-1 infection by aqueous extracts of Prunella vulgaris L. Virol J. 2011, 23: 8-12.Google Scholar
- Harikrishnan R, Kim JS, Kim MC, Balasundaram C, Heo MS: Prunella vulgaris enhances the non-specific immune response and disease resistance of Paralichthys olivaceus against Uronema marinum. Aquaculture. 2011, 318: 61-66. 10.1016/j.aquaculture.2011.05.020.View ArticleGoogle Scholar
- Lee JB, Kang TB, Choi SH, Lee U, Kim AJ, Jeong CJ, Lee HC, Cho YS, Won JG, Lim JC, Yoon TJ: Effect of Prunella vulgaris Labiatae extract on inmate immune cells and anti-metastatic effect in mice. Food Sci Biotechnol. 2009, 18 (1): 218-222.Google Scholar
- Daly JG, Moore AR, Olivier G: A colorimetric assay for the quantivication of brook trout (Salvelinus fontinalis) lymphocyte mitogenesis. Fish Shellfish Immunol. 1995, 5: 265-273. 10.1006/fsim.1995.0026.View ArticleGoogle Scholar
- Santarem M, Novoa B, Figueras A: Effect of beta-glucans on the non-specific immune responses of turbot (Scophthalmus L.). Fish Shellfish Immunol. 1997, 7: 429-437. 10.1006/fsim.1997.0096.View ArticleGoogle Scholar
- Secombes CJ, Chung S, Jeffries AH: Superoxide anion production by rainbow trout macrophages detected by the reduction of ferricytochrome C. Dev Comp Immunol. 1988, 12: 201-206.View ArticlePubMedGoogle Scholar
- Sahoo PK, Mukherjee SC: Effect of dietary β-1, 3 glulcan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton). Fish Shellfish Immunol. 2001, 11: 683-695.View ArticlePubMedGoogle Scholar
- Fang X, Chang RC, Yuen WH, Zee SY: Immune modulatory effects of Prunella vulgaris L. Int J Mol Med. 2005, 15: 491-496.PubMedGoogle Scholar
- Fang X, Yu MM, Yuen WH, Xee SY, Chang RC: Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages. Int J Mol Med. 2005, 16: 1109-1116.PubMedGoogle Scholar
- Kim SY, Kim SH, Shin HY, Lim JP, Chae BS, Park JS, Hong SG, Kim MS, Jo DG, Park WH, Shin TY: Effects of Prunella vulgaris on mast cell-mediated allergic reaction and inflammatory cytokine production. Exp Biol Med. 2007, 232: 921-926.Google Scholar
- Han EH, Choi JH, Hwang YP, Park HJ, Choi CY, Chung YC, Seo JK, Jeong HG: Immunostimulatory activity of aqueous extract isolated from Prunella vulgaris. Food Chem Toxicol. 2009, 47 (1): 62-69.View ArticlePubMedGoogle Scholar
- Yoon TJ, Yoo YC, Kang TB, Her E, Kim K, Azuma I, Kim JB: Cellular and humoral adjuvant activity of lectins isolated from Korean mistletoe (Viscum album colaratum). Int Immunopharmacol. 2001, 1: 881-889.View ArticlePubMedGoogle Scholar
- Salinas I, Cuesta A, Esteban MA, Meseguer J: Dietary administration of Lactobacillus delbrueckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish Shellfish Immunol. 2005, 19: 67-77.View ArticlePubMedGoogle Scholar
- Babior BM: Oxidants from phagocytes: agents of defense and destruction. Bolld. 1984, 64: 959-966.Google Scholar
- Bliznakov EG, Adler AD: Nonlinear response of the reticuloendothelial system upon stimulation. Pathol Microbiol. 1972, 38: 393-410.Google Scholar
- Gialdroni-Grassi G, Grassi C: Bacterial products as immunomodulating agents. Int Arch Allergy Appl Immunol. 1985, 76 (Supp.1): 119-127.View ArticlePubMedGoogle Scholar
- Kenyon CJ, McKeever A, Oliver JA, Henderson IW: Control of renal and adrenocortical function by the renin-angiotensin system in two euryhaline teleost fishes. Gen Comp Endocrinol. 1985, 58: 93-100.View ArticlePubMedGoogle Scholar
- Anderson DP, Jeney G: Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection on rainbow trout (Oncorhynchus mykiss). Vet Immunol Immunopathol. 1992, 34: 379-389.View ArticlePubMedGoogle Scholar
- Siwicki AK, Cossarini-Dunier M, Studnicka M, Demael A: In vivo effect of the organophosphorus insecticide trichlorphon on immune response of carp (Cyprinus carpio). II. Effect of high doses of trichlorphon on nonspecific immune response. Ecotoxicol Environ Saf. 1990, 19: 99-105.View ArticlePubMedGoogle Scholar
- Alexander JB, Ingram GA: Noncellular non-specific defense mechanisms of fish. Ann Rev Fish Dis. 1992, 2: 249-79.View ArticleGoogle Scholar
- Cavaillon JM: Cytokines and macrophages. Biomed Pharmacother. 1994, 48: 445-453.View ArticlePubMedGoogle Scholar
- Gopalakannan A, Aurl V: Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture. 2006, 255: 179-187. 10.1016/j.aquaculture.2006.01.012.View ArticleGoogle Scholar
- Luo L, Cai X, He C, Xue M, Wu X, Cao H: Immune response, stress resistance and bacterial challenge in juvenile rainbow trouts Oncorhynchus mykiss fed diets containing chitosan-oligosaccharides. Curr Zool. 2009, 55: 416-22.Google Scholar
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