Truong Quynh Nhu * , Bui Thi Bich Hang and Nguyen Thanh Phuong

* Corresponding author (tqnhu@ctu.edu.vn)

Abstract

Psidium guajava and Phyllanthus amarus are well-known as herbs that have been widely used in Vietnamese traditional medicines. However, studies on the effects of these plants in improving immune system of striped catfish (Pangasianodon hypophthalmus) have received less attention. Therefore, this study aimed to investigate the effect of ethanolic leave extracts from  P. guajava, P. amarus, and their mixtures (1: 1, v/v) on leukocytes of striped catfish (P. hypophthalmus). Leukocytes (5×106 cells/mL) collected from peripheral blood and head kidney were treated with P. guajava, P. amarus and their extracts combination at 2 different concentrations (10 and 100 mg/mL) for 24 hrs. The results showed that P. guajava, P. amarus and their extracts combination could stimulate positively to increased immune parameters. Specifically, lysozyme activity and total cellular Ig in treatments treated with extracts increased remarkably compared to the control (p<0.05). However, the reactive oxygen species activity statistically increased only in the treatment stimulated with 100 mg/mL of P. amarus and treatment supplemented 10 mg/mL of their combination in head kidney leukocytes. Similarly, the nitric oxide synthases activity of head kidney leukocytes only enhanced remarkably after stimulation with 100 mg/mL of P. guajava extract. Besides, the mixture of P. guajava and P. amarus at the high dose (100 mg/mL) also stimulated complement activity compare to the control (p<0,05).
Keywords: Psidium guajava, Phyllanthus amarus, leukocytes, immune response, striped catfish

Tóm tắt

Ổi (Psidium guajava) và diệp hạ châu (Phyllanthus amarus) là những loại dược liệu truyền thống nổi tiếng được sử dụng rộng rãi trong y học cổ truyền Việt Nam. Tuy nhiên, các nghiên cứu về sự ảnh hưởng của những dược liệu này lên việc cải thiện hệ miễn dịch của cá tra (Pangasianoson hypophthalmus) ít được quan tâm. Do đó, nghiên cứu này nhằm tìm hiểu sự ảnh hưởng của chiết xuất ethanol từ lá ổi, diệp hạ châu và hỗn hợp chiết xuất giữa chúng (1:1) lên tế bào bạch cầu cá tra. Các tế bào bạch cầu (5×106 tế bào/mL) thu từ máu ngoại vi và thận của cá tra được bổ sung chiết xuất riêng lẻ từ lá ổi, diệp hạ châu và hỗn hợp chiết xuất lá ổi: diệp hạ châu tại 2 nồng độ khác nhau (10 và 100 mg/mL) trong 24 giờ nuôi cấy. Kết quả cho thấy chiết xuất từ lá ổi, diệp hạ châu và hỗn hợp chiết xuất có tác động tích cực đến các chỉ tiêu miễn dịch được khảo sát. Cụ thể, hoạt tính lysozyme và tổng kháng thể của các nghiệm thức có bổ sung chất chiết xuất tăng đáng kể so với nghiệm thức đối chứng (p<0,05). Tuy nhiên, hoạt tính reactive oxygen species  (ROS) chỉ tăng khác biệt có ý nghĩa thống kê ở nghiệm thức có bổ sung chiết xuất lá diệp hạ châu (100 mg/mL) và nghiệm thức có bổ sung hổn hợp chiết xuất lá ổi: diệp hạ châu (10 mg/mL) đối với tế bào bạch cầu thận. Tương tự, hoạt tính nitric oxide synthases (NOS) của tế bào bạch cầu thu từ thận tăng đáng kể sau khi được bổ sung 100mg/mL chất chiết xuất lá ổi. Ngoài ra, hỗn hợp chiết xuất lá ổi: diệp hạ châu tại liều cao (100 mg/mL) cũng có tác dụng làm tăng có ý nghĩa thống hoạt tính bổ thể của tế bào bạch cầu thu từ máu ngoại vi và thận cá tra so với nghiệm thức đối chứng (p<0,05).
Từ khóa: Cá tra, diệp hạ châu, miễn dịch, ổi, tế bào bạch cầu

Article Details

References

Amonkan, A.K., Kamagaté, M., Yao, A.N., et al., 2013. Comparative effects of two fractions of Phyllanthus amarus (Euphorbiaceae) on the blood pressure in rabbit. Greener Journal of Medical Sciences. 3(4): 129-134.

Anbuselvi, S., and Rebecca, J., 2017. Phytochemical biochemical and antimicrobial activty of Psidium guajava leaf extract. Journal of Pharmaceutical Sciences & Research. 9(12): 2431-2433.

Awaad, A.S., and Al-Jaber, N.A., 2010. Antioxidant natural plant. RPMP Ethnomedicine: Source & Mechanism 27: 1-35.

Awad, E., and Awaad, A., 2017. Role of medicinal plants on growth performance and immune status in fish. Fish & Shellfish Immunology 67: 40-54.

Hang, B.T.B., Milla, S., Gillardin, V., Phuong, N.T., and Kestemont, P., 2013. In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus). Fish & Shellfish Immunology. 34(1): 339-347.

Hang, B.T.B., Phuong, N.T., and Kestemont, P., 2014. Can immunostimulants efficiently replace antibiotic in striped catfish (Pangasianodon hypophthalmus) against bacterial infection by Edwardsiella ictaluri? Fish & Shellfish Immunology. 40(2): 556-562.

Boyum, A., 1968. Separation of leukocytes from blood and bone marrow. Scandinavian journal of clinical and laboratory investigation. Supplementum. 21: 77.

Chakraborty, S.B., and Hancz, C., 2011. Application of phytochemicals as immunostimulant, antipathogenic and antistress agents in finfish culture. Reviews in Aquaculture. 3(3): 103-119.

Crumlish, M., Dung, T.T., Turnbull, J.F., Ngoc, N.T.N., and Ferguson, H.W., 2002. Identification of Edwardsiella ictaluri from diseased freshwater catfish, Pangasius hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam. Journal of Fish Diseases. 25(12):733-736.

Devi, T.B., Kamilya, D., and Abraham, T.J., 2012. Dynamic changes in immune-effector activities of Indian major carp, catla (Catla catla) infected with Edwardsiella tarda. Aquaculture. 366: 62-66.

Dung, T.T., Haesebrouck, F., Tuan, N.A., Sorgeloos, P., Baele, M. and Decostere, A., 2008. Antimicrobial susceptibility pattern of Edwardsiella ictaluriisolates from natural outbreaks of Bacillary Necrosis of Pangasianodon hypophthalmus in Vietnam. Microbial drug resistance. 14(4): 311-316.

Ellis, A.E., 1990. Lysozyme activity. In: Stolen, T.C., Fletcher, P.D., Anderson, B.S., Roberson, B.S., and van Muiswinkel, W.B., (Eds.) Technique in fish immunology-1. New York: SOS Publications, pp. 101-103.

Fawole, F.J., Sahu, N.P., Pal, A.K., and Ravindran, A., 2016. Haemato‐immunological response of Labeo rohita (Hamilton) fingerlings fed leaf extracts and challenged by Aeromonas hydrophila. Aquaculture Research. 47(12): 3788-3799.

Giri, S.S., Sen, S.S., Chi, C., et al., 2015. Effects of intracellular products of Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 on cytokine responses in the head kidney macrophages of Labeo rohita. Fish & Shellfish Immunology. 47(2): 954-961.

Gobi, N., Ramya, C., Vaseeharan, B., et al., 2016. Oreochromis mossambicus diet supplementation with Psidium guajava leaf extracts enhance growth, immune, antioxidant response and resistance to Aeromonas hydrophila. Fish & Shellfish Immunology. 58: 572-583.

Harikrishnan, R., and Balasundaram, C., 2008. In vitro and in vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish. Journal of Aquatic Animal Health. 20(3): 165-176.

Harikrishnan, R., Balasundaram, C., Jawahar, S., and Heo, M.S., 2012. Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila. Fish & Shellfish Immunology. 32(1): 94-100.

Hoseinifar, S.H., Zou H.K., Van Doan H., Kolangi Miandare H., and Hoseini S.M., 2018. Evaluation of some intestinal cytokines genes expression and serum innate immune parameters in common carp (Cyprinus carpio) fed dietary loquat (Eriobotrya japonica) leaf extract. Aquaculture Research. 49(1):120-127.

Ilangkovan, M., Jantan, I., Mesaik, M. A., and Bukhari, S. N., 2016. Inhibitory effects of the standardized extract of Phyllanthus amarus on cellular and humoral immune responses in Balb/C mice. Phytotherapy Research. 30(8): 1330-338.

Jahanjoo, V., M. Yahyavi, R. Akrami, and A. H. Bahri., 2018. Influence of adding garlic (Allium sativum), ginger (Zingiber officinale), thyme (Thymus vulgaris) and their combination on the growth performance, haemato-immunological parameters and disease resistance to Photobacterium damselae in sobaity sea bream (Sparidentex hasta) fry. Turkish Journal of Fisheries and Aquatic Sciences. 18(4): 633-645.

Mo, W. Y., C. Lun H.I., Choi W.M., Man Y.B., and Wong M.H., 2016. Enhancing growth and non-specific immunity of grass carp and Nile tilapia by incorporating Chinese herbs (Astragalus membranaceus and Lycium barbarum) into food waste basedpellets. Environmental Pollution. 219: 475-482.

Neumann, N.F., Stafford, J.L., Barreda, D., Ainsworth, A.J., and Belosevic, M., 2001. Antimicrobial mechanisms of fish phagocytes and their role in host defense. Developmental & Comparative Immunology. 25(8-9): 807-825.

Olivier, G., Evelyn, T.P.T., and Lallier, R., 1985. Immunity to aeromonas salmonicida in coho salmon (oncorhynchus kisutch) inducfd by modified freund's complete adjuvant: Its non-specific nature and the probable role of macrophages in the phenomenon. Developmental & Comparative Immunology. 9(3): 419-432.

Ogundele, M. O., 1998. A novel anti-inflammatory activity of lysozyme: modulation of serum complement activation. Mediators of Inflammation. 7(5): 363-365.

Phan, L.T., Bui T.M., and Nguyen T.T.T., et al., 2009. Current status of farming practices of striped catfish, Pangasianodon hypophthalmus in the Mekong Delta, Vietnam. Aquaculture. 296(3-4): 227-236.

Pierrard, M.A., Roland, K., Kestemont, P., Dieu, M., Raes, M., and Silvestre, F., 2012. Fish peripheral blood mononuclear cells preparation for future monitoring applications. Analytical Biochemistry. 426(2): 153-165.

Pratheepa, V., and Sukumaran, N., 2011. Specific and nonspecific immunostimulation study of Euphorbia hirta on Pseudomonas fluorescens-infected Cyprinus carpio. Pharmaceutical biology 49(5): 484-491.

Raa, J., Roerstad G., Engstad R., and Robertsen B., 1992. The use of immunostimulants to increase resistance of aquatic organisms to microbial infections. Diseases of Asian Aquaculture. 10: 39-50.

Rao, Y.V., Das, B.K., Jyotyrmayee, P., and Chakrabarti, R., 2006. Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish & Shellfish Immunology. 20(3): 263-273.

Ravikumar, S., Selvan, G.P., and Gracelin, A.A., 2010. Antimicrobial activity of medicinal plants along Kanyakumari coast, Tamil Nadu, India. African Journal of Basic & Applied Sciences. 2(5-6): 153-157.

Rohn, S., Rawel, H.M., and Kroll, J., 2002. Inhibitory effects of plant phenols on the activity of selected enzymes. Journal of Agricultural and Food Chemistry. 50(12): 3566-3571.

Rook, G.A.W., Steele, J., Umar, S., and Dockrell, H.M., 1985. A Simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by y- interferon. Journal of Immunological Methods. 82(1): 161-167.

Saravanakumar, A., Venkateshwaran, K., Vanitha, J., Ganesh, M., Vasudevan, M., and Sivakumar, T., 2009. Evaluation of antibacterial activity, phenol and flavonoid contents of Thespesia populnea flower extracts. Pakistan Journal of Pharmaceutical Science.s 22(3).

Secombes, C.J., 1996. The nonspecific immune system: cellular defenses. In:Kaattari, S.L., Piganelli, D., Iwama, G., and Nakanishi, T. (Eds.). The fish immune system: organism, pathogen and environment. Academic press 15. Pp. 63-103.

Sen, S.S., Sukumaran, V., Giri, S.S., and Park, S.C., 2015. Flavonoid fraction of guava leaf extract attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-κB signalling pathway in Labeo rohita macrophages. Fish & Shellfish Immunology. 47(1): 85-92.

Sirimanapong, W., Thompson, K.D., Kledmanee, K., Thaijongrak, P., Collet, B., Ooi, E.L., and Adams, A., 2014. Optimisation and standardisation of functional immune assays for striped catfish (Pangasianodon hypophthalmus) to compare their immune response to live and heat killed Aeromonas hydrophila as models of infection and vaccination. Fish & Shellfish Immunology. 40(2): 374-383.

Sirimanapong, W., Adams, A., Ooi, E.L., et al., 2015a. The effects of feeding immunostimulant β-glucan on the immune response of Pangasianodon hypophthalmus. Fish & Shellfish Immunology. 45(2): 357-366.

Sirimanapong, W., Thompson, K.D., Ooi, et al., 2015b. The effects of feeding β-glucan to Pangasianodon hypophthalmus on immune gene expression and resistance to Edwardsiella ictaluri. Fish & Shellfish Immunology. 47(1): 595-605.

Siwicki, A., and Anderson, D., 1993. An easy spectrophotometric assay for determining total protein and immunoglobulin levels in fish sera: correlation to fish health. Techniques in Fish Immnunology. 3: 23-30.

Sunyer, J.O., and Tort, L., 1995. Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effectedby the alternative complement pathway. Veterinary Immunology and Immunopathology. 45(3-4): 333-345.

Talpur, A.D., Ikhwanuddin, M., and Bolong, A.M.A., 2013. Nutritional effects of ginger (Zingiber officinale Roscoe) on immune response of Asian sea bass, Lates calcarifer (Bloch) and disease resistance against Vibrio harveyi. Aquaculture. 400:46-52.

Thy, H.T.T., Tri, N.N., Quy, O.M., et al., 2017. Effects of the dietary supplementation of mixed probiotic spores of Bacillus amyloliquefaciens 54A, and Bacillus pumilus 47B on growth, innate immunity and stress responses of striped catfish (Pangasianodon hypophthalmus). Fish & Shellfish Immunology. 60: 391-399.

Yin, X.L., Li, Z.J., Yang, K., Lin, H.Z., and Guo, Z.X., 2014. Effect of guava leaves on growth and the non- specific immune response of Penaeus monodon. Fish & Shellfish Immunology. 40(1): 190-196.