Vietnam J. Agri. Sd. 2016, Vol. 14, No. 6: 869-876 Tgp chi KH Nong nghiep Vigt Nam 2016, tap 14. sd 6: 869-8761 www.vnua edu.vn
TAC DUNG l)C CHE VI KHUAN IHVmOtAk CAO KHO DjCH CHIET LA TRAU KHONG [Piperbetlgl ofil V0IVI KHUAN Aeromonassm. VA Streptococcusagalactiae
GAY BENH XUAT HUYET THEN CA RO PHI Trjnh Thi Trang'', Nguyin Thanh Hai^
Khoa Thuy sdn, Hpc vifn Nong nghiep Viet Nam Khoa Cong nghf Sinh hpc, HQC vien Nong nghidp Viet Nam
Email • [email protected]
Ngay gui bdi: 28.04.2016 Ngdy chdp nhan: 06.06.2016 TCM TAT
Vigc sd dyng thudc khang smh de dieu tri b#nh thuy san mang lgi nhidu k i t qua kha quan nhung lai lam day len lo nggi ve viec tdn du khang sinh trong san pham thuy san cung nhu lam tang tinh khang khang smh cua vi khuan gay benh. Thao dug'c dang ngay cang chung mmh dug'c vai tro quan trgng cua chung trong ndn cdng nghiep duoc pham nhu la mdt giai phap an toan sinh hgc, thay the cho cac thuoc hoa hoc tong hap. Nghien cuu nay duoc tidn hanh nhdm kiem tra hieu suat chidt la cay tr^u khdng (Piper betle) trong 5 loai dung moi co do phan cu'c khac nhau (nude cat, methanol 80%, ethanol 96%, n-hexan va aceton 100%) ddng thdi cung danh gia kha nang dc che vi khuan in vitro cua cac cao kho djch chiet tif la cay trau khong ddi vdi 2 loai vi khuan Aeromonas spp va Streptococcus agaiactiae gay benh xuat huydt tren cd rd phi. Ket qud nghien cdu cho thay hieu sudt chidt xuat trong 5 logi dung mdi bien doi ti> 4,00% (dung mdi n-bexan) den 19,67% (dung mdi ethanol 96%). & ndng do 100 mg/ml cac cao khd dich chiet deu cd kha nang dc che vi khuan in vitro tdt ddi vdi 2 chimg vi khuan. Ddi vdi vi khuan Aeromonas spp., dudng kinh vdng vd khuan binh quan giao dgng tu 15,00 mm (dung mdi nude) ddn 28,00 mm (vdi dung mdi la Ethanol 96%). Ddi vdi vi khuan Streptococcus agaiactiae dudng kinh vdng vd khuan binh quan giao dgng tu' 17,67 mm (dung mdi nude) den 31,67 mm (vdi dung moi la ethanol 96%). Nong dg nho nhat cua cao khd dich chiet la trau khdng SLK dyng dung mdi ettianol 96% khi bd sung vao lo thach v i n quan sat thay vdng vo khuan la 0,39 mg/ml doi vdi vi khuan Aeromonas spp va 0,78 mg/ml ddi vdi vi khuan Streptococcus agaiactiae.
Tir khda: Aeromonas spp., benh xuat huyet, cao dich chief la cay trau khdng (Piper betle), ca rd phi.
Streptococcus agaiactiae, i>c che vi khuan.
In vitro Anti-BacteriaI Effect of Piper betle Leaf Extracts on Aeromonas spp. and Streptococcus agaiactiae which Cause Hemorrhagic Disease in Tilapia
ABSTRACT
The use of antibiotics for treating fish diseases has several advantages but raises the concerns about antibiotic residues in fishery products and increases antibiotic resistance of pathogenic bacteria as well. Herbs have been increasingly demonstrated their important role In the pharmaceutical Industry as a biosafety solution, an alternative to synthetic chemical dnjgs. The present study aimed to examine the efficiency of leaf extraction from Piper betle by five different solvents (distilled water.methanol 80%, ethanol 96%, n-hexan andaceton 100%), and evaluate the anti- bacterial effects of the extract on Aeromonas spp. and Streptococcus agaiactiae which cause hemorrhagic disease in tilapia The results showed that the extraction efficiency varied from 4,00% (n-hexan solvent) to 19.67% (ethanol 96%
solvent). At the concentration of 100 mg/ml, all these extracts showed good antibacterial activity against Aeromonas spp. and Streptococcus agaiactiae. For Aeromonas spp , the inhibition zone varied from 15,00 mm (distilled water solvent - DW) to 28,00 mm (ethanol 96% solvent). The inhibition zones of Streptococcus agalactiaewere varied from 17,67 mm (DW solvent) to 31,67 mm (ethanol 96% solvent). The ethanol-extract solution showed highest anU-
869
va Sfreptococcus agaiactiae gay benh xuat huyet tren ca rd phi
bacterial effect. This extract remained the anti-bactenal activity to Aeromonas spp.and Streptococcus agaiactiae at concentraton 0,39 mg/mt and 0.78 mg/ml, respectively.
Keywords: Aeromonas spp., anti-bacterial effect. Piper betle leaf extract. Streptococcus agaiactiae, tilapia.
1. DATVANDE
Hien nay, benh b ca ro nhfl b§nb loi mdt, benb den than, nha't la benh xuat huyet do vi khuan gay ra da gay nhidu thiet hai cho cac md hinh nuoi ca ro phi tham eanh. Benh do vi khuan xua't bien quanh nam va tap trung nhieu trong cac thang mua he vdi ti le chet cao. Trong so' cac vi khudn gay benh xuat huyet d ca, Aeromonas spp. la vi khuan da dUdc cong bd nhieu nhat cd kha nang gay benh tren nhieu loai ca nudc ngot tren khdp the gidi vdi dau hieu b§nh ly gom nhiem triing mau, xuat huyet. Id loet (Austin and Adams, 1996). Ben canh dd, vi khuan Streptococcus sp. gay cac trieu chflng tUdng tfl Cling dugc phat hien tren ca (Evans et al., 2006, Dang Thi Mai Thy va cs., 2012). Viec sii dung thudc, hda cha't ddng gop khong nhd cho su phat trien cua viec nudi ca rd phi noi rieng va nganh thuy san ndi chung. Tuy nhien, viec sii dung, buon ban thuo'c, hda cha't trong nudi trdng thuy san (NTTS) cdn nhieu bat hgp ly do hieu biet cua ngUdi nuoi cdn nhieu han che va nhieu hoat dgng ciia nha san xuat, nha phan phoi khdng tuan theo quy dinh cua Nha nude. Dieu dd ddn ddn viec sfl dung khdng dung cac loai thudc khang sinh. Theo dieu tra cua Mai Van Tai va cs. (2004), cd tdi 138 Ioai khang sinh da dUdc sii dung trong cac loai hinh nudi va san xuat gid'ng thuy san 6 Viet Nam. Khi viec sii dung khang sinh qua mflc, khdng dUdc kiem soat de tri benh cho ca, tom thi td't yeu se xay ra va'n de khang thudc cua cac chiing vi khuan gay benh va sfl tac ddng den sflc khoe con ngUdi. Ndi each khac, viec phdng va xii ly benh cho dgng vat thuy san ciing nhfl con ngUdi sau nay se gap ra't nhieu khd khan.
Thao dugc dang ngay cang chflng minh dUdc vai trd quan trgng cua minh trong nen cdng nghiep dUde pha'm nhu la mgt giai phap an toan sinh hgc thay the cho cac thuoc hda hgc tdng hdp. Tinh den nam 2010, cd it nhat 50 Ioai cay thao dudc da dfldc nghien cflu flng dung 870
trong nudi trdng thuy san (Citarasu, 2010).
Chiet xud't thao dudc da dUdc chflng minh chiing cd tac dung tren ca hai ddi tfldng tdm va ca nuoi vdi cac vai trd nhfl kich thich tang trUdr^
(Chitra, 1995; Rani, 1999; Citarasu et al, 1998:
2002); kich thi'cb midn dich (Minomol, 2005;
Sivaram et al., 2004); khang khuan va nhieu tac nhan truyen nhiem (Imraanuel et al., 2004;
Praseetha, 2005; Adiguzel et al., 2005). Cay trau khdng cd ten khoa hgc la Piper betle thudc hg hd tieu Piperaceae, phan bd b vung nhiet ddi, dac biet b Dong Nam A nhfl Viet Nam, Malaysia, Indonesia va vung nhidt ddi Chau My dUdc sii dung theo dan gian dd chdng viem, sat triing dfldng hd ha'p cho ngfldi (Dd Ta't Lgi, 2003;
Shameem and Thiruma, 2013). Thanh phin trong la trdu khdng co chfla betel- phenol (ddng phan ciia eugenol) va chavicol kem theo nhidu hdp chat phenohc khac, chflng co tac dung khang sinh rat manh doi vdi cac loai vi khuan, na'm va ki sinh triing (Vd Van Chi, 2000; Dd Thi Hda va cs., 2004). Hien nay. kha nang khang khuan cua cay trau khdng da budc diu dfldc nghien cflu ddi vdi mgt sd^ tac nhan gay benh tren dgng vat thuy san (Huynh Kim Di§u va Nguyin Thanh Van, 2011; Dang Thi Lua va cs., 2015). Trong nghien cflu nay, ben canh vi§c tim ra dung moi td't nhd't de chiet va danb gia higu suat chiet cua cac dung moi, tiem nang khang khuan in vitro cua la cay tr^u khong cflng dfldc tien hanh tren hai loai vi khuan Aeromonas spp. va Streptococcus agaiactiae gay benh xuat huyet tren ca rd phi.
2. VAT LIEU VA P H U O N G PHAP 2.1. Vat lieu nghien cftu
- La cay trdu khong dfldc thu bai d Yen My, Hflng Yen. Thu la banh te. lanh Ian khdng bi sau. Thu la sach vao nhflng ngay kho rao, khoang tfl 7 - 10 gig sang. La tfldi thu hai ve dugc nia dudi vdi nUdc sach ( 2 - 3 lan) rdi rita lai vdi nude cat, sau dd dUdc phdi kho trong bdng ram hoac dfldc say b 40°C. Mdu kho dfldc nghien
Tqnh Thj Trang, Nguyen Thanh Hai
thanh bgt min (< 0,5 mm). Bdt la trau khong (hdi tho, mau xanh, mui dac trflng) dflng trong till nilon bao quan trong binh hut am. Mdi loai dung moi dfldc sfl dung dd tach chiet 3 mdu bdt tr^u khdng khac nhau.
Hai chung vi khuan Aeromonas spp. va Streptococcus agaiactiae do Bg mon Mdi trfldng va Benh Thuy san, Khoa Thiiy san, Hgc vien Ndng nghidp Viet Nam cung cd'p. Cac chung nay da dfldc phan lap va dinh danh tfl ca rd phi bi bdnh xud't huyet b Nam Sach, Hai Dfldng nam 2015.
- Moi trfldng Luria-Bertani dang long (LB) dfldc bap kh^ triing trdng cac binh tam giac dd nudi cd'y thu dich khuan.
- Moi trfldng Luria-Bertani dang dac (LA) dfldc hap tiet trung, de nguoi tdi 40 - 50° C, do vao cKa petri cd dudng kfnh 10cm, v6i do day la 4 ± 0,2 mm.
2.2. PhuTOng phap
- Thu dich cao khd dich chiet la cay trau khdng. Bdt la trdu khong dfldc chidt vdi 5 dung mdi cd dd phdn cflc khac nhau (nfldc cd't, methanol 80%, ethanol 96%, n-hexan va aceton 100%) bdng pbudng phap ngdm chiet lanh b nhiet do phdng vdi cung mot ty Ie (20 g bdt la kh6/200 ml dung mdi), mdi ngay dfldc ldc dao 2 lan. Sau 72 gid, thu dich chiet, lgc qua vai man va giay lgc (bidu Whatman No. 1). Thu dich chiet dem cd quay hiit chan khong de loai bo hoan toan dung moi. Khi khdi Ifldng cua binh cd quay khdng ddi dem can de tinh hidu sud't tach chidt cua cac dung moi. Cao cd toan phan da loai bo het dung mdi bao quan trong tii mat 4''C de tien hanh nghien cflu. Bdt trdu khdng dfldc tach chidt Idp lai 3 Ian ci mdi loai dung mdi. Hieu sua't tach chiet dfldc tinh theo cdng thflc sau:
h (%) = inJmM X 100
Trong dd: h (%) la bi§u suat chidt, mc la khdi Ifldng can khd (sau kbi co quay) (g), m^ Id khdi Ifldng m l u hot trau khdng kho (g)
- Pha dich chiet nSng do 100 mg/ml
La'y Ig cao co toan phan pha vdi 10ml Dimethyl Sulfoxide (DMSO), dung dua thuy tinh khuay tan hoan toan ta dflgc dung dich cd ndng do lOOmg/ml.
- Nudi cay vi khuan Aeromonas spp. va Streptococcus agaiactiae
Vi khuan dUdc csiy vach tren dia LA, tr§n dHa. petri li 28 - 30°C/24 gid, de chgn khuan lac ddn dien hinh. Khudn lac ddn dfldc nudi long trong mdi trfldng LB, dat trdng tu bao dn b nhiet do 28°C vdi tdc dd ldc 200 vdng/phut trong 12 - 14 gid; thu dich khuan (mat do vi khuan dat 10^
te bao/ml la dat chudn).
~ Xac dinh mat do vi khu^n
Mat do vi khuan sau khi nuoi cay trong mdi trfldng long dflgc xac dinh theo phfldng phap do mdt do quang (OD) b budc sdng X = 600 nm.
- Kiim tra tac dung diet khuan cua cac dich chiet bang phtiOng phap khang sinh do khuech tan tren dia thach cua Kirby-Bauer.
Cac thao tac dugc thuc hien trong tu cd'y vd trung. Khi mat do vi khuan dat 10^ te bao/ml, lac ddu binh chfla vi khuan, diing pipet man hut 100 ^l canh khudn nhd vao gifla dia thach, dflng que thuy tinh trang deu cbo den khi mat thach khd. Sau 15 phut due 16 trdn mat thach vdi dfldng kinh 6 mm/Id due each nhau khoang 25mm. Mdi Id thach, nhd 100 jil dich chiet, dat dia vao tu a'm b S7°C/24 gid dgc ket qua bdng each do dudng kinh vdng vd khuan. Moi loai dich chidt dfldc thi nghiem lap lai 3 lan.
- Pha loang dich chiet
Chuan hi 10 diig nghiem vo triing, cho vao mdi dng 5 ml DMSO. Lay 5 ml mdu dieh chiet (100 mg/ml) cho vao diig nghiem thfl nhd't, lam ddng ddu, dfldc do pha loang 2 Ian (2'). Ld'y 5 ml dung dich b dng nghiem 2' eho vao diig nghiem thfl 2, dfldc do pha loang 4 lan (2^. Tiep tuc thflc hi&n tfldng tfl dd dugc do pha loang tidp theo: 2^, 2^... 2".
2.3.X^Iy s o l i e u
Cac tbi nghiem dfldc bo tri ngdu nhien va dfldc lap lai 3 Idn. So' lieu dUdc xii ly thdng ke bdng phan mem Excel 2007.
871
vd Streptococcus agaiactiae gay benh xuat huydt tren cd rd phi
3. KET QUA VA THAO LUAN
3.1. Hieu suat chie't xuat cac hoat chat cua la cay trau khong bang 5 dung moi khac nhau
Sau khi tien hanh ngam hot la cay trau khong trong 5 loai dung moi cd do phan cflc khac nhau sau 72 gid lgc sd bg qua vai man va giay lgc. Ket qua cho thdy, cung mgt ty Id pha loang nhflng dich chiet thu dugc tfl cac dung mdi lai cd cac mau sdc khac nhau. Cac dich chiet la cay trau khong thu dfldc eo mdu sac bien doi tfl vang nhat ddn xanh den. Khi sii dung dung mdi la nude, methanol 80% va n-hexan, dieh chiet thu dugc cd mau vang va nau nhat. Trong khi do khi sii dung dung mdi b ndng do cao la ethanol 96% cho dich chiet mau xanh den va aceton 100% cho dich chiet mau nau (Hinh 1).
Qua mau sdc khac nhau cua dich chiet co the sd bg nhan dinh rdng, cac dung mdi khac nhau c6 kha nang tach chie't cac hoat chd't trong la trau khdng la khac nhau.
Tien hanh danh gia hieu suat tach chiet ciia cao khd dich chie't la trau khdng qua 3 Idn tach chiet cho thd'y cflng mdt phfldng phap chidt ngam lanh nhflng hieu suat chidt xua't khi sii diing cac loai dung mdi la khac nhau. Khdi Ifldng cao khd dich chie't la trdu khdng tfl 20g bgt la ban dau, tuy thudc vao tflng loai dung moi cd do giao dgng kha Idn bidn ddi tfl 0,80g (dung moi n-hexan) den 3,93g (dung moi ethanol 96%) tUdng dfldng vdi hieu sua't tach chie't dat 4,00%
den 19,67% (Bang 1, Hinh 2). Nhfl vay, cd the nhan dinh rdng, cac loai dung mdi tach chiet khac nhau thi kha nang hda tan cac hdp chat trong thflc vdt khac nhau.
Hinh 1. Dich chiet la trau khong thu dflgic tii cac Ioai dung moi khac nhau Ghi chii-Dung m&i si dung- A-nitdc cat; B- methanol 80%; C- ethanol 96%; D- n-hexan; E- aceton 100%
Hinh 2. Hieu suat tach chiet la cay trdu khong sii dung cac dung mdi khac nhau
Trinh Thi Trang, Nguyen Thanh Hai
B a n g 1. Khoi liTdng cao k h o t h u dvidc cua 20g bot la t r a u k h o n g t r o n g 5 loai d u n g moi (g)
Logi dung NU'OC c i t Methanol 8 0 % Ethanol 96%
n-hexan Aceton 100%
moi Kho lu'P'ng ca 1,70'' 1,97"
3,93"
0,80"
1,73"
• kh6
Gbichu. a, b, c, d tren cung nipt cot chi ra sil sai khac cd ^ nghia vimUt thong ke P < 0,05
Bang 2. Tac d u n g ijfc che vi k h u a n in vitro cua cac dich chiet la cay t r a u k h o n g
Cao khe dich chiet sir dyng dung moi D/c DMSO Nipcrc cat Methanol 8 0 % Ethanol 96%
n-hexan Aceton 100%
Nong do (mg/ml) 0 100 100 100 100 100
Difb'ng kinh v6ng v6 khuan Aeromonas spp.
15.00 24,00 28,00 26,67 21,33 ]
± 1 . 0 0
± 1 . 0 0
± 1 , 0 0
± 1,00
± 0 . 5 8
binh quan (mm) Streptococcus agaiactiae
0 1 7 . 6 r ± 1,53 29.33= ± 0 , 5 8 31,67''±0,58 28,67'^±1,15 24,67" ± 0 , 5 8 bi cbu: a, b, c, d tren cung mgt cot chi ra sii sai khac coy nghla ve m&t thong ke P<0,05
Trong 5 loai dung moi s^ dung da phan la dung moi phan cflc, chi co n-hexan la dung mdi khdng phan cflc va lai cho hieu suat chiet nhd nha't chi dat 4,00%, do do cd the sd bg khdng dinh rang cac hoat chat trong la trau khong tan chu ydu trong dung mdi phan cflc. Kbi sit dung dung mdi nudc (phan cflc protic) cho hieu suat tach chiet tfldng dfldng khong cd sfl sai khac v l mat thdng ke so vdi dung mdi aceton (phan cflc aprotic). Ket qua thi nghiem cho thay kha nang ha tan cac boat chat cd trong la trau khdng khong chi phu thugc vao do phan cflc cua dung moi ma en phu thugc vao ban chat cua dung mdi.
3.2. Kba n a n g life che in vitro vi k h u a n A e r o m o n a s spp. va Streptococcus agaiactiae cua cac cao k h o dich chiet
Cao kho dich chidt la cdy trdu khong thu dfldc tfl thi nghiem tren b ndng do 100 mg/ml, dfldc st( dung de danb gia kha ndng flc che vi khuan Aeromonas spp. va Streptococcus agaiactiae bdng phfldng phap khuech tan tren thach. Ket qua thi nghidm cho thd'y ca 5 loai dich chie't cua la cdy trdu khong trong cac dung mdi khac nhau deu cd kha nang flc chd vi khuan Aeromonas spp. va Streptococcus agaiactiae
?2).
Doi vdi vi khuan Aeromonas spp., dfldng kinh vdng vo khuan binh quan dao dgng tfl 15,00 mm (dung moi nfldc) den 28,00 mm (vdi dung mdi la ethanol 96%). Cac cao kho dich chidt la trau khong trfl cao khd suf dung dung mdi la nude deu dat do mdn cam cao ddi vdi vi khudn Aeromonas spp. vdi dfldng kinh vdng v6 khuan > 20 mm. Cao kho dich chiet la trdu khong khi sii dung dung moi la ethanol 96% cho kha nang flc che vi khudn in vitro td't nha't. Cao khd dich chie't la trdu khdng sii dung dung mdi la ethanol 96% va n- hexan co kha nang flc che vi khudn in vitro la tfldng dUdng nhau, dUdng kinh vdng vd khuan khdng CO sfl sai khac ve mat thdng ke lan lUdt la 28,00 ± 1,00 va 26,67 ± 1,00 mm
Ddi vdi vi khuan Streptococcus agaiactiae dfldng kinh vdng vd khuan binh quan giao ddng tfl 17,67 ± 1,53 mm (dung moi nude) den 31,67 ± 0,58 mm (vdi dung moi la ethanol 96%). Tfldng tfl nhu ddi vdi vi khuan Aeromonas spp. Cac cao khd dich chiet la trau khdng trfl eao khd sii dung dung mdi la nfldc deu dat do mdn cam cao ddi vdi vi khuan Streptococcus agaiactiae vdi dfldng kinh vdng vd khuan > 20 mm. Cao khd dich ehie't la trdu khdng khi sii dung dung moi la ethanol 96% cho kha nang flc che vi khudn Streptococcus agaiactiae in vitro td't nha't vdi 873
va Streptococcus agaiactiae gay bgnh xuat huydt tren ca ro phi dfldng kinh vdng vo khuan cd sfl sai khac ve mat thdng ke so vdi cac cao dich chiet khac. Cao khd dich chiet la trau khdng sil dung dung mdi la methanol 80% va n-hexan co kha nang flc che vi khuan in vitro tUdng dUdng nhau, dfldng kinh vdng vd khudn khdng cd sfl sai khac ve mat thdng ke lan Ifldt la 29,33 ± 0,58 mm va 28,67 ± 1,15 mm.
Nhieu nghien cflu trfldc day da khang dinh kha nang flc che vi khudn in vitro cua la trau khdng, Tarun et al (2012) nhan tha'y dich chidt la trau khong sii dung dung moi ethanol, methanol cd tac dung diet khuan rat td't doi vdi vi khuan E. coli. Dd Huy Bich va es. (2004) khdng dinh, cao la trau khong co hoat tinh flc chd in vitro cac chiing vi khuan; Staphylococcus aureus, E. coH, Salmonella spp.,... Fawad et al.
(2012) nhan thay cao dich chiet la trau khong sfl dung dung mdi ethanol dm vdi ca vi khuan gram am va gram dUdng cbo dfldng kinh vdng vd khudn bien ddi tfl IS mm den 58 mm Tbeo nghi§n cflu cua Najiah et al (2011), eao dieh chidt methanol cay trdu khdng b ndng do 200 mg/ml ed kha nang fle ehd in vitro 6/9 vi khudn nhu Staphylococcus aureus. Streptococcus agaiactiae, Aeromonas hydrophila... gay benh cho dgng vat thuy san vdi dfldng kinh vong vd khuan Men ddi tfl 17,00 - 20,00mm va tha'p hdn so vdi kdt qua cua chung tdi. Theo nhan xet chu quan ciia chiing tdi sd di cd sfl sai khac nay nguyen nhan chu yeu la do dUdc lieu dUdc trdng b nhflng vung cd dieu kien thd nhudng khi hau khac nhau.
3.3. Tac dung flc che vi k h u a n in vitro cua cao kho dich chidt la cay t r a u kbdng sau khi pha loang
Theo ket qua nghien cflu kha nang flc chd vi khudn in vitro eua cae cao khd dich chiet sii dung dung moi khae nhau b ndng do 100 mg/ml cho thay, cao kho dich chiet sii dung dung mdi ethanol 96% va methanol 80% cho hieu qua flc chd vi khuan td't nhdt ddi vdi ca 2 chiing vi khudn thi nghiem. De danh gia chinh xac kha nang flc che vi khuan va xac dinh ndng dd cao khd dich chiet nhd nhat vdn cdn kha ndng flc
che vi khuan chung tdi tien hanh sti dung 2 bai cao kho sii dung 2 dung mdi ndi tren de tidn hanh danh gia kha nang flc che vi khuan khi pha loang.
Ket qua danh gia tac dung flc che' vi khuan in vitro cua cao kho dich chiet la cay trdu khdng sii dung dung mdi ethanol 96% va methanol 80%
vdi vi khuan Aeromonas spp. va Streptococcus agaiactiae bdng phudng phap he nong do pha loang dfldc tdm tdt b bang 3. Ddi vdi vi khuan Aeromonas spp., ndng dd nhd nhat cua cao khd dich chiet khi cho vao Id thach vdn cdn quan sat thay vdng vd khuan dao dgng tfl 0,39 mg/ml (vdi dung mdi la ethanol 96%) den 1,56 mg/ml (vdi dung mdi la methanol 80%). Ddi vdi vi khuan Streptococcus agaiactiae ndng do nho nhd't cua cao khd dich chiet khi cho vao Id thach vdn con quan sat thay vdng vd khuan la 0,78 mg/ml dm vdi ca hai Ioai cao khd dich chidt la trau khong (Bang S, Hinh 3). Cao kho dich chiet la trau kbdng sii dung dung moi ethanol 96% cd tac dung flc chd vi khuan tot hdn cao khd dich chiet sii dung dung mdi methanol 80% khi pha loang.
Theo nghien cflu cua Najiah et al. (2011), ndng do nho nha't ciia dich chiet sti dung dui^
mdi ethanol cdn cd tac dung flc che vi khuan Aeromonas spp. va Streptococcus sp. in vitrolaa Ifldt la 1,56 mg/ml va 3,13 mg/ml cao bdn so vdi cao khd dich chie't ciia chung toi, chflng td cao khd dich chiet cua chung tdi cd tinh khang khuan td't hdn. Ngoai ra, ket qua ciia nghign cflu tren ciing c6 tUdng ddng vdi ket qua cua chiing tdi khi cao khd dich chiet la trau khdng pha loang cd tac dung flc che vi khudn in vitro ddi vdi vi khuan Aeromonas spp. tdt hdn so vdi Streptococcus agaiactiae.
4. KET LUAN
Hieu suat tach chiet thu cao khd dich chiet la trau khdng (bdng phfldng phap ngam lanh sau dd cd chan khdng) bien ddi tfl 4,00% (n- hexan) ddn 19,67% (ethanol 96%) khi sii dung 5 loai dung mdi khac nhau. Dich chidt la cay trau khdng (Piper betle) trong 5 loai dung mdi khac nhau deu cd kha nang flc chd vi khudn in vitro tren hai chiing vi khudn gay bdnh xud't huyet tren 874
Trjnh Thj Trang. Nguyen Thanh Hai
B a n g 3 . K e t q u a He c h e v i k h u d n in vitro c u a d i c h c h i e t l a c a y t r a u k h o n g d i f d c t a c h c h i e t b a n g 2 d u n g m o i d c d c h d so' p h a l o a n g k h a c n h a u
He so pha to§ng djCh chi4t tif nong dp goc 100 mg/ml
Nong dp djch chiet (mg/ml) 50,D0
Aeromonas spp.
Ethanol 96% + Methanol 80% + StfBptococcus agaiactiae
Ethanol 96% + Methanol 8 0 % +
25,00
*
*
*
+ 12,50
*
t
+
*
6,25
+
•
*
t 3,13
*
+
*
• 1,56
*
*
•
*
0,78
*
•
• 0,39
*
0,195 0,10
Gbi chu: + Quan sat thay v< in; - Kbdng quan sat thay vdng vo khw
H i n h 3 . T a c d u n g He c h e v i k h u a n in vitro c i i a c a o k h d d i c h c h i e t l a c a y t r a u k h o n g stf d u n g d u n g m o i e t h a n o l 9 6 % k h i p h a l o a n g
t r e n ca rd p h i (Aeromonas s p p . v a Streptococcus agaiactiae). C a o k h o dich c h i e t l a c a y t r a u k h o n g sii d u n g d u n g m d i e t h a n o l 9 6 % (b n d n g do 100 m g / m l ) cd k h a n a n g flc c h e vi k h u d n in vitro t d t n h a t vdi dfldng k i n h v d n g vo k h u d n d a t 2 8 , 0 0 ± 1,00 m m (Aeromonas spp.) v a 31,67 ± 0,58 m m (Streptococcus agaiactiae). N d n g do n h o n h d t ciia cao k h d dich c h i e t la t r d u k h o n g s ^ d u n g d u n g m o i e t h a n o l 9 6 % k h i bd s u n g vao Id t h a c h v d n q u a n s a t t h a y v d n g vd k h u d n l a 0,39 m g / m l (Aeromonas spp.) v a 0,78 m g / m l (Streptococcus agaiactiae).
TAI LIEU THAM KHAO Adiguzel A., Medine G., Meryem B., Hatice U. T. C ,
Fikretnn A., Usa K (2005). Antimicrobial effects of Ocimum basilicum (Labiatae) extract. Turk J Biol., 29: 155-160
Austin, B. and C. Adam (1996), Fish pathogen:
The genus Aeromonas John Wiley and Sons, pp. 197-243
Chitra S, (1995). Effect of feeding supplemented stresstol bioencapsulated Artemia franciscana on growth and stress tolerance - in Penaeus mdicus postlarvae MPhil Dissertation, M.S University, Tiruneiveli.
va Streptococcus agaiactiae gay b|nh xuat huyet tren ca rd phi Citarasu T. (2010). Herbal biomedicmes: a new
opportunity for aquaculture industry. Aquacult Int., 18:403-414
Citarasu T., Immanuel G., Marian M. P (1998). Effects of feeding Artemia ennched with stresstol and cod liver oil on growth and stress resistance in the Indian white shrimp Penaeus indicus posdarvae.
Asian Fish Sci., 12: 65-75
Citarasu T., Sekar R. R., Babu M M., Manan M. P.
(2002). Developing Artemia enriched herbal diet for producing quahty larvae in Penaeus monodon.
Asian Fish Sci., 15:21-32
Dang Thi Lua, Nguyen Thi Hanh, Hoang Hai Ha, Tmong Thi My Hanh, Phan Thi Van (2015). Tae dung diet khudn in vitro cua dich chiet la Uau khdng (Piper belle L.) va dich chiet la di (Psidium guajava) ddi vdi vi khuan gay benh hoai tir gan tuy cap tinh Uen torn nudi nudc Ig. Tap chi khoa hgc va cdng nghe, 11:106-113
Dang Thi Mai Thy, TrSn Thi Thiiy Cue, Nguyen Chau Phuong Lam, Nguyen Dflc Hien va Dang Thi Hoang Oanh (2012), Dae diem md benh hoc ca rd (Anabas testudineus) nhiem vi khuan Aeromonas hydrophila va Streptococcus sp. trong dieu kien thvrc nghiem. Tap ehi Khoa hgc, 22: 183-193.
Do Huy Bieh, Dang Quang Chung, Biii Xuan Chuong, Nguyen Thugng Dong, Do Tmng Dam, Pham Van Hien, VQ Nggc Lg, Pham Duy Mai, Pham Kim Man, Doan Thi Thu, Nguyin T^p va Tran Toan (2004). Cay thuoc va ddng vat lam thudc d Viet Nam, Tap II, Nha xuat ban Khoa hpc va Ky thuat.
Dd Tat Lgi (2003). Nhiing cay thude va vi thudc Viet Nam, Nha xuat ban Y hoe.
Dd Thi Hoa, Biii Quang Te, Nguyin HiJu Diing va Nguyen Thi Mugi (2004). Benh hgc thuy san, Nha xuat ban Nong Nghiep, Ha Ngi.
Evans, J., P. H. Klesius and C. A. Shoemaker (2006).
Streptococcus in wann-water fish. Aquaculture Health International, 7: 10-14.
Fawad A. B., Hashmi A.N., MaUxxib A., Zahid M., Hamid B., Muhammad S.A., Shah Z.U., Afeaal H.
(2012). In-vitro antibacterial activity of Piper betle leaf extracts. J. App Phann, 3(4): 639-646.
Huynh Kim Dieu va Nguyen Thdnh Van (2011). Su thuan ehung va boat tinh khang khuan ciia cay tiau Ididng (Piper betle) va cay Lot (Piper lolot) a ddng bing song ciru long. Tap chi Khoa hgc, 17b; 282-288 Immanuel, G., Vincy Bai V. C , Palavesam A., Peter
Marian M. (2004). Effect of butanolic extracts from terrestrial herbs and seaweeds on the survival, growth and pathogen (Vibrio parahaemofylicus) load on shrimp Penaeus indicus juveniles.
Aquaculture, 236: 53-65.
Minomol M. (2005). Culture of Gold fish Carassius auratus using medicinal plants having immune stimulant characteristics. fvI.Phil Dissertation, MS University, India
Najiah M., Nadirah M. and Zahrol M. A. S. (2011).
Antibactenal activity of edible herbs agamst fi^
pathogenic bacteria. International Journal of Cuuent Research, 3(12): 84-86.
Praseetha (2005). Enrichment of brine shrimp Artemia franciscana with commercial probiotics and beibal exUacts and their resistance against shriii^) pathogen Vibrio sp. (Vibrio parahaemofylicus and V. damsela), M.Phil Dissertation, Manonmaiam Sundaranar University, India
Rani T. V. J. (1999). Fourth year annual report (CSK Research Associate ship) submitted to Council of Scientific and Industrial Research, New Delhi Shameem P. M. D., Thmimal M. B. (2013). A
preliminary antimicrobial screening on leaves of Piper betle Linn. Contemporary Investigations and Observations in Pharmacy, 2( 1): 22-26.
Sivaram V., Babu M. M., Citarasu T., Immanuel G., Murugadass S., Marian M. P. (2004). Growth and immune response of juvenile greasy groupers (Epinephelus tauvina) fed with herbal antibacterial active principle supplemented diets against Vibrio harvty I infections. Aquaculture, 237: 9-20.
Tarun A., Rachana S., Amar D. S., Imran W. and Ankita G. (2012). Comparative analysis of antibacterial activity of four Piper betle varieties.
Amity Institute of Biotechnology, Amity University, Lucknow.
Vo Van Chi (2000). Cay thudc m benh ihdng diing, Nha xuat ban Thanh Hda.
876