HQI NGHj KHOA HQC T O A N Q U O C V £ SINH THAl VA TAI NGUYEN SINH VAT L A N THLf 5
MQT SO Bi^C TRlTNG HOA SINH VA KHA NANG KHANG KHUAN CUA DICH CHIET
QUA NHAU (Morinda citrifolia L.)
vo THJ MAI HU'CWG, TRAN THANH PHONG Tnr&ng Dgi hgc Khoa hoc, Dgi hgc Hui Ngiy nay, viec tim kiem cac logi thudc cd khi nang chiia benh hieu qui, die biet la cic loai thuoc cd ngudn gdc tii' cac nguon dugc thao thien nhien la mdi quan tam die biet ciia cac nha hoa sinh vi y dugc tren the gidi. Theo Td chuc Sue khoe the gidi (WHO), cho den nay, trong khoing 20.000 cay thudc da phit hien chi cd khoang 50 cay dugc khai thae trong cdng nghiep hoi dugc. Nhilu cay thudc dugc thu hai va chl biln chi mdi theo kinh nghiem chir chua dugc tieu chuin hoi ca djnh tinh vi djnh lugng, khd cho phep tao ra nhirng sin pham ddng deu.
Chinh vi vgy nhimg khao sit thanh phan hoi hgc va tic dyng sinh hpc cua cay thudc li rit can thiet nham lao ca sd khoa hpc de danh gii ngudn dugc lieu vi irng dung vao viec chu'a benh.
Cay Nhau {Morinda cilrifolia L.) thudc chi Nhiu {Morinda L.) hp Ci phe (Rubiaeeae) li cay cd rl dugc sir dyng chifa bgnh cao huylt ap va qui dugc in vdi mudi giiip de lieu, nhuan triing, chii'a ho, cim hen, thung, dai dudng [6]. Vdi muc dich tim hieu ro hon cay dugc lieu din gian quen thupc niy, ddng thdi nhim kilm chirng tac dyng khang khuan cua nd, chimg tdi tiln hanh nghien ciru "Mpt sd die trung hoa sinh vi khi nang khing khuan cua djch chiet qui Nhau {Morinda cilrifolia L.)".
!. D 6 I TirgfNG VA PHU'ONG PHAP NGHIEN CUtJ - Ddi tugng: Qua nhau {Morinda citrifolia L.) chin.
- Cic phuang phap xic djnh thinh phin hoa sinh:
Djnh lugmg dudng khu bing phuang phip Bertrand; Djnh lugng protein bing phuang phap Lowry; Djnh lugng lipid bang phuang phap diing Soxhlet; Djnh lugng cellulose bang phuang phap thuy phin bang acid; Djnh lugng vitamin C bang phuang phip chuin dg vdi 2,6- dichlophenolinophenol (DPIP) [7, 9]; Xac djnh hogt dp enzym catalase bing phuang phip chuan dp vdi KMnO^. Xac djnh hogt dp enzym peroxidase bang phuang phip so mau. Him Iirgng glutalion dang khu dugc do bang phuang phip hda hgc vdi thudc thir Ellman [3]. Chi sd peroxit hda lipit (LP) dugc xic djnh theo phucmg phap Placer (1966),
- Phuang phap chiet flavonoid [8]:
+ Chiet va dinh lugng flavonoid toin phin: flavonoid toin phan dugc chiet qua Soxhlet bSng dung mdi ethanol. Che pham thu dugc ggi la cao loan phan (CT).
+ Chill vi djnh lugng flavonoid tdng sd theo quy trinh B.C. Talli, chl pham dugc ky hi?u li FT.
+ Quy trinh chill phan Idp: Mau dugc ngam chiet riit 3 lan/3ngay vdi hdn hgp MeOH: H2O (9:1), cit logi dung mdi dudi ip suit giim Ihu duae can tdng MeOH (ky hieu la CFn,). Chiet vi thu cin tdng MeOH tuang tu nhu tren, them khoing 100-300m! nude cit vio can va chiet phan ldp lan lugt vdi n-Hexan, EtOAC, n-Butanol, Loai dung mdi tren ndi each thuy thu dugc cao chiet tuong irng, ky hieu li CF,„ CFe va CFb
HQI NGHI KHOA HQC T O A N Q U O C V £ SINH T H A I V A T A I N G U Y £ N SINH VAT L A N THCr 5 - Dinh tinh flavonoid bing cic phin irng hoi hpc die tnmg: Phan img Shinoda; phan in vdi dung djch kilm die; phan irng vdi acid sulphuric dgc; phan ling vdi FeCls; phan img dji tinhcatechin [2j.
- Thu hogt tinh khing vi sinh vgt theo phuang phap khulch tan tren dTa thach [1], Cic chung vi sinh vat (VSV) kiem djnh dgi dien gay bgnh cho ngudi do Trung tim Kilm nghi^
Dugc, My pham-Thira Thien Hue cung cip, gdm: Vi khuan Gram (-): Escherichia coli;
Salmonella typhi va vi khuin Gram (+): Staphylococcus aureus; Bacilluspumllus.
- Cac so lieu thuc nghiem dugc xir ly thdng ke tren chuang trinh MS-Excel. Trung binh mau±sai sd chuin.
II. KET QUA VA THAO LUAN 1. Mgt 50 thanh phan hda sinh ciia qua Nhau
^
• t's.
1
2 0 1 18 • 16 • 14 • 1 2 - 1 0 - 8 . D •
4 - 2 - 9 -
5,27 2.8
8,75
1
1«.33
• 1 1 D I
Di><a'ng Pialein Lipid CelluloE
Hinh I. Ham hrgng mgt sd thdnh phdn hod sinh trong qud Nhau
Kit qua phan tich mgt sd Ihanh phin hda sinh trong dich chiet qua nhau (hinh 1) cho thfiy:' Him lugng cellulose trong qua nhau (19,33%) cao hon hin so vdi dudng khir (5,27%), protein (2,8%) vi lipid (8,75%).
2. Hoat do ciia enzyme chong oxy hoa (catalase, peroxidase) va ham Iugng mpt si chSt chong oxy hoa trong qua Nhau
Catalase (C-ase) li enzyme chira sit tim thiy trong tit ca md hiiu khi, xiic tac cho phSn irng phan giai H202 thanh H2O va O;. Trong he thong enzyme antioxidant (enzyme chdng OJQ' hda) thi C-ase la mgt trong nhtrng enzyme quan trpng nhat, cd tic dung logi trir H2O2 tich W trong te bio. Ket qui nghien ciru cho thiy hoat dp rieng cua C-ase trong qua Nhau rit cao,^t 32,626 U/mgprotein (bang 1). Hogt dp ciia enzyme niy d qui Nhau cao han so vdi mgt s6 p tupng khac nhu Dua xanh 2,38 U/mg protein, Dua leo 3,03 U/mg protein, vd Nha dam non 25,33 U/mg protein, rupt Nha dam non 9,44 U/mg protein, cii Girng 0,06 U/mg protein, li?t Tieu 1,17 U/mg protein [4].
HOI NGHI KHOA HOC T O A N Q U O C V £ SINH T H A I V A TAI NGUYEN SINH VAT UkN THLf 5 Bdng 1 Hoat do catalase va peroxidase trong qua Nhau
Hoat dg enzyme
• " Catalase Hogt dg Chung
1,420 ±0,281
Hogt dg rieng (U/mg protein) 32,626 ±0,316
Peroxidase Hogt do chung
2,271 ± 0,042
Hoat dg tieng (U/mg protein) 68,818 ±0,012 Enzyme peroxidase (P-ase) cd mat d dpng vgt va thuc vat, tic ddng len H2O2 va peroxide hihi ca trong su hien dien cua chat nhan oxy. Peroxidase ngan chin su nhiem dgc cua te bio biing cich phan huy H2O2 dugc tao thinh trong qua trinh trao ddi chit. Ngoii ra enzyme niy c6n xuc tic cho cac phin ung oxy hda nhieu logi polyphenol va amime tham, tao thanh cac san pham khac nhau.
Hogt d$ peroxidase ciia qui Nhau rat cao (68,818U/mg protein). So sanh vdi boat dp ciia enzyme nay d vd nha dam non (0,44 U/mg protein), d rugt nha dam non (0,12 U/mg protein) va mOt so doi tugng cay dugc lieu khac thi boat dp en^me P-ase trong nghien ciru nay ciia qui Nhau cao han rat nhieu [6].
* Mpt so hgp chht chong ojcv hod
Trong qua trinh logi gdc t\r do, cac chit chdng oxy hoa tu nhien cd trgng lugng phan tir thip c6 mat trong cac he thong sinh hgc cung thudng dugc chu y, trong dd glutalion dang khu (GSH) d6ng vai trd quan trpng. Khi ndng dp H2O2 trong t l bao thip, glutalion peroxydase la he thdng bao V? chinh cua le bio. Khi ndng dp H2O2 Idn gay dgc hai thi C-ase budc vio boat dpng. Ben c?nh dd vitamin C (VTMC) dugc xem nhu li mpt thudc cd tie dyng chong oxy hoi, chdng goc t\rdo. Chi so peroxid hoa lipid (LP) dugc xic djnh nhu la chi sd ndi len tic dpng ciia gdc oxy phan tir len mang t l bio ma cd the md ta nhu phan img giira cac gdc phit sinh tir O2, H2O vi ciia acid b6o khdng no [5]. Ket qui djnh lugng mpt sd chit cd tic dung chdng oxy hda tren trong qui nhiu nhu sau:
- Him lugng glutalion dang khd (GSH) trong qua Nhau khoang 2,270 ^M/g mau, cao ban nhilu so vdi Ihanh phin nay d Cam thao dii (0,37), Sai dit (1,13), Che xanh (0,87)...
- Ham lugng vitamin C chiem I21mg/I00g.
- Chi sd peroxid hda lipid (LP) dgt 79,392 nM MDA/g mau.
Nhin chung boat do cac enzyme chdng oxy hda trong qui Nhau tuang ddi cao, ciing vdi sy c6 mgt mpt so thinh phin chdng oxy hda khac phin inh khi nang chdng oxy hda ciia ddi lugng nay ludn dugc duy tri d mirc cao.
3. Ham lirg'ng cac che pham tach chiet tiir qua nhau bang cac quy trinh khac nhau Ket qua chill CT bang dung mdi ethanol thu dugc ham Iugng cao nhit (8,35%); Him Iirgng flavonoid long sd (FT) chill theo quy trinh Talli thu dugc 4,12%; Chill phan ldp vdi cac dung mdi khac nhau (CF), lan lugt thu dugc: Dung mdi MeOH (CFn,); 8,06%; dung mdi n-Hexan (CF|,): 2,15%; dung mdi EtOAC (CF^): 1,76% vi dung mdi n-BuOH (CFb): 1,81%.
Nhu v§y ty le chl phim thu dugc tir chill phan Idp li khac nhau, trong dd chl phim MeOH chiem ham Iugng cao nhai.
HQI NGHI KHOA HQC TOAN QU6C V ^ SINH THAl VA TAl N G U Y E N SINH VAT LAN THCf 5
• • •
FT CT CFm CRi Cfe CFb
Hinh 2. Hdm hrgng chi phdm thu dirge bdng cdc phirang phdp chiet khde nhau 4. Djnh tinh flavonoid bang cac phan ling hda hoc dac trung
Flavonoid cho phin img die trung vdi mpt sd chit thu nhu: Dung djch NaOH, H2SO4, FeCb, vanilin 1% trong HCl. Phin ung duong tinh the hien qua miu sic dgc trung de dang quan sil bing mat Ihudng. Thi nghiem dinh tinh (bing 2) cho phep du doin thanh phin flavonoid fin trong djch chiet phin Idp vi dich chiet flavonoid tdng sd ciia qui Nhau.
Ket qui dinh tinh tren cho thay djch chiet long MeOH qui Nhiu cho phin ung duang tinh vdi ca 4 logi thudc thu chimg td su cd mat ciia flavonoid. Che pham chiet phin Idp bing dung mdi EtOAC cho phin irng duang tinh mgnh han ca, mau phin irng ciia chl phim vdi thulc thit cho phep du doan su cd mat ciia mpt so flavonoid. Chl pham chilt phan Idp bing dung m6l n-BuOH khdng cd phin ung tao miu dgc trung ciia flavonoid vdi H2SO4 va FeCb va cd phSn itng ylu vdi NaOH vi dung djch vanilin 1% trong HCl die.
Bang 2 Dinh tinh flavonoid trong cac phan djch chiet qua nhau
Djch c h i l t
MeOH n-hexan EtOAC n-BuOH FT
Thudc thi?
NaOH + + + + + + + + + +
H2SO4 + + + + + +
-
+
FeCb + + + +
+
Vanilin/HCI + + + +
Du' doan s v cd m^t cda flavonoid
Flavonoid Flavonoid Flavonoid Flavonoid Flavonoid S. Hoat tinh khang khuan cua cac dich chiet qua Nhau
Tinh khing khuin la mpt trong nhifng tic dyng sinh hgc dugc quan tam ciia cac cay thuoc.
Nghien ciiru boat tinh khang khuin ciia cac chl pham CT, FT vi CF tir qua Nhau cho thay ck che pham deu cd kha nang khang vdi 4 loai vi sinh vat kilm djnh, trong dd S. aureu Ii chiinB nhay cim nhat.
HQI NGH! KHOA HQC T O A N Q U O C V £ SINH THAI V A TAI NGUYEN SINH VAT L^N THlJ 5 Bdng 3
Chfe ph^"i
CF„
CFB CF.
CFB CT FT
Hoat tinh khang khuan cua ch^ pham C T , F T va C F tir qua Hifu s6 vong
Staphylococcus aureus 18,33±0,33 10,33±1,20 19,67±1.20 18.76±0.75 15,03±0,68 19,17±0,44
Nhau
vo khuin ddi v6i cac VSV kilm djnii D-d (mm) Salmonella typhi
16,33±0,88 5,67±0.33 22,33±1.20 16,00±1.00 12.67±0.67 20,83±2.09
Escherichia coll 15,02±0,57
8.75±0,25 15,67±1,20 17,75±0,25 6.67±0,88 13,75±0,75
Bacillus pumilus 16.70±0,33 4,25±0,75 13,33±0,88 13,25±1,25 6.33±0,88 10,60±1,50 Hogt tinh khing khuan ddi vdi cac logi vi khuan gay benh cua cac che pham cy the nhu sau:
+ Che phim CFm khang mgnh ddi vdi ci 4 nhdm VSV kiem djnh, nhat la ddi vdi S. aureus.
+ Che phim CF^ khang manh ddi vdi ci 4 nhdm VSV kiem djnh, die biet ddi vdi S. typhi.
Che phim CFb khing manh nhit ddi vdi S. aureus, S. typhi va E. coli. FT khing manh vi khuan S. aureus va S. typhi. Trong cic che pham thu dugc, che pham CF^ cd tic dung khang B.piimilus manh nhit; che pham CFb khang E.coli mgnh nhat.
+ Nhin chung djch chiet qui Nhiu the hien hoat tinh khing khuin khi cao.
Hinh 3. Vong vo khudn ciia dich chiil CFm ddi v&i B. pumilus
J vd khudn ciia che phdm FT doi v&i E. coli
Hinh 5. Vdng vd khudn CT (2 giing bin phdi) va cua FT (2 giing ben trdi) ciia qud Nhctu
did v&i S. typhi
PTinh 6. Vdng v6 khudn cua CT doi v&i vi khudn kiem dinh S.lypht
HQI NGHj KHOA HQC T O A N Q U O C V E SINH T H A I V A T A I N G U Y £ N SINH VAT L A N THCf 5 m . KET LUAN
1. Q u i Nhau chin chua 5,27g/100g dudng khir; 0,033g/100g protein; 8,750g/100g lipid;
19,33 (%) cellulose:
2. Hoat dp mdt sd enzyme v i c i c chat chdng oxy h o i trong qua Nhau k h i cao. Hogt d?
rieng cua C-ase la 32,626 (U/mgprotein), hogt dg rieng cua P-ase l i 68,818 (U/mg protein), him Iugng vitamin C la 0,121 (g/1 OOg mau). Chi so LP: 79,39 ( n M M D A / g mau)
3. H i m lugng cao toan phan vdi dung mdi elhanol (CT) chiem 8,35%; flavonoid ldng so (FT): 4,125%; c i c c h l p h i m c h i l t phan ldp vdi dung mdi M e O H ( C F J : 8,06%; n-Hexan (CFh);
2,15%; dung mdi EtOAC (CF^): 1,76% v i dung mdi n-BuOH (CFb): 1,81%. D u doan sy conit ciia flavonoid trong t i t c i cac dich chiet qua Nhau thu dugc.
4. Cac che p h i m CFm, CFh, CFe, CFt,, C T , FT (flavonoid tdng sd) deu the hien hogt tinh khing khuan manh vdi c i c logi vi sinh vat kiem dmh. Trong dd d i n g chu y la 2 loai VSV kilm dinh Staphylococcus aureus v i Salmonella typhi I i nhay cam nhat ddi vdi tat ca cic che pham nghien ciru CFb, CFe va FT ndi tren.
TAI LIEU THAM KHAO
1 Gomez-Flores R., P. Tamcz-Guerra, C. Rodriguez-Pad ilia, E. IVIonreal-Cuevas, 2006, Journal of Infectious Diseases, 1: 1-8
2. Keoliman J., K. H. Roehn, 2005. Color Atlat of BiochemisUy, Thieme Stuttgart New York 3 Bo Quy Hai, 1986. Nghien cuu so sanh cac enzyme antioxidant d trong cac h? thing sinh hpc khic
nhau, Luan i n Phd Tifin sT (tieng Hungari) Szeged, Hungari.
4. Do Quy Hai, Do T i n Thao, Phan VSn Cu, 2006. Tgp chi Khoa hgc, Dai hgc Hue, Sd 33: 51-59.
5. Va Thj Mai Huong, Tran Thanh Phong, 2007. Nghjen cim hogt dgng chdng oxy hda ciia cay Nha dam {Aloe vera). Bao cio Khoa hgc Hgi nghj toin qudc 2007. Nghien ciru co ban trong khoa hgc sil sdng. NXB. KHKT, tr.: 883-886.
6. Do Tat Loi, 2001. Nhimg cay thulc v i vj thuoc Viet Nam. NXB. Y hgc.
7. Nguyen VSn M i i i , 2000. Thyc hanh hda sinh hgc. NXB. KHKT, Ha Ngi.
8. Placer Z. A., LCushman., B. C. Johnson, 1986. Biochemical system. Analyt Biochem. 16: 359-364.
9. Ho Viet Quy, 2002. Chilt tich, phan chia xac djnh cic chat bang dung moi hiJu ca. NXB. KHKT, HaNgi, tap 1.
10. Phan Van S5, Biii Thj Nhu Thu^n, 1975. K i l m nghiem luong thirc, thyc phim. NXB. KHKT, Hi NgL
SOME BIOCHEMICAL COMPOSITION AND THE ANTI-BACTERIAL ACTIVITY OF Morinda citrifolia L. EXTRACT
VO THI MAI HUONG, TRAN THANH PHONG
SUMMARY
Morinda citrifolia L. is used as a traditional medicine to treat many diseases. The ripe faiit contains . reducing sugar (5,27g/100g), protein (0.033g/100g), lipid (8.750g/100g), cellulose (19,33%), vitamine C (0,121 g/1 OOg). The activity of antioxidant enzyme C-ase and P-ase is rather high (32.626 U/mg proton and 68,818 (U/mg protein), LP (79.39 nM MDA/g). High levels of whole fruit as solvent extraction with ethanol (CT) is 8.35%, total flavonoid content (FT) is 4.125%; layered preparations with solvent extraction MeOH (CFm): 8,06%, n-hexane (CFn): 2,15% solvent EtOAC (CFe): 1.76% and solvent n-BuOH (CFt):
1.81%. The presence of flavonoids was predicted in the fruit extract. The products obtained showed strwj activity againsl the tested microorganisms, particularly the preparation of CFB, CFB and FT- Staphi^ococcus aureus and Salmonella typhi are the most sensitive species to the all studied preparalions.
