DSpace at VNU: Investigation of Proteinase Protein Inhibitors (PPIs) from Seeds of Certain Plants belonging to Moraceae Family and other Ones

DSpace at VNU: Investigation of Proteinase Protein Inhibitors (PPIs) from Seeds of Certain Plants belonging to Moraceae...

VNU JOURNAL O F SCIENCE N at Set., t.xv, n^3 - 1999

IN V ESTIG A TIO N OF PROTEINASE PROTEIN IN H IBITO RS (PPIs) FRO M SEEDS OF CERTAIN PLANTS BELONGING

TO M ORACEAE FAMILY AN D OTHER ONES*

P h a m T ra n C h a u , N g u y e n H o n g A n h , Le T ro n g Q u a n g

P h a n T h i H a , N g u y e n T u y e t M a i, D ao T ro n g A n h

Biotechnology Center, College o f Nãtìiraì Sciences - VNU

I INTRODUCTION Proteinase protein inhibitors (PPIs) plav an important role in living system aiid have several practical applications [7, 9, 11] Therefore, the investigation of PP Is ill plants lias both scientific and roalistic significance

However, ill Victiiaiii, the study on PPIs has started in our laboratory only since

1986 During th a t period, most of studies were focused on PPIs from Cucurbitaceae seeds.

As far as wo know, there arc luoro than 90 published papers about protein from

Artocarpus seeds, b u t no one reported about PPIs.

This work presents some prcliuiiiiarv results of PPIs from seeds of ccrtaiu Moraccac plants and from some plants of other faiuilios

II MATERIALS AND METHODS

1 M a te ria ls

T i u ‘ o f A i to c u i ]}UƠ i t i c l u i o j y l a G u g li, ÁrioctnpXLO c ĩ i a p ỉ a s h a RoxV>; A v t o r n r

-pus intei'ger ; Artocai'jms lakoocha Roxb; Steblus asperLoni; Annona squamosa L; Diospyjvs kaki Linn f; Abeimoschus esculentns L; Syzygmm, jambos L ; Delavaya toxo- cai'pa; Ciừ^ts gm ndis Osbock; Cifnts sinensis L; Pynts pyTi-folia Nakai; CaHca papaya L; Sechixmi edule Sw; Clirụsophyllmìì cainito L; Persivim.on kahi soft type ripe were colloctccl

fioiu Iiiatme fruits

' (-h) Chemicals

a - chyiuotrypsiii from Lab Lciirquiii (France); casciu from BDH clioiuicalH Ltd,

Scpliadox - G 75 from Phanuacia Fine chemical (Sweden); tr\psiii, albuiiliu wore supplied

by Sigiiia Ltd (USA)

*) This work was financially sponsored by ” Basic scientific program m e - project", subproject 6.4.4

A b b re v ia tio n s : PA: proteolytic activity; TIA; trypsin inhibitor activity; ChIA; chym otrypsin in­ hibitor activity; DChIA (D TIA ): Tl or Chi peak.

- Chemicals for electrophoresis: acrylaniide, bis - acrylaiiiidc, Tris - basc ctc., from Sigina Ltd (USA) The other chemicals reached the PA giade

(-h) Methods

The finely ground seeds were extracted with distilled w ater (with ail exception

for Diospyros kaki Linn seeds, where pH of 4.5 Britton and Robinson buffer was used)

The ớbtained suspensions were stirred for 30 minutes at room tem perature aiid claiified

by centrifugation (8000 rpm) for 10 miuutes The supem ataiit was collected for further analysis

- Protein concentration was determined by the Lowry m ethod [6], using bovine serum albumin as staiidiqrd

- Determination of proteolytic activity (PA) was carried out by Ansou modified

m ethod [8] PA was determined at 35.5^c using pH 7.6 Srrensen buffer with 1% casein

as substrate for 20 minutes One unit of proteolytic activity was defined as the amount

of enzyme after one Iiiinute of action at 35.5°c generating TCA soluble products, giving colour reaction with Folin Ciocalteau reagent equivalent to one micromole of tyrosiiie

Inhibitory activity was deteniiiued from the residual enzyiuatic activity after 10 mill pre-iucubation of the Guzyiue with the iiứiibitoi solution Oue unit of inhibitorv activity was defiued as th at anioimt of inhibitor, which reduces the activity of two mg of eiizyiiie

by 50%

- PA, ChIA, TIA were also detenninểd by diffusion m ethod [5,10

Heat stability of TI and Chi was estimated as follows: the cnidc extract was treated

at lOO^C for 15 luiii, cooling iimnediately in icc The activity was measured in standard conditions The remainiug activity was calculated in coiripariiig with the initial activity

- Polyaciylaiiiido gel electrophoresis for identification of protein bands was pci- fomicd according to Laemiiili method [4] Protein pattern was analysed by Sliaip JX 330 scaiiiior

Gel Ịìlừution by Sephadex~G75 cohirrin

The gel filtration cohiimi witli a size of 1.7 X 92cni was equilibrated by pH 6.5

Srreuscu buffer for AHocarpus melinoxyla Gagii and AHocarpus chaplasha Roxb pH 7.6 for Abelm.oschus esculentiLS L aiid Delavaya toxocarpa Protoiiis were ohitcd by the equi­

librated buffer The fraction volume was 3ml The niiiiiiiig speed was 20inl/houi

III RESULTS 1- S c re e n in g p ro te in a s e in h ib ito ry a c tiv ity (P IA ) o f s e e d c r u d e e x tr a c ts

Seventeen seed samples of Moraceacy Rutuceae, Annonaceae, Rosaceae, Ebenaceae, Caricaceacj Malvaceae, Sapindaceacj Myrtaceae, Sapotaceae, fam ilies were subjected to PIA investigation Nine of them showed iiiliibitory activity (Table 1) blit in the right

others: pomelo, orange, peai’, papaya, diayote, star apple, pcrsiiiunon, kaki soft ripe neither TI nor Clil wOvS discovered

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P ham Tran Chau, Nguyen Hong Anh, Le Trong Quang,

As shown ill table 1, Artocai'pus nielinoxyla Gagii, Artoairpus chaplasha Roxb,

A b e h w s c h u s excnlentĩLS L and DeẦavaya toxocarpa co n tain b o tli T IA a n d CliIA However

ill the first two samples ChIA was 2-3 times higher tliaii TIA, while ill Abelmoschus escidenius L and Delavaya toxocar'pa, there was no clear distinction between TIA and

ChlA

In 1 cniaincd samples, TIA was only discovered in seeds of Artocarpĩis lakoocha Roxb Dtospyros kaki Liliii f, Delavaya toxocai'pa, Annona squamosa L while KIA existed in Artocarpĩís melinoxyla Gagii Among the Moraccae soods owing PPI, the highest TIA lo\rl (5675 n iiu /g of dry uiattor) was foiiiid in Artocai'jms Melinoxyla Gagii In opposition

Sfeblus aspei' Lour sam ple showed prot.oolytic a ctiv ity [ I J S l U / g of d ry Iiiatto r) blit, was

lacking in PA, PPIs Tims in comparison with the Moniordica seeds, TIA of Artocaiytis

was 10 tiiiies lower [12, 13] but it was higher tliaii punipkiii, squash, w atrr melou TIA

Basing on this oxpcriuioiit, W(' scloctcd 4 saiiiỊ)l(‘s of Al'tocarpas iiidinoiyyla Gagn; ArtocaiyxLS chaplasha Roxb, Delavaya toxocarpa and Abelviochus escideiitus L for fiuthoi

analysis

At first, SDS - PA G E was u sed for clctoctiiig th e p io to u l coinpoiiouts

Number

Rm

Artocarpus melinoxyla Gagn

Artocarpus chaplasha

Abeim oschus esculentus L

Delavaya toxocarpa

Table 2: Tlio ralativc mobility (Riii) of protein bands from 4 samples.

In ve stig a tio n o f P roteinase P ro te in Inhibitors (P P Is)

2 E le c tro p h o re s is p a t t e r n o f se e d e x tr a c t o f A r to c a r p u s m e lin o x y la G ag n ;

A r to c a r p u s c h a p la s h a R o x b ; A b e lm o c h u s e s c u le n tu s L; D elav ay a to x o c a r p a L

For convenience, the Rin of protein bands

was calculated and listed ill table 2

The d ata in photo 1 showed th a t the

protein pattern of seed extract of AHocaryus

melinoxyla Gagii and Artocarptis chaplasha

Roxb was rather similar, including 10 protein

bauds in Artocarpus melinoTtjla Gagii and 11

ones in Artocarpus chaplasha Roxb Among

these bands, the protein ones with Rin=0.39;

0.44; 0,58; and 0.62 were inajor bands

It was iiiterestiug to note that there

was only one difference ill protein band with

Rju=:0.39 between Artocarpus vielinoxyla Gagii

a n d A r fo c a r p u s ch a p la sh a R o x b M a y b o tlic

differentiation between Artocai'pus nielinoxyla

Gagii and Artocarpus chaplasha Roxb was de­

fined by this protein

The rate ratio of main protc'in bands

(Rni = 0.39; 0.44; 0.58 and 0.62) was calcu­

lated and presoiitecl ill table 3 In

Artocar-jn i s m eiiT w :ry la G a g ii, tlio p r o t e in b a n d w it h PI 4 1 • r I

Rill = ,0.39 a c c o u n ts foi a p p io x iia a t o ly 6 IG ^ ^ tr o iu A r t o c a i p i i s liK 'linoxyla G a g a ( 1, 3 )A * I 1 - /1

of tlio to tal pioteiii coiitnit 6.16 % whilo for , I A 1 1 1 r 1 /0

’ aiul A i t o c a i p u s cha])lasha R d x I) (2, 4)

tlio R n i = 0.4 4 p io t o iii h a ix l it w as r>‘) % , ,, , , , , ,

1, r iie li l a iif ('(Jilla m lu I I I ()1 sa iu

{)l(-lowt'i' Mian that of tlir coriPspoiKliiie, one ill r, , , , „ , • , r ; r ,

.5, 4: ('Hcli laiK' coulaiii 1-) ỊIÌ C)i saniplr Artocarfius cliaplashaRoxh Thus, a(iucstioii

arises; d o c s tlir p r o te in band w ith R m = 0 3 9 o rigin ate ÍI 0111 thí' one wi t h Hni ^ 0.44? aiul

the coriTspoiidiug goii(s) klmtifviiig the (lifỉ'('i('iic(' l)('l\v('('n t!i('s(« two Artixarfiis spades!

The major protein bands (Km)

Artocarpus chaplasha Roxb

Artocarpus melinoxyla Gagn

Table 3: The porcoiitago ratio of luajoi proti'iii bauds ill c()iiiỊ)ai isoii

with the total of dotcctod bauds bv Sharp JX-330 scaiuior

The SDS - PAGE results also showed tlio difforeiicr hi clcctrophorosis pattoni aaoiig

Artocai'pus ĩneẦỉnoxyla Gagii; AHocarpus chaplasha Roxb and Delavaya toxocarpa The

shiiplost protein pattern with 5 protein bands ill which the major one with Riii = 0.Í6 was

foiiiic] in Abelniocfms escĩdenÌMS L Although, ton protein bauds were dctectcd ill p.'Otciii

pattern of Delavaya toxocarpa, almost tliese proteins wore different from the concspciidiiig

ones of the other families ill Riu value

To preliminary separate PPIs from crude cxt,ract, Sephadex G75 coliuuii was used

for chromatography

3 C h ro m a to g ra m o f se e d e x tr a c t o f sa m p le s th r o u g h S e p h a d e x - G 75 colum n

As shown in iig 1 and fig 2, the chroniatogxaiii patterns of Artocaryus rn,tli'ioaryla

Gagii and Artocarpus chaplasha Roxb [5, 10] were siinilai' ill shape, consisting of 2 peaks

(D i,D9), in which D \ was an active peak Ill AHocarpiis vielinoxyla Gagii, D i naclied

52.78 % of the total protein loaded oil the coluiiiii while it W£LS 55.75 % in AHocarpxLS

chaplasha Roxb Thus, at the back of separation, the puriiiod level of Chi hi Artocarpus

melinoxyki Gagii was equivalent to th a t of Artocarpus chaplasha Roxb After chroiaatog-

raphy, since TIA both of AHocarpiis melinoxyla Gagn, Artocarpus chaplasha Roxb was

very low, it was not possible to dctcct by Anson uiotliod, TIA was only discoveiod by

diffusion niothod

It should be noted th at TIA fraction shared the same peak with CliIA OIIC- This

pheiiomenoii may be the cause of the similarity ill Rill between TI and Chi in these

samples? or there existed the double headed inhibitors ill Artocarpus: one doinaiii iiiiibits

trvpsiii, the other is completed witli chyuiotrypsiii, following the typo of BBI fcrni in

soybean seed [2, 3]?

Under the same couditiou, thcic wore SOUIO differences ill chroiuatogiaiu pattern

of Delavaya toxocarpa and Abelmodms esơidentĩLS L PPIs fractious were eluted slowlicr,

thus it documoiitod th at luoloculai weight of PPI ili these samples was lower than that of

A rtocarpĩis.

I"ig 1 showed two main piotoin peaks {Dị, Do) ill chroiiiatograiu pattern of sood

cxtract of Abelniock'us esculentus L TIA, CliIA wore shown ill D\ As the protein contciit

of Di roachod 35.71 % of total studied protein, the specific activity of TIA and CliIA

iiicrcased rrspcctivoly 3.15 and 3.35 tiiiios

As shown ill fig 1, there wore 3 peaks {Dị D2 and Ơ3) in chroinatography pattern

of Delavaya toxocarpa Since D2 had the lowest protein coiitciit (about 26.5 %) and was a

TI peak, thus the specific activity of TI increased 3 tiiiios more than that of initial extract

4 E ffect o f lie a t tr e a tm e n t o n C h IA (o r T IA ) o f s tu d ie d sa m p le s

It is known that high PPI content ill footl might affect unfavorably digestion ill

aiiiiiials and humans duo to the docrcasr of cnzyinc activity by inhibitors.

- 4.9 Artocarpiis vielinoxyla Gagii and Abebnochus escnlentus L has been used as a

good initiiont, wo studied the impact of heat troatiiiont at lOO^C for 15 luiuutos oil TI,

Chỉ in those saiiiplos

Fig 1: Chromatogpaphy p attern of seed extract of studied samples

by Sephadex G 75 column

In ve stig a tio n o f P rotein ase P ro te in Inhibitors (PPIa) .

Pf actions

(b)

12

10

ÍÍ ÍỈ s 5! s? s R

Fractions

f k M i %

Practions

(đ)

Pham Tran Chau, Nguyen H ong Anh, Le Trong Quang,

Aíỉocarpus Arỉocâíípus Abdfnochus chđplâshd meiitioxyld eaciientưs L

C tixle exffact

D ela v ay a A tto c a ip u s Aiỉocafp*,Ji A tfcca iip u s

ỉoxocàĩpâ chaplasha mdmũxylâ fr.ei*no5<ylô

R o x b G ag n G d g n

Active peak (CWA 0{ TiA) oòỉ^-ied Sepha«jex chforn^ogidphy

Fig.2: The cffcct of heat treatiueiit on CliIA (or TIA) of studied samples.

- The obtained results showed th at when the seed extract was treated at lOO^C

for 15 iniuutes, TIA arid CblA of Abelmochus esculentiis L were almost destro^tĩd Ill

Artocarpus melinoxyla Gagli, Artocarpus chaplasha Roxb, CliIA ill both cnidc extract

and active peak (DchiA,-Dtia) were reduced by aroiilid 70 % but the remained TIA of

seed extract ill these samples was still high This phenomenon might be due to the fact

th a t sample concentration for deteniiiiiiiig TIA was 5 times higher than th at one for Clil

detection Therefore, TIA, ChIA ill boiled seeds were detcimined in a further step

After boiling, TIA and CliIA rcdiiced to 10 tiiues ill coiuparisoii with initial activity,

correspoudiug to 0.1 - 0.2 lU The ininiuuuu of trvpsiii coiitcut released from hiiiiian

pancreas per clay is 782 irig (782 Ư) [1], thorcfoio the roiuainhig TI, Chi in AHocaTpiLS

seed after boiling has no considerable iinpact on trvpsin and chvinotrvsin activities in

liuiiiaii body

IV CONCLUSION 1- Among 17 iiivostigatod samples, TI aiid Clil are found in 9 samples, consisting of

ArtocaTpus w.elino:ujla Gagii, Artocarpus chaplasha Roxb; Artocaiyus integer Moir;

tocarpĩis lakoocha Roxb; Streblus asper Lour; Annona sqttaniosa L; Dispyros kaki Linn f;

Diospyros kaki I ll which 4 samples of ArfocaiyiLs nielinoxyla Gagii, ArtocarpĩLS chaplasha

Roxb Syzygiiiin jauibos L., Abchnosclms escuiciitus L., had both TI aiid Chi

2- pH 8,3 SDS - PAGE 12,5 % has enabled the idcutificatioli of 10 protein bands from seed

extract of Artocarjms ĩìieỉinoxyla Gagn, Artocai'pvs chaplasha Roxb However, there was

another protein band with Riu = 0.39 iu Artocarpus melinoxyla Gagn, at the saiuc time,

the protoin with Riii — 0.44 was decreased

At least, 5 protoin bands were found ill Abehwchus escĩãentus L and 9 protein ones

were discovered ill Delavaya toxocaiya Almost those baiids difforod ill tenus of Rrn value

to the coiTOspondiiig other samples

3- Chiomatograni pattoni of AHocaiyus nielinoxyla Gagii; Artocaiyus chaplasha Roxb

wore rathoi siuular, consisting of 2 peaks (D i, D ,) hi which D , had both TIA aiul ChlA.

- I I and Chi of seed cxtiact of Artocarpus Tri.elinoxyla Gagn; Artocarpus chaplasha were

After heat treatm ent at 100°c for 15 m im Z s the K'liiaiiK'tl activity was over 30%

V REFERENCES

1] R.M Bonio , M N N Levy Physiology , Mosby - Yoai' Book, Inc., USA (1993)669-

[2] Y Bilk , A Gorier Proc Int Res Conf Inhibitor., Int ed H fritz H Tes chesche 142-1Ị8 Berlin; De Gruftcr (1971)304.

[3] D.E Bowman Diffcrcutiatioii of Soybean antitryptic factors, Proc Soc Exp Biol.

Med 63(1946) 547-550

4] V.K Laeiiuiili Cleavage of stnictm o protein during the assoiiibly of the lioad of

bactcrophagc T4, Nature 227(1970) 680-685.

5] Lohik Jacok, Pham Till Trail Chau, Kicloczawa Jacck Zastosowanio edcstviiy do

vadaiiia aktywiio’sc’ proteiiiaz i icli inhibitorow, X X I Zjazd P.T.Bioch K m L Z Streszczenie 140(1985).

|6| Q H^Lowry, N.J^ Rose b.cugl,, AX Fan , R.J^ Rai.dall p.otain with

tlio Foliii phenol reagent, J Biol Chem 193(1951) 265-270.

|7] Ng,,v<-„ Khoa Dion Tl,„, PI,a,,, Tl.i Tia Cl,an, Prclimmary roscarchcs on tl.o

s'-paiatii.g from Mo.uodica seed to R a t i

reproduction, Journal o f Biologs-Soil science: Hanoi National University (1989)

w M.M W incjunajtc OpreJelcute piotcolitichcskoi aktivuosti k n m M -Iiykh prcparatov niikrobiologichoskovo proiskliachdenia, Pnklad Biochern Mikro-

bUu. 2 (1 9 0 0 ) 232.

Chau TVi/psm inhiUtors of white bush (CucnrbUa pepo var, patisso-nia) jn n t anh seeds (Doctor babilitatus thesis), Acta Uiiivorsitatis Wratislavion^s

(198o) 912

[10] Pham Thi Tran Chan, L Koiiopska, J Lehik TiyiJsin inhibitors ill tlio alouroiio

gian io so fC u cu rb itap ep o v ai patissonia (White bush) cotylodous, Brochem Pi>z

*o/., paaiizoii, 181 ( 1986) 565-569.

[11] Phani Tin Tran Chan, Nguyou Tiiyct Mai, Phan Thi Ha Purification, cliaractoriza- tioii of autilysin fioin bovine Imig - preliminary study regarding the detonuhiatiou

ot Its practical uses, Journal o f Biology 15(4)(1993) 8-10 (ill Viotiiamcs).

,12] Pham Thi Tian Chau, V.T.Hion, P.T.Ha, N.H.M.Q„yon, N.T.Mai Proliniiiiary

data of trypsin inhibitors (Tls) from squash seeds of Virtnaui, 10th FAOB syvipo-

13| PUa„ T„a„ Nghia, Pl.au, Thi T,a Cl.ãũ (1987): a Z g c s to trvpsh i„l,ibito.y ac-

t vitv (TI A) aiKl proteolytic activity of develop,uct seeds ami fii.its of Momoulica

chaiaiitia L, Jowmal of Biology 9(3), 12-17 (ill Victuaiiios).

TAP CHÍ KHOA HOC ĐHQGHN KHTN, t.x v n°3 - 1999

ĐIỀƯ TRA CÁC PROTEIN ỨC CHỄ PROTEINAZ (PPI)

o HẠT MỘT SÓ CÂY THƯỘC HỌ MORACEAE VÀ MỘT VÀI HỌ KHÁC.

P h a m T râ n C h â u , N g u y ễ n H ồ n g A n h , Lê T ro n g Q u a n g

P h a n T h ị H à , N g u y ễ n T u y ế t M a i, Đ ào T rọ n g A n h

7>uíjg Tàin Công nghệ Sinh bọc Đại học K H T ụ nhiên - ĐHQG Hầ Nộí

Chúng tôi đã tiếu hàiih điều tra sa bộ PPI của 17 mẫu hạt thuộc các họ Dâii tằiu, Bỏng, Siiu, Bồ hòn, Tliị và Na Tioiig đó 4 uiẫu hạt Mít rnật, Mít dai, Roi, Đậu bắp có chứa cả chất ức chế tripxiii và kiinotripxiu Năiu niẫu hạt khác: Chay, Mít tố nữ, Hồiig

đỏ, Na có chứa TI hoặc KI Tír các kết quả trên cúng tôi đà cliọii hạt Mít luật và Mít dai

để nghiên cứu tiếp Kết quả điệu di dịch chiết hạt 2 Iiiẫiỉ Mít m ật và Mít dai cho thấy pliổ điệii di proteiii 2 mẫu Iiày khá giống uhau, trừ băiig protein có Riu = 0.39 (Mít dai kliỏiig có băug proteiii uày) Phải cliăiig băng proteiii trên qưi định Hự sai khác giữa Mít

m ật và Mít dai sắc ký qua cột Sephadex G75 cũiig cho thấy phổ sắc kí 2 m ẫu này khá giống nhau gồm 2 đỉiih protein chính, troug đó đỉnh tliứ Iihất có cả KIA và TIA uhưng

ờ mức độ th ấp liơii TI và KI từ hạt luít khá bều vói uliiột, sau khi sử Iv ờ 100'^c trong

15 phút, hoạt độ kìm hãin chỉ còn lại khoảng 30 % so với hoạt độ ban đầu.