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

VNU. JOURNAL O F SCIENCE. Nat. Set.,

t.xv, n^3

- 1999

INVESTIGATIO N OF PROTEINASE PROTEIN INH IBITO RS
(PPIs) FROM SEEDS OF CERTAIN PLANTS BELONGING
TO M ORACEAE FAMILY AND OTHER ONES*
P h a m T ra n C h a u , N g u y en H ong 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 en T u y et M ai, D ao T rong 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 PPIs 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, but 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 iu ‘

o f A i to cu i ]}UƠ i t ic lu i o j . y l a G u g li, Árioctn-pXLO 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 toxocai'pa; Ciừ^ts gmndis 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 programme - 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 (DTIA): Tl or Chi peak.


2

Pham Tran Chau, Nguyen Hong Anh, Le TYong Quang. . .

- 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 water (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 that 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 pcifomicd 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 se in h ib ito ry a c tiv ity (P IA ) o f se e d c ru 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.

Investigation o f P rotein ase Protein Inhibitors (PP Is) . . .
Table 1, 4. .
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Pham 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 sc h u s excnlentĩLS L and DeẦavaya toxocarpa contain b otli T IA an 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 1cniaincd 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 activ ity [ I J S l U / g of d ry Iiiattor) 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 used for clctoctiiig th e piotoul coinpoiiouts.

Number

Rm

Artocarpus
melinoxyla Gagn

Artocarpus
chaplasha

Abeim oschus
esculentus L

Delavaya
toxocarpa

1

0,14

+

-

+

-

2

0,18

+

-

+

-

3

0.2

4

0,21

+

-

+

-

5

0,23

+

-

+

-

6

Ị

0.24
0.2t)

+

-

+

-

8

0,28

9

0.29

+

-

+

-

10

0,32

+

-

+

-

11

0,36

12

0,39

13

0,44

14

0,45

15

0,50

16

0,54

17

0,58

18

0.62

19

0,70

+++

20

0,83

+

21

0,85

22

0,90

+
+
+

+++
+

+++
++

+

+

+
+
++
+++

+

-

+++

+

-

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

+


Investigation o f Proteinase P rotein Inhibitors (P P Is) .
2.

E le c tro p h o re s is p a tt e r n o f seed e x tr a c t o f A rto c a rp u s m elin o x y la G agn;
A r to c a r p u s c h a p la s h a R oxb; A b elm o ch u s e scu len tu s L; D elavaya to x o c a rp a L
For convenience, the Rin of protein bands
was calculated and listed ill table 2 .
The data 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 rp 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 Artocarjn 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

4 1
. •
JPIliofo
1. piotriu
iJattnn ofr

Rill = ,0.39 a cco u n ts foi a p p io x iia a to ly ^6. IG
^

Ar to
*. c a i p iis liK'linoxyla
I
1 G a g- a /1
tro iu A
( 1, 3 )

of tlio total pioteiii coiitnit.’ 6.16 % whilo for

aiul A ito c a ip u s cha])lasha R d x I) (2, 4)

'

tlio R n i = 0.44 p io to iii h a ix l it was

, I A.

r>‘) % 1, , ,,

lowt'i' Mian that of tlir coriPspoiKliiie, one ill
Artocarfius cliaplashaRoxh. Thus, a(iucstioii

r, ,
.5, 4:

1

, ,
r iie li l a iif
,

,

1 1

('(Jilla m

,
lu

„

, r

,

•

r.

I

.
.
('Xtiact

1 /0

, .
,
I I I ()1 sa iu {)l(;

r

,

('Hcli laiK' coulaiii 1-) ỊIÌ C)i saniplr

arises; d o c s tlir p ro tein band w ith R m = 0.39 originate ÍI0111 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)
0,39

Samples

0,44

0,58

0,62

09

23,65

36,47

18,6

24,65

29,39

'

Artocarpus chaplasha Roxb
(column2 )
Artocarpus melinoxyla Gagn
(column 1)

6,16

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.


6

Pham Tran Chau, Nguyen Hong Anh, Le Trong QuariỊ. . .

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 of seed e x tr a c t o f sam p les th r o u g h S ep h ad ex - 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,D 9), 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 at of Artocarpus chaplasha Roxb. After chroiaatography, 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 that 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 that luoloculai weight of PPI ili these samples was lower than that of
Artocarpĩis.

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. D 2 and Ơ 3 ) in chroinatography pattern
of Delavaya toxocarpa. Since D 2 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 lieat tr e a tm e n t on C h IA (or T IA ) of s tu d ie d sam p les
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.


In vestigation o f Proteinase P rotein Inhibitors (PPIa) .
Fig. 1: Chromatogpaphy pattern of seed extract of studied samples
by Sephadex G 75 column
a) Artocarpus mekmoxyla Gagn
c) Delavaya toxocarpa
b) Ratocarpus chaplasha Roxb
d Abelmochus esculentus

12

10

f k

M

i

%

^ R S ? S 8 ỉ 5 8 8
Pf actions

Practions

(b)

(đ)

ÍÍ ÍỈ s

5! s? s

R

Fractions

TIA

KIA

Piotein

.

8 g 8


Pham Tran Chau, Nguyen Hong Anh, Le Trong Quang,

Aíỉocarpus Arỉocâíípus Abdfnochus D ela v ay a A tto c a ip u s
chđplâshd meiitioxyld eaciientưs L ỉoxocàĩpâ chaplasha
Roxb
Roi
Gốgn
Ctixle exffact

Aiỉocafp*,Ji

A tfccaiip u s

G ag n

G dgn

mdmũxylâ fr.ei*no5
Activepeak(CWA0{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 that 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 at sample concentration for deteniiiiiiiig TIA was 5 times higher than that 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. Ill 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


Investigation o f Proteinase Prxitein Inhibitors (P P Is)

g

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
K'liiaiiK'tl activity was over 30%.

After heat treatm ent at 100°c for 15 m im Z s the

V. REFERENCES
1] R.M. Bonio , M. N. N. Levy. Physiology , Mosby - Yoai' Book, Inc., USA (1993)66967G.
’
[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
tlio Foliii phenol reagent, J. Biol. Chem.. 193(1951) 265-270.

with

|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 ti
reproduction, Journal of Biologs-Soil science: Hanoi National University (1989)

w

M.M Wincjunajtc. OpreJelcute piotcolitichcskoi aktivuosti k n m M Iiykh prcparatov niikrobiologichoskovo proiskliachdenia, Pnklad. Biochern. MikrobUu. 2 (1 9 0 0 ) 232.

Chau. TVi/psm inhiUtors of white bush (CucnrbUa pepo var, patissonia) 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
gianiosofC ucurbitapepovai. 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, cliaractorizatioii 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 syviposruvi on protein Research
7-10/12/1993
13| PUa„ T„a„ Nghia, Pl.au, Thi T,a.. Cl.ãũ (1987): a Z g c s to trvpsh. i„l,ibito.y act 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).


10

Pham Tran ChaUf Nguyen Hong Anh, Le Trong Quang, . .

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 ồng A n h , Lê T rong Q u an g
P h a n T h ị H à , N g u y ễ n T u y ế t M ai, 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 mật 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 mẫu này khá
giống nhau gồm 2 đỉiih protein chính, troug đó đỉnh tliứ Iihất có cả KIA và TIA uhưng
ờ mức độ thấp 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.