isolation and indentification of antiviral principles from plant extracts

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INAL REPORT OF ADHOC SCHEME
ON
ISOLATION AND INDENTIFICATION OF
ANTIVIRAL PRINCIPLES FROM PLANT EXTRACTS
Financed by
Indian Council of Agricultural Research
Neio Delhi
Principal Investigators : Dr. R. Jeyara jan and Dr. S. Sadasivam
Departments of Plant Pathology & Biochemistry
Tamil Nadu Agricultural University
COIMBATORE - 641 003
FINAL REPORT OF RESEARCH SCEMES
1. Project Title ; Isolation and identification, of antiviral
principles from plant extracts
2. Sanction No. i P.No.3-8/85 PP dt. 19.3.87.
3. Report ; Final
4. Period ; 10.2.88 - 9.2.1993
5. Date of termination; 9.2.93 *
6. Name of Institute/
Department i Tamil Nadu Agricultural University
Biochemistry and Plant Pathology
Location. i Coimbatore 64t,003
Tamil Nadu.
7. Principal Investigator ;
"p^Qt'n T ^
i. Dr.S. Sadasivam r-r
Prof essor and Head
Dept. of Biochemistry
TNAUT Coimbatore. 3. 10.2.1988 22.5.91
ii. Dr.R. Jeyarajan
Professor and Head
Dept. of Plant Pathology
TNAU, Coimbatore.3. 23.5.91 9.2.93
8. Technical personnel employed ;
Name vjith Designation Date of Joinir^g Date^of^JLeji.yinj
r
A Tlimlp s"i
ResiAssociate~(Biochem. ) 10.2.'1988 1.10.1989
ii. Miss. S.Rajamaheshv;ari
Res.Associate (Biochem.) ,11 = 10.1989 9.3.91
iii. Miss. P.Muthulakshmi
Res. Assoc. (Plant Path. ) 23.5.91 11.6.91
iv. Mr. C. S elv ara j
Res. Assoc. (Plant Path. ) 20.6.91 3Q.11.91
v. Mr. R.Thangavelu
Res. Assoc. (Plant Path.) 20.12.91 9.2.93
9. Total outlay : Rs. . 1,90,800-00
10. Total amount spent s Rs. 1,17,458-55
11. Objectives >. ^
i. Identification of suitable extrnctdnts for antiviral
principles.
ii. Screening of various viruses inhibited by these plant a
extracts by local lesion test individually.
iii. Biochemical changes in host plants during virus infection
and recovery phase due to antiviral principles.
iv. Identification of antiviral principle as protein, polysaccha-
rlride, polypeptide, tannin, alkaloid etc.
v. Purification of sntiviral agent, if it is a protein "by colounm
ohrotoatography, electrophorosis electrofocasing and testing
at every stage. ^
vi« Characterization of antiviral principle, molecular weight
determination, chemical composition and special char act eristics.
vii. Physico chemical properties, viral dilution, therm.?.! in^cti-
vation and lungivity of inhibitor, shelf life, sensitivity
sunlight and duration of efficiency.
viii.Effect of antiviral .principles af molecular level, iclc yi>"i~
.cation of nucleic acid protein synthesis inhibitor ;„
12. Progress of research :
Leaf powders of Eougainvillca spec tab ills,, ^Qj^oderidron
i
aeuleatum and Sorghuta, root powder of Boerhaavia diffusa and
homeopathic drug Thuja-30 were tested for their, efficacy against
three viruses viz., cowpea aphid borne mosaic virus, tomato
spotted wilt virus and tobacco mosaic virus under glass house
condition.
Screening of antiviral principle :
The plant extracts (10$) was sprayed on the test hosts.
The Imves of control plants were sprayed with water. After 24 hrs
the plants were washed with distilled water and inoculated with
the test virus after dusting 600 mesh carborundum powder. Obser-
vations were taken for the appearence of disease symptoms.
1. Chilli : TSWV
Complete inhibition was observed in plants treated with
plant products (Table 1)
ii, Cowpea : Cowpea aphid - borne mosaic virus
o,
In all the test materials there was complete in hibltion
upto 10 days (Tab le 2).
iii. Groundnut : TSWV ; (In pot culture)
All the 4 extracts showed complete inhibit! onjof infection
upto 10 days in all the 3 different experiments carried out in
June, October and'December 1988 (Table 3).
iv. Tobacco : TMV
The efficacy of Bougainyillea spectabilis was found to
be much greater than the other plant extracts and Thuja 30 (Table 4).
Physical and chemical, properties of AVP from Boji^aiiwill;^ and
sorghum.
i. Extraction with organic solvents :
The organic solvents used for the extraction wvr: > troloun
ether, diethyl ether and chlorofom. The organic layer ol>;cained
after extraction of Bpjj£^.nvi.ll_ea ^£e£t£blli.s and sorghru :>xtraqts
did not inhibit ,CPMV. This indicates that the AVT i.y U'Ot a .li;;;id
(Table 5).
ii. Effect of Dilution ;
Serial dilutions were done v.1.3. 1 s5 to 1j100 and tested for .
antiviral activity. .Both the plant 'extracts (j:ougainvilleav and
sorghum) were found to have complete inhibitory activity even at a
dilution of 1:100 (Table 6).
iii. Effect of Dialysis :
In order to find out whether the inhibitory principle is
dialysable the crude extracts were dialysed against distilled
water at 4*G for 24 hr. The inhibitory activity was retained in
the contents of the dialysis bag. In other words, the ,WP in
both the extracts are non-dialysable (Table 7).
iv. Effect of pH :
The pH of extracts were adjusted to 2 to 11.5. .after 30
min. the pH was readjusted to 7.0 and the extracts was then sprayed
on to the cowpea plants. The inhibitory activity of Boj^a^jvilJ^ea
was retained in pH 4-10 and sorghum in pK 4.5 - 9.0. The inhibitory
activity was lost at very low and very high pH (Table 8).
v. Thermo sensitivity ;
Bougainvil1ea extract was heated in a water bath for 10 min.
to 60*C - 90"G and 100*0, cooled and tested on cowpea. Fhen heated
beyond 60*0 the extract lost its inhibitory effect (Table 9).
vi. Effect of absorbants s
To 50 ml of crude extract, 3 g of activated charcoal was
added and centrifuged at 5000 rpm for 15 min. The supernatant was •
filtered through Whatman Ho.1 filter paper and the clear filterate
was used for testing the efficacy of £VP. . The results showed
that" the antiviral principle we* g&td absorbed to charcor-1
( Table. 10,11). /
vii. Sensitivity to trypsin ;
Leaf extracts were mixed with trypein solution (1 mg/ml)
and incubated for 1 hr. at 37"C and tested. The digested extract
lost its inhibitory property.
Isolation of antiviral principle :
1» Precipitation with ammonium sulphate :
To the extract ammonium sulphate was added and the preci-
pitate was dialysed. The dialysatolwas tested for antiviral acti-
vity. The results showed that the precipitate had higher inhibi-
try effect than the crude extract (Table 12).
ii. Gel filtration :
Sephadex G.200 column was prepared and it was elutod with
0.1 M sodium phosphate buffer pH 7.0. About 48 fractions of 1 ml
each were collected. The absorbance for each fraction was measured
in a spectrophotometer at 210 nm. Two peaks were obtained. The
biological activity of the 2 fractions of the extract was tested.
The results showed that fraction I was more inhibitory than
fraction II (Table 13).
Field trials :
1. Cowpea Aphid borne mosaic virus ;
A field trial was conducted during August to November,
1989 to test the efficacy of plant products onthe incidence of
cowpea aphid borne mosaic virus in cowpea. Randomized block design
was followed. .Three sprays were given 10, -20 and 30 days after
sowing and the number of infected plants was counted every 5 days
from 10 days after sowing. The results showed that the inhibitory
effect was seen upto 30 days (Table 14).
2. Groundnut i TSWV s
Experiment No. 1
This experiment was ci onducted as r menti.oned above during
i
July to .November 1989. The results showed that, .all the plant
products tested effectively reduced the spread of TSWV upto 50
days as compared to the control (Table 15).
Experiment No.2
This second field experiment was conducted during
Kharif'91 Three sprays were given at 15, 25 and 35 days.-agftor
followed by Thuja (6.9$) and Sorghum (8$) leaf extracts, where
as control plots recorded 43.8$ disease incidence. Thuja Bougain-
villea and sorghum leaf extracts increased the yield by 20,7, 15.6
and 11.7 per cent respectively over control (Table 16").
Effect on fungal diseases
In the same plots, fungal diseases namely late leaf spot and
"rust were also recorded. These diseases were not significantly
influenced by the treataants (Table 17, 18).
3. Tomato TSKV °,
A' field experiment was conducted during the TQiarif'91
using tomato variety Go. 3. Monocrotophos (170 was used as
standard check. The plant products were sprayed on 15, 25 and
35 days after planting and TSW disease incidence was recorded.
The results showed that Bougainvillea leaf extract showed minimum
disease incidence of 8.9 per cent followed by sorghum, Thuja and
monocrotophos which recorded 9.4, 12.2 and 15.7 per cent respecti-
vely and increased yield by 3.44 to 27.74 per cent. The control
plots recorded'the maximum disease incidence of 20.9 per -cut
(Table 19).
*
Effect on leaf curl of tomato :
In the same field experiment, the incidence of leaf curl
/
disease was also recorded. The disease incidence was very low
during the season and the treatmental differences were not signi-
ficant (Table 20).
Effect of nematode population J
For this study soil samples (200 gm) were collected before
planting and 30 days after the treatment and.analysed for the
presence of nematodes. The population of Meloidogyno an'.V Tvl.cn-
chorynchus were not influenced by the treatment (Taolo 2" ).
13- Publications :
i. Sadasivam, S., Rajamaheshwari, S., and Jeyarajan, R.(1991).
Inhibition of certain plant viruses by plant extracts.
J. Scobiol. 2 (1) -. 53-57.
ii.Sadasivam, S. and Rajameheshwari, *S, one! Jeyarajaii, R* (1991).
Antiviral Principles in plant extract. Proc. i;irau~il
n-f thp A«wirn n"IH rm of mi r»T>nh-i nl orH a-h-c» .--,-? t-n.'T-i-o,
ill, Jeyarajan, R, and Thangavelu, R. 1993. Effect of certain
plant products on the incidence of tomato spotted wilt
virus (TSWV) in tomato and Bud necrosis virus (BNV) in
Groundnut.
20. Signature :
,-,'.*--'
Pr in c ip al In v e s-t i gat or / ecutive authority of the
Institute/organisation
Director of Research,
Place : Coimbatore 641 003.
Date :.5.8.1993.
i'A'..<IJ': -1. EFFECT OF PLAj-T PRO WOTS Oil JJIFF^RTilNT DATS AFTER IttOOULA <'IOH (T^T'/O
Fu-raber of Plants Infected *
particulars
5 10 15 20 25 30 35
pays aftar __
R1 L2 R3 HI R2 R3 R1 R2 E3 R1 i^2 R3 H1 B2 H3 R1 B2 R3 R1 R2 R3 R1 ^2 R3
E--crh?.avia 0 00 0 00 1 11 22 2 3 ^ M- ? 66 8 89 9 91 r,
Q CO 0 00 1 ~ 1 1 22 23 '-f 5 ^ 5 ' -f 7 6 6 88 7
Glcrb.djpndron 0 00 0 00 1 11 32 1 5 M- > 5 55 1 11 88 8
r
^-a-'O^O O^O'O iZi'1 2 2 2 3 3 35 5 ^7 7 78 78 ?
TTTiil.ja-30'' 1 1 1 ' 1 3 . 2 3 ^3 ^ ^ ' ^ 5 55 7 7 7 1 0 8 8 1 2 1 1 10
c ntrti / 7 810 91111 15 15 15 1515 15151515 151515 151515 -'515 15
* 15 plants wore inoculated for all treatments
NOTfi: 0 number rf plants infected = 100^ inhibl^ -n
All 15 pl.rvtits infected - 05^ inhibition
CD ia caloulctcd biiss:! ,,n the n.br>vc
1 T „ 4- r: i'7
Oayr? 2.892
i'rpatmcrit = 2.236
Tabl» 2 EFKBC'i1 OP PLAN? PRODUCTS OH DIFfriHBN'i' DAYS AFTKK INOCUIATM (COWPEA.- AEHI'D BOKME
1.10 SAI0 VIWJ S/CGWPEA )
particulars Number of plants infsctfto*
Bays after" * -j 5 20 2< 30 3^
10
In :c il-liai _t»i on j/ '^ ^
R1 1^2 R3 Rl"^2 R3 ~R1 ~R2 R3 K1 R2 R3 m~R2 aj R1 H2 H3 HI 12 ?-
0 00 0 00 1 11 1 21 2 3 .2 ^ 3 ^ 5 55 /
0 0 0 0 0 0 1 11 2 11 »f 2 2' 5 2 3 6 if V 3 55
0 00 0 00 1 1 1 . 1 21 >+ £ 1 '6 > 2 7 53 9 66
: 0 00 0 0 0 1 21 1 21 2 3 * f' 5 ^ 5 - 5 56 6 77
o oc 0 00 1 21 2 32 if 5 if 7 76 8 87 10 1 ) 10
Control 7 8 10 111213 151515 151515 151515 151515 15 15 15 151515
* 15 plrnus incculfeted in all treatments
NOTE; 0 number rf plants infected -V = 1005^ inhibitirn
All 15 pl-nts infc-ctaa C^lnhijbtti .a
CD in calculated basuJ .:n tho ab'. <va
OV) ph ^ ItiV'vjl
TA"i,.U 3. JS OF PLANT PMWJC'i'G Oil DAYS AFTER Ih'OClJLATIOfl (TSW/GROUNDNUT )
Numbta of plants infected *
Parti cul ITS
T/ayg ?.ft>?r 10 o r, o c 35
inocul?.ti?n
"tMil l-its-i UtiJ " > Itf )-|1 J.Tv_) j.Tl? J1.-5k0L -J>".3O r)J-JLlt
0 00 2 13 ^ ^ 5 6 66 7 78 98 o
' Bo UK ain ville a o ro o
0 10 11 2 2 2- 3 33 '"5 5 5 7 ;6 6 8 .8 8
Cle TO gengron ^
»..^ ..VJ.,'!,'-.""-' o o. o oo o 2 2 3 3 - 5 V7' • " 77
Thula 0 1 .1 2 11 3 56 • 71 8 7 8 98 9 9 10 10 io 10
8 8 ' 9 ill 3 1 M- 15 1? IP 15 15 15 15 15 15 15 15 15 15 115 15.
. ,15 pl.ants inpculatod in all treatments - •-.
' > ' O niTnhiTT ' .^'f -n"!->n^~ •?• n f ^p f r -1 - 1 ^r^ -t IT? >.-;-!-' •-->
• AV lili tui1i »5J w J;p _ lwsXn ^t-3'-L ilni'Jf'-o' c-it-loi-li- l- --^Q wfo -• j—. nhib—i tjI L- j-tyi- ^-iinl-i-Li—i-w^i—. ;^
GL is calculstod b^s^d .-.n the r.b:,vs ;
it-Lf ni / "i/'o