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6.0 ANTIVIRAL PROPERTY OF BIOACTIVE CRUDE EXTRACTS FROM

NATIVE TRADITIONAL MEDICINAL PLANTS AGAINST WSSV

INFECTED SHRIMP PENAEUS MONODON AND PENAEUS INDICUS

6.1 Introduction

White Spot Syndrome (WSS) is one of the most damage causing viral

diseases in penaeid shrimp characterized by 100% mortality within 3-10 days

(Lightner, 1996). Antiviral research using plant extracts has gained

momentum since 1950. Scores of medicinal herbs have already been tested

and used with good results in the control of viral and bacterial diseases in

shrimp and fish. The ethanol extract of Psidium gugajava leaves was tested for

antiviral activity against various fish pathogenic viruses namely, Infectious

Haematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus

(IPNV) and Oncorhynchus Masou Virus (OMV) using CHSE-214 cell lines by

plaque reduction assay. The ethanol extract of Phyllanthus amarus and

Phyllanthus gugajava have been found to have antiviral activity against

yellow head baculovirus in P. monodon (Direkbusarakom et al., 1993).

Sixteen species of Thai traditional plants have been tested against fish

and shrimp pathogenic bacteria and among these plants P. guajava and

Momordica charantia displayed the highest activity against Vibrio harveyi and

Vibrio parahaemolyticus (Direkbusarakom et al., 1998a). The extract of

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Clinacanthus nutans has been tested against Yellow Head Virus (YHV) of

shrimp and the results indicating that, this plant could effectively control

YHV infection in shrimp (Direkbusarakom et al., 1998b). Hence, the present

study is aimed to findout the antiviral property of crude extracts from native

medicinal plants (both marine and non marine) of India.

6.2 Materials and Methods

6.2.1. Preparation of WSSV stock

Infected wild Penaeus monodon were collected and 1:10 dilution of gill

suspension was prepared in phosphate-buffered solution (pH 7.4) and

injected intramuscularly (50 µl) into SPF P. monodon to amplify the virus.

Moribund and dead shrimp were collected at 48 h post inoculation (hpi).

Carcasses without hepatopancreas, gut and exoskeleton were minced. A 1:10

dilution of the suspension was made and centrifuged subsequently at 3,000 g

and 13,000 g at 4ºC for 20 min. The supernatant was filtered (0.45µm) and

made upto 250ml and stored at -70ºC. PCR analysis of viral pathogens in all

the samples was carried out by following standard methodologies as

described in the previous chapter. The results confirmed the presence of only

WSSV DNA. The median virus titer of infection was 106.6 SID 50 ml–1 by i.m.

route based on Reed and Muench (1938).

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6.2.2. Collection of traditional medicinal plants

The present study was chosen 3 non-halophytic traditional medicinal

plants and 1 halophytic traditional medicinal plant (Table.7) which are

already proved to posses antiviral activity were collected from different

locatioins in Tamil Nadu and used for the antiviral assay (Fig.3).

Table 7. Name of the plants chosen for the present study

Scientific name

Vernacular name

Plant parts used

Family Geographical

location Origin

Sphaeranthus indicus

Sivakaranthai Leaves, Bark, Stem and Flower

Asteraceae

Cinnamomum camphora

Pachai karpuram

Whole plant Lauraceae

Ocimum sanctum

Tulsi Whole Plant Lamiaceae

Tenkasi Lat. 8°57’ N; Lon. 77° 18’ E

Non-Halophyte

Avicennia marina

Grey Halophyte

Leaves Avicenniaceae

Karangadu Lat.9°43’N Lon. 79°00’E

Halophyte

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Fig.3 Map showing the collection site

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6.2.3 Description of plant species

(a) Cinnamomum camphora (L. Sieb)

Taxonomical position Kingdom : Plantae

Angiosperms : Magnoliids

Order : Laurales

Family : Lauraceae

Genus : Cinnamomum

Species : camphora

Camphor is a white crystalline substance, obtained from the tree

Cinnamomum camphora. Camphor has been used for many centuries as a

culinary spice, a component of incense, and as a medicine. Camphor is also

an insect repellent and a flea-killing substance. In the ancient and medieval

Middle East and Europe, camphor was used as ingredient for sweets but it is

now mainly used for medicinal purposes. For example, camphor was used as

a flavoring in confections resembling ice cream in China during the Tang

dynasty (AD 618–907). An anonymous Andalusian cookbook of the 13th

century contains a recipe for meat with apples, which is flavored with

camphor and musk. A 13th century recipe for "Honeyed Dates" is also

flavored with Camphor. By the time of the Renaissance, camphor as a

culinary ingredient had fallen into disuse in Europe.

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Today, camphor is widely used in cooking (mainly for dessert dishes

such as kheer or paal paayasam) in India where it is known as pachha

karpooram (literally meaning "green camphor"). It is widely available at

Indian grocery stores and is labeled as "edible camphor". In Hindu poojas

and ceremonies, camphor is burned in a ceremonial spoon or plates for

performing aarti. This type of camphor is also sold at Indian grocery stores

but it is not suitable for cooking. The twigs and leaves of the camphor plant

are used in the smoking and preparation of Zhangcha duck, a typical

banquet and celebratory dish in Szechuan cuisine.

(b) Ocimum sanctum

Taxonomic position

Kingdom : Plantae

(unranked) : Asterids

Order : Lamiales

Family : Lamiaceae

Genus : Ocimum

Species : sanctum

"The Queen of Herbs" - is the most sacred herb of India. Tulsi

(Ocimum sanctum), although also known as Holy Basil, is a different plant

from the pesto variety of Basil (Ocimum basilicum). Tulsi has been revered in

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India for over five thousand years, as a healing balm for body, mind and

spirit and is known to bestow an amazing number of health benefits. Organic

India is pleased to offer organic Tulsi, for the first time, as a stress-relieving,

energizing and delicious tea. Tulsi Tea collection we utilize a proprietary

combination of 3 varieties of Tulsi: Rama Tulsi (Ocimum sanctum), Krishna

Tulsi (Ocimum sanctum) and Vana Tulsi (Ocimum gratissimum). Each variety

lends its own distinct and characteristic taste that contributes to the delicious

flavor and aroma of our blend.

Tulsi is rich in antioxidant and renowned for its restorative powers.

Tulsi has several benefits: relieves stress adaptogen; bolsters immunity;

enhances stamina; provides support during cold season; promotes healthy

metabolism; a natural immuno-modulator. Modern scientific research offers

impressive evidence that, Tulsi reduces stress, enhances stamina, relieves

inflammation, lowers cholesterol, eliminates toxins, protects against

radiation, prevents gastric ulcers, lowers fevers, improves digestion and

provides a rich supply of antioxidants and other nutrients. Tulsi is especially

effective in supporting the heart, blood vessels, liver and lungs and also

regulates blood pressure and blood sugar.

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(c) Sphaeranthus indicus Linn.

Taxonomic position

Kingdom : Plantae

Subkingdom: Viridaeplantae

Phyllum : Tracheophyta

Subphyllum : Euphyllophytina

Infraphyllum: Radiatopses

Class : Magnoliopsida

Subclass : Asteridae

Superorder : Asteranae

Order : Asterales

Family : Asteraceae

Genus : Sphaeranthus

Species : indicus

Sphaeranthus indicus Linn. (Asteraceae) is widely used in ayurvedic

system of medicine to treat vitiated conditions of epilepsy, mental illness,

hemicrania, jaundice, hepatopathy, diabetes, leprosy, fever, pectoralgia,

cough, gastropathy, hernia, hemorrhoids, helminthiasis, dyspepsia and skin

diseases. There are reports providing scientific evidences for

hypotensive, anxiolytic, neuroleptic, hypolipidemic, immunomodulatory,

antioxidant, anti-inflammatory, bronchodialatory, antihyperglycemic and

hepatoprotective activities of this plant. A wide range of phytochemical

constituents have been isolated from this plant including sesquiterpene

lactones, eudesmenolides, flavanoids and essential oil.

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(d) Avicennia marina (Common Name: Grey mangrove) Taxonomic Position

Division : Spermatophyta

Sub division : Angiospermae

Class : Dicotyledonae

Sub Class : Gamopetalae

Series : Bicarpellatae

Order : Lamiales

Family : Acanthaceae

Genus : Avicennia

Species : marina

Avicennia marina, commonly known as grey mangrove or white

mangrove, is a species of mangrove tree classified in the plant family

Acanthaceae (formerly in the Verbenaceae or Avicenniaceae). As with other

mangroves, it occurs in the intertidal zones of estuarine areas. Grey

mangroves grow as a shrub or tree to a height of three to ten metres, or up to

14 metres in tropical regions. Avicennia species has aerial roots

(pneumatophores) in which grow to a height of about 20 centimetre and a

diameter of one centimetre. These allow the plant to absorb oxygen, which is

deficient in its habitat. These roots also anchor the plant during the frequent

inundation of seawater in the soft substrate of tidal systems.

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Grey mangrove is a highly variable tree, with a number of ecotypes

and forms closely resembling other species. It has been reported to tolerate

extreme weather conditions, high winds, and various pests and diseases. It is

a pioneer in muddy soil conditions with a pH value of 6.5 to 8, but is

intolerant of shade.

6.2.4. Collection and maintenance of experimental animals

Shrimp, Penaeus monodon and Penaeus indicus (10–15 g body weight)

were collected from the sea and were maintained in 1000 litre fibreglass

tanks with air-lift biological filters at room temperature (27–30 °C) with

salinity between 30 and 35 parts per thousand (ppt). Natural seawater was

collected from the Bay of Bengal, (Thondi region) and allowed to settle to

remove the sand and other suspended particles. The seawater was first

chlorinated by treating it with sodium hypochlorite at a concentration of 25

parts per million (ppm) and then dechlorinated by vigorous aeration, before

being passed through a sand filter and then used for the experiments. The

animals were fed with artificial pellet feed (CP feed, Thailand).

6.2.5. Extraction of bioactive principles (Percolation Method)

Four species of traditional medicinal plants which already proved to

posses antiviral activity were selected for this study (Zaidi et al., 1988;

Direkbusarakom et al., 1997; Parida et al., 2002; Vimalanathan et al., 2009;

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Dhar et al., 1968; Margaret Beula, 2010). Three non- halophytes viz. Siva

karanthi (Sphaeranthus indicus), Pachaikarpuram (Cinnamomum camphora),

Tulsi (Ocimum sanctum) and Grey halophyte (Avicennia marina). Collected

plant samples were thoroughly washed thrice with tap water and twice with

sterile distilled water to remove adhering debris and associated faunal

species and then shade dried under room temperature (280C) and pulverised

by using electric mixer. One kilogram of each plant species of the pulverised

sample is mixed with 3 litres of ethanol and water mixture (3:1) in a glass

bottles at room temperature and kept for 20 days with occasional shaking.

Then the extracts were carefully filtered through Whatman filter paper No.1.

The clear extracts were separately subjected to distillation by using rotary

flash evaporator (Superfit, India) to recover back the solvent and a pasty

residue containing the plant extract was obatined. The residues obtained

from the samples were then allowed to dry. To get solvent free residues, the

dried residual samples were then lyophilized.

6.2.6. Preparation of plant extracts

Crude extracts (100 mg) were first dissolved in 10 ml of DMSO (SRL)

and Tween 80 (Qualigens) was used as an emulsifier at a concentration of

0.05% in the final test solution. The extract negative control consisted of

DMSO and PBS.

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6.2.7. Challenge studies

Eleven groups of 6 shrimps (MBW =14± 4.92 g, n = 66) of P.monodon

and P. indicus were inoculated with 40µl of WSSV stock through

intramuscular injection (Table 8). In addition, 2 groups of 12 shrimp were

mock-inoculated with 40 µl of PBS and 40 µl of DMSO were used as controls.

Shrimp were injected between the 3rd and 4th segments of the pleon. Before

and after injection, this surface was wiped with 70% ethanol. These

experiments were carriedout until all the infected shrimps died. Control

shrimps were sacrificed at 120 h after post inoculation (hpi).

Table 8. Treatment schedule

Treatments Schedule

T-1B Peneaus monodon treated with Sp.indicus leaf + WSSV

T-1W Peneaus indicus treated with Sp.indicus leaf + WSSV

T-2B Peneaus monodon treated with Sp.indicus root + WSSV

T-2W Peneaus indicus treated with Sp.indicus root + WSSV

T-3B Peneaus monodon treated with Sp.indicus flower + WSSV

T-3W Peneaus indicus treated with Sp.indicus flower + WSSV

T-4B Peneaus monodon treated with Sp.indicus stem + WSSV

T-4W Peneaus indicus treated with Sp.indicus stem + WSSV

T-5B Peneaus monodon treated with Camphour + WSSV

Contd….

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T-5W Peneaus indicus treated with Camphour + WSSV

T-6B Peneaus monodon treated with Ocimum sanctum + WSSV

T-6W Peneaus indicus treated with Ocimum sanctum + WSSV

T-7B Peneaus monodon treated with Avicennia marina + WSSV

T-7W Peneaus indicus treated with Avicennia marina + WSSV

T-8B Peneaus monodon treated with mixed non halophytes + WSSV

T-8W Peneaus indicus treated with mixed non halophytes + WSSV

T-9B Peneaus monodon treated with mixed halophytes and non halophytes + WSSV

T-9W Peneaus indicus treated with mixed halophytes and non halophytes + WSSV

T-10B Peneaus monodon treated with DMSO and PBS solution

T-10W Peneaus indicus treated with DMSO and PBS solution

T-11B Peneaus monodon treated with DMSO, PBS solution and + WSSV

T-11W Peneaus indicus treated with DMSO, PBS solution + WSSV

6.2.8. Assessment of antiviral activity

Mortalities were recorded for each day and the experiment was

carried out up to 30 days after post infection with WSSV. The confirmation

and the level of WSSV DNA occurence was assessed through RT- PCR.

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Plate 1. Picture showing the viral load in WSSV infected P.monodon.

(a) Primer control (b) Wild infected shrimp

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Plate 2. Picture showing the viral load in uninfected shrimps treated with negative and positive control

(a) Negative control- P. monodon

(b) Negative control- P. indicus

(c) Positive control- P. monodon (d) Positive control- P. indicus

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Plate 3. Picture showing viral load in WSSV infected shrimps treated with

halophytes and non halophytes

(a) Mixture of non halophytic extracts- P.monodon

(b) Mixture of non halophytic extracts- P.indicus

(c) Mixture of non halophytic and halophytic extracts- P.monodon

(d) Mixture of non halophytic and halophytic extracts- P.indicus

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Plate 4. Picture showing the viral load in WSSV infected shrimps treated

with different plant extract

(a) Avicennia marina-P.monodon (b) Avicennia marina-P.indicus

(c) C. camphour-- P.monodon (d) C.camphour-- P.indicus

(e) Ocimum sanctum-- P.monodon (f) Ocimum sanctum-- P. indicus

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Plate 5. Picture showing viral load in WSSV infected shrimps treated

different plant parts of S. Indicus

(a) S. indicus leaves- P.monodon

(b) S. indicus leaves- P.indicus

(c) S. indicus root- P.monodon (d) S. indicus root- P.indicus

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(e) S. indicus flower- P.monodon (f) S. indicus flower- P.indicus

(g) S. indicus stem- P.monodon (h) S. indicus stem- P.indicus

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Table 9. Quantity of viral DNA and thermal cycle value in different

treatements

Treatments Quantity Thermal cycle value

T1B 4.18×109 14.43

T1W 5.14×1011 14.93

T2B 3.14×107 21.68

T2W 2.12×108 15.25

T3B 1.34 29.88

T3W 1.34×107 20.85

T4B 0.02 32.25

T4W 0.05 31.77

T5B 1.44×1012 14.36

T5W 0.04 31.81

T6B 3.12×107 20.12

T6W 5.23×109 18.42

T7B 8.85×1011 14.63

T7W 1.14×1013 13.2

T8B UD UD

T8W 8.75×1011 14.64

T9B 2.65×1013 12.73

T9W 0.01 32.64

T10B UD UD

T10W UD UD

T11 B 2.12x 1013 12.18

T11 W 1.72×1012 13.14

Mean ± SD Value

2.7579±2.65779 19.9426±7.70073

± SD- Standard Deviation

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6.3 Results

The antiviral property of the chosen halophytes and non halophytes in

P.monodon and P.indicus infected with the WSSV were carried out by the

present study. The viral load and its corresponding thermal cycles after

treatments were represented in Table 9. The viability of the WSSV virus in

the infected wild shrimps were checked with the control primer and

confirmed the inoculated viral load of 5.9×106(plate 1a & b).

Infected animals treated with non halophytic plant reveals that, the

S.indicus leaves decreased the viral load in P. indicus and P. monodon when

compared with the control (Plate 5 a & b) and the viral load was recorded as

5.14×1011 (T1W)and 4.18×109 (T1B) with the thermal cycle value of 14.93 and

14.43 respectively. It is interesting to notice that, the Camphour drastically

decreased the viral load (Plate 4 c & d) up to 0.04 (T5W) with the thermal

cycle value of 31.81 in white shrimp P.indicus and tiger shrimp P.monodon

with the viral load of 1.44 ×1012 with the thermal cycle value of 14.36 (T5B).

In Ocimum sanctum treated animals, the viral load was recorded by

5.23×109 (T6W) and 3.12×107 (T6 B) in P.indicus and P.monodon. The

corresponding thermal cycle values were recorded as 18.42 and 20.12

respectively (Plate 4 e & f).

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In the case of halophytic plant Avicennia marina, the viral load was

decreased by 8.85×1011 in P.monodon and the thermal cycle value is 14.63

(T7B). Controversely, the viral load was increased with 1.14×1013 in P.indicus

(T7W) when compared with control with the thermal cycle value 13.2

(Plate 4 a & b).

Various plant parts (root, flower and stem) of the non halophytic plant

S.indicus treated WSSV infected shrimps reveals that, the root extract

decreased maximum (2.12×108) viral load with the thermal cycle value of

15.25 in P.indicus (T2W) and decreased 3.14×107 in P.monodon with the

thermal cycle value of 21.68 (T2B). Moreover, the flower extract drastically

decreased the viral load in P.monodon (1.34) with the thermal cycle value of

29.88 (T3B). But in the case of P.indicus, the viral load was decreased with

1.34×107 and the thermal cycle value of 20.85 (T3W) (plate 5 e & f). The

results of the S.indicus stem extract reveals that, the maximum reduction of

viral load was recorded by 0.02 with the thermal cycle of 32.25 (T4B) and

minimum 0.05 with the thermal cycle of 31.77 (T4W) in P. monodon and P.

indicus respectively (Plate 5 g & h).

The synergetic activity of the crude extracts from non halophytes in

WSSV infected shrimps were also carried out by the present study. It reveals

that, the complete removal of viral load was recorded in P.monodon (Plate 3

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a) (T8B) and 8.25×1011 level of viral load was recorded with the thermal cycle

value 14.64 in P.indicus (Plate 3 b) (T8W). Further, the synergetic activity of

the crude extracts from halophytes and non halophytes reveals that,

negligible amount (0.01) of viral load was recorded in P.indicus (T9W) with

the thermal cycle value 32.64 and the viral load was slightly decreased

(2.65×1013) when compared with the control and the thermal cycle value was

recorded as 12.73 (T9B) in P.monodon (Plate 3 c & d).

In addition to that, the animals were also treated with the positive

control and negative control. It reveals that, the animals treated with the

DMSO and PBS did not have viral load ( plate 2 a & b) in negative control

(T10 W &T10 B). But in the case of positive control, the viral load was

recorded by 1.72×1012 in P.indicus (T11W) and 2.12×1013 in P.monodon (T11B)

with the thermal cycle values 13.14 and 12.18 respectively (Plate 2 c & d).