Title: Extraction of Salicin from Flacourtia Indica (bitungol) plant and its effects against

Dysdercus Cingulatus Infestation

Introduction

Plants and Insects have been present for the past 350 million years, throughout those

years, both have developed strategies to avoid each other’s defense system. (Howe G.A, Jander

G., 2008) This evolutionary arms race between plants and insects has resulted in the

development of an elegant defense system in plants that has the ability to recognize the nonself

molecules or signals from damaged cells, much like the animals, and activates the plant immune

response against herbivores. (Hare JD, 2011).  An example is the phytohormone, salicylic acid

that plays an important role in transducing the activation of plant defense systems against

pathogen attacks. (Bari R, Jones JD, 2009). Systematic acquired resistance (SAR) is a type of

induced and long-lasting response, (Hammerschmidt, 2009), that is very effective against

biotrophic and hemibiotrophic pathogens and some insect herbivores (Freeman, B.C., G.A.

Beattie, 2008). Worldwide, there is a vast majority of insects that feed on cotton crops, causing it

to be unusable in cotton harvests. An example of this is the Dysdercus Cingulatus.(wikipedia,

2020)Throughout the years, there has been multiple sources of the Salicylic acid in plants all

over the world, Particularly in the willow family, The willow family of flowering plants is also

known as the traditional family (Salicaceae sensu stricto) included the willows, poplar, aspen,

and cottonwoods. Genetic studies summarized by the Angiosperm Phylogeny Group (APG) have

greatly expanded the circumscription of the family to contain 56 genera and about 1220 species,

including the Scyphostegiaceae and many of the former Flacourtiaceae. (Chase, Mark W., Sue

Zmarzty, M. Dolores Lledo, Kenneth J. Wurdack, Susan M. Swensen, Michael F. Fay ,2002)
(Christenhusz, M. J. M. & Byng, J. W. 2016) (Stevens, P. F. 2001 onwards). The Flacourtiaceae

family is a genus of flowering plants in the willow family, it is considered as a classification of

the Salicaceae, which are the known sources of Salicin.

Dysdercus Cingulatus (Red Cotton Stainer) is a common species of true bug in

the family Pyrrhocoridae. This is a serious pest of cotton crops and as the adults and older

nymphs feeding on the emerging bolls and the cotton seeds as they mature, will cause a

transmission of cotton staining fungi. (Jaleel 2Waqar, Saeed Shafqat,  Naqqash,

Muhammad Nadir ,2013). Dysdercus Cingulatus is mainly red in color and has white

collars and three black spots along its body. This insect feeds on Cotton (gossypium) and

a number of crops as well, including silk cotton tree (Bombax ceiba), and also okra

(Abelmoschus esculentus) (Dysdercus cingulatus , 2020). Like most bugs, Dysdercus

Cingulatus sucks the fluids of the host plant and the parts that it targets are the

development of the cotton and also the seed. This causes the cotton to turn an indelible

brownish color causing it to be unsafe for use in textiles and in agricultural purposes.

This type of insect is evident in most parts of Asia, Africa and America. In fact, This

Insect was spotted in Pili, Camarines sur and was infesting most of the cotton fields in

those areas. These tiny Insects may seem harmless to humans but when it comes to cotton

plants, they are considered as one of the deadliest predators.

In most cases, Dysdercus Cingulatus infestation is treated with synthetic

pyrethroids, carbamates such as carbaryl, or organophosphates such as dimethoate.

However, some of these synthetic insecticides such as Carbamates, causes an increase in

the acetylcholine levels at ganglionic synapses of the parasympathetic and sympathetic

nervous system. In addition, It also targets the muscarinic receptors on the

 parasympathetic nervous system, the organs in the central nervous system and the

nicotinic receptors in skeletal muscle tissue. (Silverman, & Taylor, 2021)

The plant Flacourtia Indica is one of the native plants present in the rich terrains

of Luzon and is one of the few members of the Flacourtiaceae family. By finding great

utility of this plant, this research aims to give importance to the Cotton crops in our

country. By using the Flacourtia Indica Tree Bark as a source of salicin, there is a higher

chance of acquiring a high concentration of salicylic acid, which is an organic compound

that can be used instead of these synthetic insecticides. The Flacourtia Indica is related

and included in the family of Saliceaece, thus there is a possibility that this plant contains

a salicin with high concentrations, which could then be converted into a concentrated

salicylic acid, That would be beneficial in controlling the infestation of cotton eating

insects such as the Dysdercus Cingulatus. The results of this study will be useful in

promoting new sources or agents for pest control.

Methodology
Collection of Dysdercus cingulatus

The Collection of the The Red cotton stinger (Dysdercus cingulatus) will be collected at

the Zambales or Laguna because these are the common locations and are most abundant

for these types of insect.The insect would then be transported to the University of The

Philippines, College of Public Health for Certification.

Collection of Flacourtia indica Tree Bark

 The Flacourtia indica Tree Bark will also be collected at Zambales or Bataan because

these are the places in which the plant is cultivated and abundant. It will then be

transported to the University of The Philippines, College of Public Health for

Certification.

Sample of Cotton(gossypium) Plant

The sample of the Cotton gossypium plant will be collected at the Cotton Development

Administration (CODA) Located at Alacan, Pangasinan.

Preparation of the Plant

The collected samples of the cotton plant would be positioned in a place with enough

sunlight. Each plant would be placed in a separate pot and labeled from 1-4. Each plant

should have a corresponding spray bottle that should also be labeled from 1-4 and should

also be watered before the treatment. 

 Extraction of the Salicin from the Flacourtia indica Bark

The process of extracting the Flacourtia indica bark is done with the help of a mortar and

pestle. Upon starting, the collection of the bark is achieved with the help of a scientist, to

ensure that the needed parts of the bark are taken without additional harm to the plant

itself. The collection of the Tree bark is collected with the help of a scalpel or a sharp

blade at the inner and outer bark of the tree. This process is the most advisable among all
 the different methods since other methods of collection in tree barks cause damage to the

tree itself. (Helmenstine, 2020). The collected sample of the bark would then be separated

into different parts based on the different sections of the bark. The pink and light section

of the bark would then be placed in a mortar and turned into a powderized form, The

process of grinding the bark would be done 3 times and would pass through a sieve to

remove unwanted properties. This powderized form is the extracted salicin. The extracted

salicin would then be transported to a coffee filter with the help of a spatula. The coffee

filter acts as an added protection that could reduce the risks of unwanted transmission of

dust and dirt. (ThoughtCo, 2019)

Process of Turning Salicin into acetylsalicylic acid

The method that would be used to turn the extracted salicin into acetylsalicylic acid is

distillation and evaporation. The collected powderized salicin will be mixed with

methanol or ethanol in a 50 to 20 ratio. The solute and the chosen solvent will be

combined in a beaker and evaporated until the mixture is combined. The salicin should

also be added in small parts to prevent undissolved powder. The solution should stand for

about an hour to let all the starch and cellulose sink to the bottom of the beaker. The next

is the presence of a vacuum filter, to wash the starch and cellulose solid twice with 100

milliliters of the chosen solvent either, methanol or ethanol. Then the solution would be

transferred to a large open top container and placed at a well-ventilated area to let the

solvent evaporate and to recover the acetylsalicylic acid. The solution could also be dried

in an oven overnight for faster results.( Kirchhoefer, Reepmeyer,1979)

 
  Conversion of Acetylsalicylic acid to Salicylic acid

This procedure requires the assistance of a scientist, for this part of the experiment

requires the presence of both acids and bases. The procedure that would be done is

hydrolyzing the acetylsalicylic acid by using an ester acid hydrolysis. The only material

that would be used to extract the acetylsalicylic acid is hydrochloric acid at 31.45%. For

the extraction, 22.3 grams of acetylsalicylic acid would be added to a round bottom flask

that was pre sanitized with distilled water. Upon starting, the flask should have a stir bar

followed by the 22.3 grams of ASA. Next is the addition of 300 milliliters of distilled

water and 5 milliliters of Hydrochloric acid to the flask. Acids should be added after the

water to minimize the risks for unwanted contact with acids. Then is the presence of a

cold-water condenser and a burner to evaporate the solution. 30 minutes is the estimated

time for the solution to evaporate. The solution should be placed at rest to cool down and

would stop refluxing at 180 degrees. Once the solution is fully cooled, Thestir bar would

be disposed of and crystals will eventually form on the sides of the flask. When the

crystals fill the flask, the solution would be vacuum filtered and washed with 250

milliliters of distilled water for 3-4  times.( Kirchhoefer, Reepmeyer,1979)

Experimental Setup

This study would be carried out during summer because these are times that Dysdercus

cingulatus is most abundant. The 4 cotton plants should be placed in 4 different pots and

are labeled randomly from 1-4. One plant should be labeled as the control group and the
 Other 3 would be labeled as the experimental group or the one that would be receiving

the treatment. Three plants would be exposed to the Dysdercus cingulatus and would be

checked upon the next day. Each plant would be receiving different doses of SA to check

the plants response to the acid. (Souri, Tohidloo, 2019). The experimental setup is

presented in figure 1. 

Figure 1:https://www.ontrack-media.net/gateway/biology/g_sm0l3es2.html

 
Treatment Application

This study would be done during the daytime and in a completely randomized design

with Four replications. The treatments were (1) control (No exposure to Dysdercus

cingulatus and SA application), (2) Exposure to Dysdercus cingulatus + SA Application

to the roots(10 ml) (3) Exposure to Dysdercus cingulatus + SA Application to the roots

(15ml). (4) Exposure to Dysdercus cingulatus + SA Application to the roots (20ml). For

all the plants, it was given equal amoun of sunlight and water to ensure the growth of the

plants. (Souri, Tohidloo, 2019) Salicylic acid was supplied to cotton plants by direct

application to the plant’s roots, Treatment with salicylic acid also was applied with two

sprays, the first spray was on the day after the exposure to Dysdercus cingulatus and

another spray was on 5 days later. Foliar sprays of SA were done with a portable sprayer,

at the early morning and 1 h after sunrise. (Ali, El-Sherbeni, Khaled,2016)

Statistical Analysis

All data will be statistically analyzed by analysis of variance (ANOVA) using the

Statistical Analysis System (SAS Institute, Inc., 1996) or through a chart representation

for an easier way of understanding the effects of the collected and extracted acid. 

Risk and Safety

A Scientist should be present before the experimentation to assess certain risks, such as

ingestion, inhalation, or any other dangerous contact with hazardous chemicals associated
 with experiments that will be performed and during certain parts of the experiment in

which corrosive acids and bases are present. Laboratory rules and regulations should also

be followed strictly, such as always wearing protective equipment, the use of google,

gloves, masks, and proper wardrobe should be followed to ensure that spills and

accidents would not cause much harm to the researcher and scientist present.

Disposal Method

Proper waste disposal is critical due to the fact that certain types of wastes can be

hazardous and can contaminate the environment if not handled properly. These types of

waste also have the potential to cause disease or get into water supplies. After Extracting

the acid that were used in the treatments, The researcher would be separating the

hazardous from the non-hazardous wastes were used for the experimentation. Masks,

gloves, and protective gears should be disposed properly in their corresponding trash

bin, this also includes broken equipment and fractured laboratory gears. The Insects that

were used should be returned to their natural habitat as soon as possible. Chemicals

should be completely removed from the site and transported to another location for

treatment and proper disposal. On the other hand, used laboratory equipment should be

washed properly with distilled water and should be returned to their designated

containers.