The Quantification of Carotenoids from

Chamaecostus Cuspidatus

leaves

A Research Paper

Presented to the Faculty of the

Senior High School Department

Cebu Institute of Technology - University

Cebu City, Philippines

In Partial Fulfillment of

The Requirements for the Course

Research 4 - Capstone Project

by

Baguinda, Janifah G.

Ducoy, Analyn O.

Dumaguit, Josephine A.

Gaña, Kate Angelie W.

Pila, Mary Ella Mae C.

Root, Crystal Jane A.

 Sarmiento, Claire Dean A.

ABSTRACT

Before the introduction and development of modern medicinal practices,

herbal medicine has always existed. Different kinds of herbs and plants are used

for medical treatment. ​Chamaecostus cuspidatus,​ also called insulin plant, is a

member of the ​costaceae family and is widely known for its anti-hyperglycemic

effect therefore it is a good treatment for diabetes. ​The purpose of the study was

to quantify the amount and type of carotenoids present in the plant’s leaves

through solvent extraction and using a UV-visible spectrophotometer. Solvent

extraction involved extracting the carotenoids from the leaves of the plant with

the use of different solvents namely diethyl ether and acetone. The extract or the

fresh sample was transferred to a 10 cm cuvette filled with the solvent to perform

spectrophotometric analysis using a double beam UV-vis spectrophotometer and

the same procedure was also applied for the air dried sample. Upon finishing the

procedures of the experiment, the researchers were able to obtain five types of

carotenoids based on the absorption coefficients of common food carotenoids:

astaxanthin, lycopene, zeaxanthin, violaxanthin, and lycoxanthin with the

respective amounts: astaxanthin - 2.7x10​5​, ​lycopene - 1.7x10​5​, zeaxanthin -

2.4x10​5​, violaxanthin - 2.3x10​5​, and lycoxanthin - 1.9x10​5​.

 ACKNOWLEDGMENT

The researchers would like to express their deepest gratitude towards the

following people for their contribution/partial contribution that lead to the success

in the completion of the study:

To Mr. Robert Martin Sim, the researchers’ research adviser, who was a

huge contributor to the success of this study because of his expertise in

Chemistry and because of his insights and criticisms. He has been there all

throughout the way and made time for the researchers despite his busy

schedule.

To Mr. Ian Neil Virtudazo, the researchers’ statistician, who was also a

huge contributor and of huge help to the completion of the study because of his

expertise in Statistics. The information that he shared with the researchers were

both valuable and of paramount importance in analyzing the gathered data.

To Mr. Joshua G. Vidal, the researchers’ research teacher, who gave

constant support, attention, and encouragement to the researchers all throughout

the research process. He constantly provided them with suggestions and

criticisms to make what was already a good paper, a better one.

 To Ms. Sharajen A. Julasiri and Ms. Charita N. Gevana from the University

of San Carlos - Talamban Campus Chemistry Department for allowing the

researchers to conduct the study in their laboratory and for being so

accommodating throughout the course of the research.

To the researchers’ parents and guardians for being patient,

understanding, and for being mentally and emotionally supportive with all the

expenses and restless days and nights the researchers’ spent in conducting the

research and making the paper.

To the researchers’ classmates and friends for providing the researchers

with assistance and encouragement which helped increase their confidence in

completing the study. The study would not have been possible if not for their

peers’ support and if not for their share of information.

And most importantly, to the Almighty Father whose guidance and wisdom

helped the researchers gain courage to surmount numerous challenges in

completing the study.

 CHAPTER 1

THE PROBLEM AND ITS SCOPE

INTRODUCTION

1.1 ​Rationale of the Study

Herbal medicine has always existed even before the introduction and

development of modern medicinal practices. About 75-80% of the world’s

population is still using and practicing herbal medicine for primary health care

because of lesser side effects it gives to the body and better compatibility.​1

There are different herbs and plants that are used for medical treatment.

​ nd malunggay ​for treatment of wounds,

Examples of these plants are ​bayabas a

another is garlic that is used for lowering cholesterol and blood pressure, and

many more. There are numerous examples of herbal medicine proven to be

effective medical treatments and one of these is the insulin plant.

Chamaecostus cuspidatus​, commonly known as fiery costus,​ spiral flag,

costus igneus ​or insulin plant, is a member of the ​Costaceae family and a newly

introduced plant in India from South and Central America. It is a perennial,

upright, spreading plant with a height of two (2) feet, with its tallest stems lying on

the ground. Its leaves are alternate and oblong, four to eight (4-8) inches long
spirally arranged around the stems. Its flowers are orange and are 1.5 inches in

diameter and its fruits are green in colour and are less than 0.5 inch.​2

In Southern India, the plant usually grows as an ornamental plant until

recently, it has been introduced from America as an herbal cure for diabetes and

hence commonly called “insulin plant” in India and in the Philippines.​3

In an episode of Salamat Dok on October 19, 2018, ​costus igneus grew

increasingly popular as the “insulin plant” because the public believes that it is an

effective natural treatment for diabetes because consumption of its leaves is

believed to help lower the blood glucose levels because of their

anti-hyperglycemic properties. Multiple studies have been carried out to evaluate

its anti-diabetic potential; a cross-sectional human study found out that regular

consumption of insulin plant leaves by diabetic subjects resulted in a “statistically

significant reduction in the fasting and postprandial blood sugar levels in all the

patients who consumed the leaves”.​4

This study aims to discover additional benefits of the insulin plant through

its leaves’ carotenoids. Carotenoids are pigments found in a plant’s leaf that are

responsible for a plant’s color. There are many types of carotenoids and people

who consume these in food get certain health benefits. Since it is already known

as a treatment for diabetes, the researchers want to find out what other medicinal

treatment/s the insulin plant can contribute. Furthermore, this study also aims to

become a basis for future studies.

1.2 Statement of the Problem

The study aims to quantify the amount and type of carotenoids extracted

from an insulin plant leaf as basis for future studies.

Specifically, the research aims to answer the following questions:

How many carotenoid​/s is/are present in the insulin plant leaves?

What type of carotenoid/s is/are present in the insulin plant leaves?

What is/are the additional benefit/s of the insulin plant based on the amount and

type of carotenoid/s present in the plant’s leaves?

1.3 Objectives

The study aims to quantify the amount and type of carotenoids extracted

from an insulin plant leaf as basis for future studies. It also aims to discover the

additional benefits of the insulin plant through the amount and type of

carotenoids that it has. Furthermore, it aims to contribute to the field of medicine

and biology, and other research studies related to the study.

1.4 Significance of the Study

The researchers believe that the results and information acquired through

the study on the quantification of carotenoids from ​chamaecostus cuspidatus

(insulin plant) are beneficial to the following:

 ​ he study can serve as a basis for a future
Future Researchers​. T

 
research ​ n determining common carotenoids. It could also contribute to several
o

existing works related to the study.

​ he study mirrors the

Cebu Institute of Technology–University​. T

school’s v​ ision in producing innovative graduates. It can also promote the

school’s prominence or popularity within Cebu City.

Medical Students. The study can serve as a reference to medical

students to gain information about carotenoids and/or the insulin plant.

1.5 Scope and Limitations

The scope of the study only covered the quantification of carotenoids from

Chamaecostus Cuspidatus ​(insulin plant)​. The study covered the extraction,

measurement and identification of the amount of carotenoids from insulin plant. It

was inspired by previous studies regarding carotenoids extraction, the choice of

solvent, its benefits, and the popularity of insulin plant. It examined the quality

and benefits of carotenoids that an insulin plant has. The extract was obtained

through solvent extraction and results were obtained through the use of a double

beam UV-vis spectrophotometer.

The study did not tackle on the certain diseases that each carotenoid

fights but rather, it only quantified what type/s of carotenoid/s is/are present in the
insulin plant and it limits to the identification of the amount of carotenoids from

the insulin plant.

1.6 Definition of Terms

In order to have an easy and better understanding for the readers, the

researchers define the following terminologies according to how it is used in the

study:

Carotenoids

  is a pigment found in the insulin plant leaf that will be extracted.

It

Chamaecostus Cuspidatus

It is a medical term for insulin plant; a plant which the carotenoids will be

extracted.

Diabetes

It is an illness or disease that occurs when there is lack of insulin in the

body.

Extraction
 It is the process of getting or pulling out carotenoids from an insulin plant.

Insulin

It is a hormone that allows cells to use glucose; if the body is not making

or using insulin correctly, a person can take man-made insulin, the insulin plant's

leaves for example, to help control blood sugar.

Quantification

It is the separation, extraction, analysis and identification of the amount

and type of carotenoids present in the plant.

 CHAPTER 2

THEORETICAL BACKGROUND

2.1 Theories

Extraction Theory

Extraction Theory, also known as liquid-liquid extraction, is the most

common technique in extracting carotenoids. The determining parameters of an

efficient extraction is the polarity of a solvent. Like attracts like, thus, nonpolar

carotenoids are used for nonpolar carotenoids (e.g., hexane – β-carotene), while

polar carotenoids are used for polar carotenoids (e.g., acetone – lutein). Hexane

and ethanol are the most frequent applied solvents in carotenoid extraction. In

the last decade, new techniques have raised, becoming popular because of its

green features and environmentally friendly practice. These include the

microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE),

pressurized liquid extraction (PLE), pulse electric field-fluid assisted extraction

(PEF), supercritical fluid extraction (SFE), and the enzyme-assisted extraction.

The development of these techniques is mainly motivated by the specific

difficulties for the extraction of lipophilic content from microalgae and seaweed

species (Perez-Galvez & Roca, 2018). Solutes dissolve in pieces into both layers

when a solution that is placed in a different funnel is being shaken with an

unmixable solvent. The components then “partition” or “distribute themselves”

between two layers. The ratio of the concentration of the solute is constant in

every layer for every system whenever the equilibrium has been fixed. This is

represented by a value “K” (partition coefficient or distribution coefficient) [5].

(Nichols, 2019)

2.2 Review of Related Readings

Since 1993, the increase in the sales of ready-made herbal remedies and

interest from the conventional medical profession in using herbal extracts as an

alternative to powerful drugs can be observed. This interest was rooted from the

increased number of antibiotic-resistant micro-organisms that conventional

treatments find it hard to tackle. In our constantly developing world, a lack of

budget to pay for pharmaceutical imports is encouraging a re-establishment or

wide restoration in using traditional folk remedies [6]. (Ody, 1993).

 Plants make up one of the two main kingdoms of living things. Animals

make up the other kingdom. The first plants appeared on the Earth more than a

billion years ago. They were simple, single-celled organisms called algae, some

similar to the seaweeds we find on seashores today. They, too, lived in the

oceans [7]. (Kerrod, 1998).

According to Oram (1994) there are three pieces of evidences that prove

that all plants on Earth came from green algae. First, both green algae and plants

have cell walls containing cellulose. Second, both their cells contain the same

kinds of chlorophyll and carotenoid pigments. And third, the product of

photosynthesis in both green algae and plants is starch, which is stored in

plastids in the cytoplasm [8].

Organisms have evolved a variety of pigments. The two groups of

pigments that are important to the process of photosynthesis are the carotenoids

and the chlorophylls. Carotenoids absorb photons of green, blue, and violet

wavelength and reflect photons of red, yellow, and orange. The latter, on the

other hand, absorb photons of violet-blue and red wavelengths and reflect green

and yellow [9] (Gottfried, 1993).

Carotenoids possess special properties that other group of substances do

not have. The functions of carotenoids rely on these special properties that are

discovered in the structural features of the molecules. In understanding the

relevance of carotenoids, properties and functions of the carotenoids are

important to determine the relationships between the structures, also for

optimizing commercial applications [10] (Britton et al., 2008).

Presently, over 1178 natural carotenoids have been properly

characterized and reported, which present a huge structural diversity and

physicochemical properties. These carotenoids contain a wide distribution of

about 700 source organisms that includes plants, fungi, bacteria, and algae.

Carotenoids do not only produce the applicability as natural dyes, some, such as

β-carotene are also a source of provitamin A. However, there are still diverse

biochemical, structural diversity of the molecules, and the physiological functions

of carotenoids that enable this class of compounds to drive in the global market

[11] (Nascimento et al., 2018).

2.3 Review of Related Studies

In the study that was conducted by Bramley, Burns, & Framser (2003), the

carotenoid content of a selection of fruits and vegetables found usually in a diet

have been determined using a rapid RP-HPLC technique with on-line PDA

detection. For the dietary intakes of micro-nutrients, plant metabolites are

needed. This method can also be applied to routine screening to determine the

variation of metabolic differences. The identity and levels of carotenoids in the

selection of fruits and vegetables have been achieved with on-line PDA detection

[13].

CHAPTER 3

RESEARCH METHODOLOGY
 This chapter presents the research methodology of the study, which

includes the proposed method, description of the experiment and description of

subject.

3.1 Proposed Method

In this chapter, the methods were applied to attain the data needed for the

study. The study used quasi-experimental research method as the study aims to

evaluate interventions since it ​involves the manipulation of the independent

variable but without the random selection of participants to conditions​. The

researchers are focusing on demonstrating causality between the intervention

and the outcome. Specifically, the researchers studied the carotenoid leaf extract

of ​chamaecostus cuspidatus using solvent extraction. The researchers utilized

the experiment inside a laboratory with the help of an official laboratory

personnel. The treatment in this study is c​hamaecostus cuspidatus leaf extract.

The researchers determined the results by extracting its pigments then analyzing

it using a double beam UV-vis spectrophotometer.

3.2 Description of the Experiment

This research study used a quasi-experimental research method to

identify the different kinds of carotenoids present in ​chamaecostus cuspidatus

leaves. This was done by extracting the carotenoids from the leaves of the plant

with the use of different solvents namely diethyl ether and acetone. In

determining the amount of carotenoids present in the plant, we used a process

 called solvent extraction; and to identify the kinds of carotenoids present in the

plant, we used the device, double beam UV-vis spectrophotometer.

3.3 Description of subject

The carotenoid extracted from chamaecostus cuspidatus leaves, also

known as insulin plant, is the subject of the study. The extract will be used to

identify the amount and type of carotenoids present in the plant. The plant is

widespread due to its efficiency in treating diabetes and hypertension.

EXPERIMENTAL PLAN

Objectives

General Objective

The study aims to quantify the carotenoids extracted from the

chamaecostus cuspidatus l​ eaves to specifically know and discover its additional

benefits only from its carotenoids.

Specific Objectives

To know the amount of carotenoids present in the ​Chamaecostus

cuspidatus l​ eaves.

To identify what type of carotenoid/s is/are present in the ​chamaecostus

cuspidatus l​ eaves.
 To identify and/or discover additional benefits of the ​chamaecostus

cuspidatus l​ eaves.

Materials/Equipment

Laboratory Equipment

10-cm cuvette

Beaker

Centrifuge

Centrifuge tubes

Cork

Droplet

Graduated cylinder

Micro spatula

Mortar and pestle

Pipettor

Stirring rod

Test tubes

Test tube rack

UV-vis Spectrophotometer

Watch glass

Weighing scale

Weigh boat
 Volumetric pipette

Chemicals/Reagents

100% acetone

Diethyl ether

Distilled water

MgO (magnesium oxide)

Procedure

The researchers based their experiment on the solvent extraction of

photosynthetic tissues since it is most convenient to them. The first step

before proceeding to the experiment proper is to gather and prepare all

the materials and chemicals needed, including the plant sample. After

preparation, the researchers then started the extraction by punching

samples from the leaf (​both air dried and fresh) ​specifically 0.04 grams

each and pounded the leaf samples separately with 0.6 grams of MgO

(magnesium oxide) with a mortar and pestle.

The sample was then added with 5mL acetone for the fresh sample

and 5mL diethyl ether for the air dried sample and was mixed together.

Using a pipette and a pipettor, the samples were transferred to 5mL

centrifuge tubes and placed into the centrifuge for 5 minutes with slight

cooling. Upon finishing, the fresh sample was then transferred to a 10cm

cuvette with the other cuvette filled with the solvent used in the fresh
 sample to perform spectrophotometric analysis using a double beam

UV-vis spectrophotometer. The same procedure applies for the air dried

sample.

CHAPTER 4

RESULTS AND DISCUSSIONS

This chapter presents the analysis and interpretation of the results of the

study conducted by the researchers.

4.1 Results

The present study aims to quantify the carotenoid content of

chamaecostus cuspidatus.​ The plant samples used are fresh leaves extracted

with 100% acetone and air dried leaves extracted with diethyl ether. Table 1

shows the plant samples with its respective solvents and its final extract volume

for the quantification of carotenoids.

Table 1. Plant Samples with its solvents

Plant Sample Weight of the Solvent Final Extract

Plant Sample Volume

 Fresh leaves 0.04 grams 100% Acetone 5ml

Dried leaves 0.04 grams Diethyl ether 5ml

Carotenoids are generally extracted with organic solvents. One should

always apply purified solvents such as spectrophotometric grades. This is an

essential requirement in using spectrophotometer in reading the absorbance of

your solution. Organic impurities in standard-grade organic solvents considerable

change absorption coefficients and wavelength maxima of the pigments.

Different plant samples require different solvents because of its different level of

water content. Fresh leaves have higher level of water content than the air dried

leaves, thus it is suitable to use a polar solvent such as 100% acetone for it can

take up water. Air dried leaves can be extracted with diethyl ether for it contains

traces of water and is more polar than any solvents.

According to Harvest Plus, the most important characteristic of

carotenoids is the absorption spectrum that is the function of the chromosphere

of carotenoids. The most important identification process of carotenoids is the

determination of its absorption spectrum is visible in different solvents.

Characterizing with precision of carotenoid chromosphere is possible due to the

correlation between the electronic structure and absorption, position of

absorption maximums being a function of the conjugated double bonds in the

molecule.

Table 2 shows the total carotenoids present in Insulin plant extracted

using different polar solvents analyzed by their absorbance maxima through

double beam Ultraviolet Visible Spectrophotometer.

Table 2. Different types of carotenoids and its amount

Plant Solvent Carotenoids Absorbance Extinction Total

Sample Present Maxima Coefficient Amount of

(nm) Carotenoid

Fresh 100% Zeaxanthin 452 nm 2340

leaves Acetone
Lycoxanthin 474 nm 3080

Violaxanthin 432 nm 2400

Air dried Diethyl Lycopene 470 nm 3450

leaves ether
Astaxanthin 470 nm 2100
 The absorption maxima of extracted pigments strongly depend on the type

of solvent and, to some degree, on the type of spectrophotometer used.

According to Beer-Lambert Law, there is a relationship between the attenuation

of light through a substance and the properties of that substance. Absorbance is

a dimensionless quantity and should therefore, be unit less.

Figure 1 shows the absorbance maxima of diethyl ether processed by a

double beam Ultraviolet Visible Spectrophotometer. The main absorbance region

of carotenoids is at 470 nanometer as supported by Lichtenthaler and

Bauschmann (2001).

Figure 1. Absorbance maxima using diethyl ether

According to Harvest Plus, the absorbance maxima values of carotenoids

in hexane or petroleum ether are practically the same as in diethyl ether. As

supported by Rodriguez-Amaya (2001), the carotenoids found in Insulin Plant

using diethyl ether are lycopene and astaxanthin with an absorbance of 470 nm.
Identification of extracted carotenoids base on their absorbance was done based

on related literature.

Figure 2 shows the list of carotenoids extracted with different solvents with

their absorbance maxima as supported by Rodriguez-Amaya (2001). The list had

been used as a reference for carotenoid pigment studies.

Figure 2. A Guide to Carotenoid Analysis in Foods by Della B. Rodriguez-Amaya,

Ph D. (2001)
Using the formula for the total carotenoid content for astaxanthin:

Where A = Absorbance;

V = Total Extract Volume;

P = Weight;

= 2100 (Astaxanthin extinction coefficient in Diethyl ether)

There are astaxanthin in insulin plant.

Using the formula for the total carotenoid content for lycopene:

Where A = Absorbance;

V = Total Extract Volume;

P = Weight;

= 3450 (Lycopene extinction coefficient in diethyl ether)

There are lycopene in insulin plant.

Figure 3 shows the absorbance range of the sample extracted by 100%

acetone processed by a double beam ultraviolet-visible spectrophotometer. The

absorbance range was acquired using the spectrum of the double beam

ultraviolet-visible spectrophotometer before the final extract was processed for

the carotenoid content.

Figure 3. Absorbance Range of the final plant extract using 100% acetone

As stated in A Guide to Carotenoid Analysis in Foods, the carotenoids

found in Insulin Plant using 100% acetone within the absorbance range of 488

nm to 431 nm are zeaxanthin, violaxanthin, and lycoxanthin. The absorbance

646 nm wasn’t included in the range for carotenoid content for according to

Rodriguez-Amaya (2001), carotenoids are found within the range of 400 nm to

500 nm.

Using the formula for the total carotenoid content for zeaxanthin:

Where A = 452 nm;

V = Total Extract Volume;

P = Weight;

= 2340 (Zeaxanthin extinction coefficient in 100% acetone)

There are zeaxanthin in insulin plant.

Using the formula for the total carotenoid content for violaxanthin:
Where A = 442 nm: V = Total Extract Volume: P = Weight: = 2400

(Violaxanthin extinction coefficient in 100% acetone)

There are violaxanthin in insulin plant.

Using the formula for the total carotenoid content for lycoxanthin:

Where A = 474 nm;

V = Total Extract Volume;

P = Weight;

= 3080 (lycoxanthin extinction coefficient in 100% acetone)

There are lycoxanthin in insulin plant.

 CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

This chapter presents the summary of findings, the conclusions made

based on the results, and the recommendations offered.

5.1 Conclusions

The primary objective of the study was to quantify both the type and

amount of carotenoids present in the insulin plant leaves. Based on the results,

the researchers were able to obtain five types of carotenoids: astaxanthin,

lycopene, zeaxanthin, violaxanthin, and lycoxanthin. These carotenoids have

different benefits to the well-being of an individual. The amount of these

carotenoids obtained are as follows:

Astaxanthin - 2.7x10​5
 Lycopene - 1.7x10​5

Zeaxanthin - 2.4x10​5

Violaxanthin - 2.3x10​5

Lycoxanthin - 1.9x10​5

Astaxanthin is an antioxidant and can contribute to a healthier skin and

heart and also joint pains. Lycopene can contribute to a healthier heart and can

protect the skin against sunburn and the body from free radical stress.

Zeaxanthin can protect the body’s proteins, fats and DNA from stressors and it

can also help the body recycle glutathione. Lycoxanthin, on the other hand, have

been proven to be present in the plant but no study has been able to prove its

benefits yet. Same goes with violaxanthin but it cannot be incorporated into and

used by the body.

5.2 Recommendations

After thorough analysis, the researchers recommend to freeze dry the

plant samples in the experiment for better results. Freeze dried samples can

yield more carotenoids and the researchers only used fresh and air dried

samples. They also recommend using three replicates of each sample for definite

data because more samples can give average and accurate results. In addition,

they recommend to try another method of extraction such as Thin-Layer

Chromatography (TLC) if it would yield another or with the same results. Proper
planning should also be observed to have a smooth flow of the experimentation

for the time of the execution is limited and with this, the researchers should find a

research adviser who specializes in the field of the study of the researchers to

speculate during the experiment if ever the researchers undergo difficulty during

the process and, for instance, if one cannot find specific chemicals, researchers

could consult their adviser for alternatives. The researchers also suggest trying

other polar solvents such as hexane, petroleum ether, methanol, and ethanol for

more variation of results. Since the researchers have identified different

carotenoids in the insulin plant leaves, future researchers can try to prove the

discovered remedies and they could try to use the same plant but bred within

different environments.
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