CHAPTER 4

RESULTS AND DISCUSSIONS

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

conducted by the researchers.

Results

The present study aimed to quantify the carotenoid content of

Chamaecostuscuspidatus. The plant samples used were 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

Weight of the Final Extract

Plant Sample Solvent
Plant Sample Volume

Fresh Leaves 0.04 grams 100% acetone 5mL

Dried Leaves 0.04 grams Diethyl ether 5mL

 18

Generally, carotenoids are extracted using organic solvents that is purified such

as spectrophotometric grade solvents. This is a requirement for the absorbance

reading using the UV-vis spectrophotometer. Organic impurities are contamination

already present in the raw materials. Once present in the extraction process, they

can result in considerable change in the absorption coefficients and wavelength

maxima. 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 using diethyl ether since it is
[21]
considered more polar than other solvents.

According to Harvest Plus (2001), the most important characteristic and

identification process of carotenoids is the absorption spectrum which is visible in

different solvents. Absorption spectrum is the frequency or wavelength of the

interaction of light with a sample. Precise characterization of carotenoid

chromosphere is viable due to the relationship that exists between the state of

motion of the electrons and absorption with the position of the absorption maxima
[21]
being a function of the conjugated double bonds in the molecule.
 19

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 Carotenoids Absorbance Extinction Total Amount

Solvent
Sample Present Maxima (nm) Coefficient of Carotenoid

Zeaxanthin 452 nm 2340 2.4x105 μg/g

Fresh 100%
Lycoxanthin 474 nm 3080 1.9x105 μg/g
leaves Acetone

Violaxanthin 432 nm 2400 2.3x105 μg/g

Lycopene 470 nm 3450 1.7x105 μg/g

Air dried Diethyl

leaves ether
Astaxanthin 470 nm 2100 2.8x105 μg/g

The absorption maxima and range of extracted pigments is based on the type

of solvent and on the type of spectrophotometer used. According to Beer-Lambert

Law, there is a relationship between the gradual loss of light intensity through a

substance and its properties. Absorbance is a dimensionless quantity and should,

therefore, be unit less. [20]

 20

Table 3 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). [20]

Table 3: Absorbance maxima using diethyl ether

Wavelength (nm) Absorbance

470 nm 0.036

According to Harvest Plus (2001), the absorbance maxima of carotenoids in

hexane or petroleum ether are basically the same as the absorbance maxima of

carotenoids 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. [21]

Table 4 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. [21]

Table 4: A Guide to Carotenoid Analysis in Foods [22]

 21

Some Physicochemical Properties of Carotenoids

Table 8. Absorption coefficients (A1%1cm) of common food carotenoids

Carotenoids Solvent Λmax,nm A1%1cm
Antheraxanthin Ethanol 446 2350
Astaxanthin Hexane 470 2100
Auroxanthin Ethanol 400 1850
Bixin Petroleum Ether 456 4200
Canthaxanthin Petroleum Ether 466 2200
Capsanthin Benzene 483 2072
Capsorubin Benzene 489 2200
α-Carotene Petroleum Ether 444 2800
Hexane 445 271
β-Carotene Petroleum Ether 450 2592
Ethanol 450 2620
Chloroform 465 2396
β-Carotene-5,6- epoxide Hexane 444 2590
β-Carotene-5,6,5’,6’- Hexane 440 2690
diepoxide
δ-Carotene Petroleum Ether 456 3290
γ-Carotene Petroleum Ether 462 3100
Hexane 462 2760
ζ-Carotene Hexane 400 2555
Crocetin Petroleum Ether 422 4320
α-Crypoxanthin/zeinoxanthin Hexane 445 2636
β-Cryptoxanthin Petroleum Ether 449 2386
Hexane 450 2460
Echinenone Petroleum Ether 458 2158
Lutein Ethanol 445 2550
Diethyl Ether 445 2480
Diethyl Ether 445 2600
Lutein-5,6-epoxide Ethanol 441 2400
Ethanol 441 2800
Lycopene Petroleum Ether 470 3450
Lycoxanthin Acetone 474 3080
Mutatochrome Diethyl Ether 428 2260
Neoxanthin Ethanol 438 2470
Ethanol 439 2243
Neurosporene Hexane 440 2918
Phytoene Petroleum Ether 286 1250
Phytofluence Petroleum Ether 348 1350
Hexane 348 1577
Rubixanthin Petroleum Ether 460 2750
Violaxanthin Ethanol 440 2550
Acetone 442 2400
α-Zeacarotene Petroleum Ether 421 2450
Hexane 421 1850
β-Zeacarotene Petroleum Ether 428 2520
Hexane 427 1940
Zeaxanthin Petroleum Ether 449 2348
Ethanol 450 2480
Ethanol 450 2540
Acetone 452 2340
 22

Using the formula for the total carotenoid content for astaxanthin:

A ×V (ml)×10 4
carotenoid content :
A 1%
1 cm × P( g)

Where A = Absorbance;

V = Total Extract Volume;

P = Weight;

A11 %cm = 2100 (Astaxanthin extinction coefficient in Diethyl ether)

470 ×5 ml ×10 4 8
carotenoid content : =2.8 x 10 μg /g
2100 ×0.00004 g

There are 2.8 x 108 μg/ g astaxanthin in insulin plant.

Using the formula for the total carotenoid content for lycopene:

A ×V (ml)×10 4
carotenoid content :
A 1%
1 cm × P( g)

Where A = Absorbance;

V = Total Extract Volume;

P = Weight;

A11 %cm = 3450 (Lycopene extinction coefficient in diethyl ether)

470 ×5 ml ×10 4 8
carotenoid content : =1.7 x 10 μg /g
3450 ×0.00004 g
 23

There are 1.7 x 10 8 μg/ g lycopene in insulin plant.

Table 5 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.

Table 5: Absorbance Range of the Final Plant Extract using 100% Acetone

Abscisa Absorbance

646.0 0.020

488.0 0.027

431.0 0.029

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. [18]

Using the formula for the total carotenoid content for zeaxanthin:
 24

A ×V (ml)×10 4
carotenoid content :
A 1%
1 cm × P( g)

Where A = 452 nm;

V = Total Extract Volume;

P = Weight;

A11 %cm = 2340 (Zeaxanthin extinction coefficient in 100% acetone)

452× 5 ml ×10 4 8
carotenoid content : =2.4 x 10 μg/ g
2340 ×0.00004 g

There are 2.4 x 108 μg /g zeaxanthin in insulin plant.

Using the formula for the total carotenoid content for violaxanthin:

A ×V (ml)×10 4
carotenoid content :
A 1%
1 cm × P( g)

Where A = 442 nm: V = Total Extract Volume: P = Weight: A11 %cm = 2400 (Violaxanthin

extinction coefficient in 100% acetone)

442× 5 ml ×10 4 8
carotenoid content : =2.3 x 10 μg /g
2400 ×0.00004 g

There are 2.3 x 108 μg/ gviolaxanthin in insulin plant.

Using the formula for the total carotenoid content for lycoxanthin:
 25

A ×V (ml)×10 4
carotenoid content :
A 1%
1 cm × P( g)

Where A = 474 nm;

V = Total Extract Volume;

P = Weight;

A11 %cm = 3080 (lycoxanthin extinction coefficient in 100% acetone)

474 ×5 ml × 104 8
carotenoid content : =1.9 x 10 μg /g
3080 ×0.00004 g

There are 1.9 x 108 μg/ g lycoxanthin in insulin plant.

Amount of carotenoids with its absorbance maxima

472

468

464

460

456

452

448

444

440
0 5000 10000 15000 20000 25000 30000
Lycoxanthin Astaxanthin Lycopene Violaxanthin Zeaxanthin

Figure 1: Amount of Carotenoids with its Absorbance Maxima