RESEARCH ARTICLE

Fluorescence Studies of the Seeds of the Pumpkin(Cucurbita pepo L.)

P. R. Borthakur • Anurup Gohain Barua

Received: 14 July 2013 / Revised: 8 October 2013 / Accepted: 30 October 2013 / Published online: 5 May 2014

� The National Academy of Sciences, India 2014

Abstract A violet diode laser of wavelength 405 nm is

used to excite fluorescence of seeds of the pumpkin at raw

and ripe stages of growth. Emission bands appear at

wavelengths 630 and 670 nm at both the stages. Another

band in the region 460–550 appears in the ripe stage only.

Fluorescence intensity ratio F630/F670 calculated for the

two stages show marked variation—indicating the process

of ripening of the fruit. Time–resolved spectra at this

excitation wavelength show two decay times at both the

stages at emission wavelength 500, 630 and 670 nm.

Keywords Seeds of pumpkin �Fluorescence intensity ratio � Riboflavin � Decay time �Protochlorophyllide

Introduction

Pumpkins are gourd squashes of the genus Cucurbita and

the family Cucurbitaceae. These have been known since

the dawn of time. Native to the Americas, pumpkins are

found across North America, South America, and Central

America. Today, these are widely cultivated all over the

world for food and decorative purposes. Pumpkins have

long been used for traditional medicine in many countries,

such as China, Argentina, India, Bangladesh, Mexico,

Brazil, and Korea. Pumpkin seeds are a good source of zinc

[1], polyunsaturated fatty acids [1, 2], and phytosterols [3,

4], which can prevent chronic diseases. Nowadays,

pumpkin seed oil is used successfully in preventing and

alleviating prostate and bladder problems [5]. Pumpkin

seed oil is also being studied for its role in lowering cho-

lesterol levels. Composition and nutritional profile of the

pumpkin seed could be found in USDA nutrient data base.

Pumpkins are very versatile in their uses for cooking. Most

parts of the pumpkin are edible, including the fleshy shell,

the seeds, the leaves, and even the flowers. In India,

pumpkin is an important vegetable during summer.

In this communication, 405 nm-excitation of steady-

state and time-resolved fluorescence emission spectra of

seeds of pumpkin in raw and ripe stages are presented. In

recent times, spectral signatures of ripening of seeds of the

bitter gourd and spiny gourd are obtained with the help of

their fluorescence spectra in raw and ripe stages [6, 7].

Very recently, recording the fluorescence spectra of raw

and ripe lemon juice, the intensity ratio of green and red

fluorescence bands has been shown to vary, indicating

completion of the process of ripening of the fruit [8].

Materials and Methods

Pumpkin (cucurbita pepo L.) cultivated in Hajo, a sub-

urban area of Assam, around 60 km from the city of Gu-

wahati were collected for the experiment (10 raw and 10

ripe). Raw pumpkins were green and fully ripe ones were

orange in colour. Fruits were cut and seeds were separated

from the pulp with clean hands. The removed seeds were

washed with distilled water and then crushed in an agate

mortar, forming paste; 100 mg of the paste was dissolved

in 3 ml of acetone in a quartz cuvette. As the fluorescence

P. R. Borthakur (&)

Faculty of Science and Technology, The ICFAI University

Tripura, West Tripura 799210, India

e-mail: priyangshu.rana@gmail.com

A. G. Barua

Department of Physics, Gauhati University, Guwahati 781014,

Assam, India

e-mail: agohainbarua@yahoo.com

123

Natl. Acad. Sci. Lett. (May–June 2014) 37(3):275–279

DOI 10.1007/s40009-014-0248-1

intensity of the sample was reasonably low, acetone was

used as a solvent to enhance the intensity. The solution in

the cuvette was excited with a 50 mW violet diode laser

(Pegasus) of wavelength 405 nm. The laser was placed at a

distance of 8 cm from the cuvette. This distance as well as

the mixing ratio (100 mg of the paste was dissolved in 3 ml

of acetone) of the paste and acetone were maintained very

carefully. The cuvette was held fixed with the help of a

retort stand. Fluorescence spectra were collected at an

angle of 90� with respect to the incident light, with the help

of an optical fiber cable connected to an Ocean Optics

HR2000 series spectrometer. To minimize the contribution

from the reflected light, the excitation beam was allowed to

incident at an angle of 30� to the plane of the cuvette,

which contained the sample. The spectrometer was

equipped with data acquisition and display software

(Ocean Optics Spectra Suite, OOI Base 32). The sensitivity

of the spectrometer can be increased by adjusting the

integration time.

Fully integrated fluorescence lifetime spectrometer

LifeSpec II (Edinburgh) was used to record the time

resolved spectra. EPL 405 nm, of repetition rate 10 MHz,

pulsed diode laser having pulse duration of 90 ps was used

as the excitation light source. Emission slit width was

20 nm. The detector was a Micro Channel Plate-Photo-

multiplier Tube of response width \25 ps and the instru-

ment response function was \130 ps. After reconvolution,

the shortest recoverable lifetime was approximately 1/10 of

instrument response function. The goodness of fit quality

parameter (v2) for all the recorded spectra was approxi-

mately 1. The experiments were performed at a room

temperature of 28 �C.

Results and Discussion

Figure 1a and b display the 405 nm-excited steady-state

spectra of raw and ripe pumpkin seeds, respectively. For

Fig. 1 Steady state

fluorescence emission spectra of

seeds of the pumpkin a in the

raw stage, b in the ripe stage

276 P. R. Borthakur, A. G. Barua

123

raw pumpkin seeds, bands appeared approximately at

wavelengths 630 and 670 nm. For ripe pumpkin seeds, a

broad band appeared in blue-green (460–550 nm) region,

along with other two bands appearing approximately at

wavelengths 630 and 670 nm. Blue-green fluorescence

(460–550 nm) has a heterogeneous origin with a number of

candidate fluorophores, such as hydroxycinnamic acid

derivatives (ferulic acid, p-coumaric acid), flavonoids

(quercetin, kaempferol) and flavins (riboflavin) [9]. Ferulic

acid absorbs with maxima at 285 and 310 nm. It can be

seen that p-coumaric acid absorbs with a maximum at

285 nm. Flavonoids, such as quercetin, have low absorp-

tion at wavelength 405 nm, as well as have very low

intensity of fluorescence [10]. On the other hand, flavins

(riboflavin) have high absorption between 375 and 450 nm,

and high intensity of fluorescence at 500–520 nm [11].

This band, almost missing in raw stage (Fig. 1a), appeared

in the blue-green region at ripe stage (Fig. 1b). This could

be considered as a spectroscopic evidence of the increase

of the content of riboflavin in the ripe stage. Fluorescence

of two different forms of protochlorophyllide could be the

reason for the bands at 630 and 670 nm, as attributed to

similar types of bands in nearly the same wavelength

regions in maize, wheat, and wild-type pea [12], dark

grown plant leaves [13], and seeds of bitter gourd and spiny

gourd [6, 7], when excited by wavelengths of 405, 440, and

460 nm. As the fruit approaches maturity, the intensity of

the bands in the region 460–550 and at 630 nm increased in

many folds, while that of the band at 670 nm increased a

little. The intensity variation of the bands at 630 and

670 nm with the growth of the fruit indicates a variation of

relative amounts of two components of protochlorophyllide

present, at raw and ripe stages. Chlorophyll(ide) fluores-

cence emission has been reported to decrease at room

temperature during completion of protochlorophyll(ide)

reduction, the reason given as regulation by a conjunction

of factors such as energy transfers and photobiochemical

activities [14]. The intensity ratio F670/F630 at two stages

of growth is determined for 10 specimens in each of the

two stages. The average intensity ratio F630/F670 in stage

1 comes out as 1.27 and in stage 2 as 4.47. Standard

deviations in the ratio for the two stages are 0.08 and 0.03,

respectively. The intensity ratio F630/F670 in stage 1 and

stage 2 of the seeds presents a signature of maturity of the

fruit.

Time-resolved spectra for emission peaks of 500, 630

and 670 nm are shown in Figs. 2, 3 and 4, respectively.

Discrete component analyses are given in Table 1.

405 nm-excitation of time-resolved spectra reveals two

decay times in each of the raw and ripe stages for each of

the emission wavelengths 500, 630 and 670 nm. For the

emission wavelength of 500 nm, the two decay times at the

two stages are 3.428 and 9.611 ns in raw stage (Fig. 2a)

and 3.654 and 9.973 ns in ripe stage (Fig. 2b). Thus, decay

times of the involved fluorophore almost remain unchan-

ged at these two stages. The component with decay times

Fig. 2 a Time-resolved spectra at emission peak of 500 nm of seeds

of the pumpkin in raw stage. b Time-resolved spectra at emission

peak of 500 nm of seeds of the pumpkin in ripe stage

Fig. 3 a Time-resolved spectra at emission peak of 630 nm of seeds

of the pumpkin in raw stage. b Time-resolved spectra at emission

peak of 630 nm of seeds of the pumpkin in ripe stage

Fluorescence Studies of the Seeds of the Pumpkin 277

123

of 3.428 and 3.654 ns gives a strong indication for the

presence of flavins, in particular riboflavin or flavin

mononucleotide (FMN) rather than flavin adenine dinu-

cleotide (FAD). Presence of hydroxycinnamic acid deriv-

atives could be ruled out as these give rise to peaks at the

blue-green region upon excited by ultraviolet radiation

(285–310 nm), and use to have very fast decay times.

Intensity decay revealing two decay times may originates

with a single fluorophore or multiple fluorophores. Multi-

exponential decay originates with a single fluorophore

indicates the presence of more than one conformational

state. However, intensity decay of protein bound flavins

are typically complex with multi-exponential decay times

ranging from 0.1 to 5 ns [15]. Thus, decay times of 9.611

and 9.973 ns definitely indicate that the concerned fluo-

rophore is not flavin and decay at this wavelength orig-

inates with multiple fluorophores. Two decay times at

raw and ripe stages were obtained for the emission

wavelengths of 630 and 670 nm. For the emission

wavelength of 630 nm, the two decay times at the two

stages are 1.823 and 5.984 ns in raw stage (Fig. 3a), and

2.034 and 6.14 ns in ripe stage (Fig. 3b). For the emis-

sion wavelength of 670 nm, two decay times at the two

stages are 1.306 and 6.523 ns in raw stage (Fig. 4a) and,

1.708 and 6.93 ns in ripe stage (Fig. 4b). Protochloro-

phyllide, an immediate precursor of chlorophyll a, could

be the reason for giving rise to two decay times in the

raw and ripe stages, as a double exponential model has

been proposed to describe the protochlorophyllide fluo-

rescence decay [13]. The slow component of decay time

around 6 ns red fluorescence could also be ascribed to

free chlorophyll [16].

Fractional components of the fluorescence intensity

(f) could be used to determine the contribution of each

fluorophore in the fluorescence spectra. For the emission

peak at 500 nm (Fig. 2) changes in fractional intensity,

from approximately 58–79 %, and 42–21 % in the two

stages, appear to be very significant. We could safely

conclude that the contribution of the ‘bound’ fluorophore

increases while that of ‘free’ fluorophore decreases at

maturity. At the emission peaks of 630 and 670 nm,

the ‘slow’ components of the fluorophores contributes

approximately four to ten times more than the ‘fast’ one in

both the stages. We could conclude that the free form of

chlorophyll is mostly present at raw and ripe stages.

Fig. 4 a Time-resolved spectra at emission peak of 670 nm of seeds

of the pumpkin in raw stage. b Time-resolved spectra at emission

peak of 670 nm of seeds of the pumpkin in ripe stage

Table 1 Discrete component

analyses (reconvolutions)

Experiment number 1, 2 for

Fig. 2a; 3, 4 for Fig. 2b; 5, 6 for

Fig. 3a; 7, 8 for Fig. 3b; 9, 10

for Fig. 4a and 11, 12 for

Fig. 4b

Exp No Goodness of fit quality

parameter

Pre-exponential

factor

Fractional

intensity

Life time

(ns)

v2 B f s

1 1.066 1.812 41.51 3.328

2 0.002 58.49 9.611

3 1.062 0.113 16.32 1.823

4 0.026 83.68 5.984

5 1.063 0.029 8.463 1.306

6 0.054 91.54 6.523

7 1.091 1.005 20.88 3.654

8 0.057 79.12 9.973

9 1.027 0.037 24.29 2.034

10 0.048 75.71 6.14

11 1.03 0.016 12.13 1.708

12 0.066 87.87 6.93

278 P. R. Borthakur, A. G. Barua

123

Conclusion

Fluorescence spectra of seeds of the pumpkin contain

information about fluorophores (riboflavin/or FMN, and

chlorophylls). The spectra allow monitoring changes in the

relative content of fluorophores present in the seeds during

growth. A distinct increase in fluorescence features ascri-

bed to riboflavin was observed in seeds during growth of

the fruit. Changes in fluorescence intensity ratios of the

pumpkin seed demonstrate the progress of ripening of the

fruit. It could be taken as a spectral fingerprint of ripening

of the fruit. Further investigations are required to determine

the changes in intensity of the fluorescence bands of dif-

ferent species of the fruit during the ripening process,

which will enable us in drawing a general conclusion.

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