Report for Summer Research Workshop, 2023

On

“Molecules, Metabolism and Biological Systems”

submitted to
School of Life Science
Jawaharlal Nehru University
New Delhi-110067

Supervisor
Prof. Nirala Ramchiary

Submitted by
Sounak Bagh

Student Supervisor

Sounak Bagh Prof. Nirala Ramchiary

Dean, SLS
Prof. Supriya Chakraborty
 Acknowledgement

I would like to thank Prof. Nirala Ramchiary, for providing access to his laboratory for carrying out
short term workshop. I would also like to thanks Prof. Supriya Chakraborty for providing the
opportunity for the summer research workshop-2023 at School Of Life Sciences, JNU, Delhi,
110067. I would like to thank the organizers Dr. Devinder Kaur, Dr. Karunkar Kar and Prof. Nirala
Ramchiary for their initiative and support throughout the entire programme. I would like to thank all
the faculty members of SLS for all interactive and inspiring lectures related to the theme of the
workshop.
I would like to thank Ms. Khusbu Islam for assisting me in the lab during my entire workshop. I
would like to thank Dr. Abdul Rawoof, Dr. Ilyas Khan, Dr Shabari Sarkar, Mr. Nitin Kumar, Smt.
Meenakshi Dubey, Mr. John Momo, Mr. Souparna Biswas, Smt. Vishesh Jangra, Ms. Mona Kumari
for their support during the entire programme.
I would like to also thank Smt. Saraswati Devi and Mr. Tiwari for maintaining a clean lab and
watering the plants in the field and glass houses.
 Jawaharlal Nehru University
School of Life Sciences
New Delhi 110 067

July 30, 2023

Funding Support Acknowledgement

The research work entitled “XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX” is sponsored by

ZZZZZZZZZZZZZZZZZZZZ vide grant no. YYYYYYYYYYYY.

We also acknowledge the facilities/laboratories supported by DBT BUILDER [grant no

BT/INF/22/SP45382/2022] and DST FIST-II [grant no. SR/FST/LSII-046/2016(C)].

Signature of Student Signature of Supervisor

Signature of Dean, SLS

XXXXXXXXX : title of thesis/research topic

ZZZZZZZZZ : name of funding agency, for example : DBT/DST/SERB/CSIR/ICMR/UGC etc.
YYYYYYYYY : Grant number as provided by respective funding agency.

The second paragraph is for acknowledging umbrella grant support from BUILDER/FIST etc.
Introduction
Capsicum is a genus of flowering plants within the family, Solanaceae. The fruits of Capsicum are
commonly used as spice and a supplement of flavor in diverse dishes all over the world. The unique
pungency trait in Capsicum is preferred by Asians, Southern Americans and those living in the
Mediterranean. The origin and domestication of Capsicum has been dated back around 7500 B.C in
the Western Hemisphere. Domestication was earlier traced in South America probably around
Mexico, and is believed to have spread from there to Central America, Europe and then to the whole
world. In India, it is reported that Spanish conquistadors brought Capsicum for cultivation in the year
1542 as a vegetable and also for use as spice trade . Today, China contributes the highest production
value of Capsicum worldwide followed by Mexico.
The fruits of Capsicum represent remarkable diversity in shape, size, color and pungency. Around
~400 species of Capsicum with roughly 50,000 varieties have been reported in the world today. But
only four main species have been dominantly cultivated viz. C. annuum, C. frutescens, C. chinense
and C. baccatum. The fruits are rich in beneficial compounds such as pro-carotenoids, vitamin c,
minerals and essential amino acids. Some species of C. annuum have rich source of carotenoids and
antioxidants that impart stress tolerance to the plants in extreme environmental conditions, such as β-
carotene, β-cryptoxanthin, zeaxanthin, capsanthin and capsorubin. Other species of Capsicum
belonging to varying genotypes and cultivars have purple color that are rich flavonoids such as
luteolin and quercetin. Some of the purple manifestations are dependent on the developmental stages
and is inconsistent in fruit development throughout.
The presence of these rich bioactive compounds renders Capsicum an important crop plant for
consumption as these compounds possess pharmaceutical properties. Chili have been proven to be
effective in ameliorating health conditions such as bronchitis, rheumatoid arthritis, obesity, weight
loss, hypertension, diabetes and digestive complications. There have also been reports of positive
effects on certain cancer such as pancreatic, colon, liver and lung cancer. Capsaicin also renders anti-
bacterial and anti-fungal activities against pathogens like Salmonella typhi, Bacillus cereus,
Staphylococcus aureus and Candida albicans. Evidently, Capsicum has become a majorly relevant
crop preferred and consumed by people all over the world today given its significance to health.
Objective 1-
Extraction of RNA using Trizol (RNA isoplus).
Requirements:
Chemicals required:
1. RNA isoplus (Trizol)
2. Isopropanol
3. Chloroform
4. 70% Ethanol
5. RNase Free water
6. DEPC H2O
7. 1xTAE
Apparatus required:
1. Mortar & pestle
2. Micro centrifuge tubes
3. Table-top centrifuge
Plant material:
Total fruit of Capsicum annuum ‘NB6’.
Principle
Ribonucleic acid (RNA) are single stranded nucleic acids found in living organisms. They constitute
mRNAs which are codes for protein synthesis, tRNAs and rRNA which transfer amino acids for
protein synthesis, snRNA, hnRNA (involved in splicing) and other regulatory RNAs (miRNA,
siRNA).
Trizol is a chemical component widely used in molecular biology to isolate RNA from eukaryotic
organisms. They constitute a monophasic solution of phenol and guanidinium isothiocyanate which
solubilizes the membranes and denatures proteins. The RNA isolated using Trizol is phase separated
by using chloroform. During phase separation, RNA is present in the upper transparent liquid phase.
The RNA isolated is then precipitated using isopropanol at room temperature. After precipitation, the
RNA is then washed in 70% ethanol solution. The RNA is finally dried and dissolved in DEPC
treated H2O. The quality of the isolated RNA is using in 0.8% agarose gel (in 1xTAE). The amount
of RNA isolated is finally quantified using nano spectrophotometer.
Protocol:
The Capsicum fruit samples were grinded to powder in Liquid Nitrogen.

RNA isoplus is added to homogenize the powder.

The powder is transferred into fresh MCTs & centrifuged at 12000g for 5mins @ 4℃

The supernatant was transferred into a fresh tube.

To the supernatant 0.2ml of Chloroform per mL of RNA isoplus was added

& mixed until solution turns milky.

Then stored for 5min at Room Temperature

Centrifugation was done at 12000g for 15min at 4℃

The upper layer was transferred to a fresh tube

Then 0.5-1ml of Isopropanol was added. Stored at Room Temperature for 10min

Again, centrifugation was done at 12000g for 10min at 4℃

The supernatant was removed and washed with 70% ethanol (prepared in DEPC).
 Centrifugation was done at 7500g for 5min at 4℃.

The pellet was dried for 10-15 min and the RNA was dissolved in DEPC water.

The quality of RNA was checked in 0.8% agarose gel, 1X TAE.

The quantity of the RNA was estimated using nanodrop spectrophotometer
For DNA contamination in the sample, the RNA was treated with DNase as mentioned in the
following protocol.
Protocol for DNase Treatment:
We prepared a stock of 50ul
Chemicals Volume
Total RNA 20-50 µg
10X DNase Buffer 5 µl
DNase 2 µl (10U)
Ribonuclease inhibitor 20U
DEPC treatment Upto 50µl
Total volume 50µl

The samples along with the stock were incubated for 20-30 min at 37℃

To the sample 2.5 µl of 0.5 M EDTA was added and incubated at 80℃ for 2 min.

The reaction volume was increased to 100 µl with DEPC treated water.

Then 10 µl of 3M Sodium acetate and 250 µl of chilled EtOH were added. It was mixed well and
kept for 20 min at -80℃.
 Centrifugation was done at 12,000 rpm for 10 min at 4℃. After that supernatant was removed.

The precipitate was with chilled 70% EtOH.

Again, centrifugation was done at 12,000 rpm for 5 min at 4℃. The supernatant was removed and
the precipitate was dried.

The dried precipitate was dissolved in suitable amount of DEPC treated water.

Results and Discussion

Table 1: Concentration of RNA isolated using Trizol
Sample Concentration (ng/µl) Abs at (260/280) nm
1 265.0 2.00
2 222.5 1.83
3 354.6 1.88
4 431.7 2.04
5 714.6 1.99
6 113.3 1.75

1 2 3 4 5 6

28S
18S

Fig 1. Bands showing RNA isolated using Trizol

The quality of the RNA samples was observed on the agarose gel (in 1xTAE) (Fig. 1). Two bands
that were observed in the gel correspond to 28S and 18S RNA species. The quality of the samples 1,
2 and 4 although showing good concentration in the nanodrop did not show good quality in the gel,
because the RNA could have degraded during the isolation process. RNA samples at absorbance
260/280nm usually show 2.0-2.2 whereas for DNA, the absorbance usually show at 1.8-2.0.
Objective 2
Extraction of Vitamin E from Capsicum fruits
Requirements:
Capsicum fruits: The fruits were harvested from plants of different Capsicum species.
Chemicals required:
1. Acetone.
2. BHT (Butylated hydroxytoluene)
Apparatus required:
1. Mortar & pestle
2. 50 ml falcon tubes
3. Vacuum drier
4. Table top centrifuge
5. 0.45 µm membrane syringe filter.
Principle:
Vitamins are essential organic compounds required in small amounts for the proper functioning and
maintenance of the human body. They are crucial for various physiological processes and are
involved in energy production, metabolism, immunity, and overall health. Based on their solubility,
vitamins are classified into two categories: water-soluble vitamins and fat-soluble vitamins. Water-
soluble vitamins, such as vitamin C and the B vitamins (B1, B2, B3, B5, B6, B7, B9, and B12),
dissolve in water and are not stored in significant amounts in the body. They need to be replenished
regularly through diet or supplements. On the other hand, fat-soluble vitamins, including vitamins A,
D, E, and K, dissolve in fat and can be stored in the body's fatty tissues and liver.
In this report, the focus is on vitamin E, which is a fat-soluble vitamin known for its potent
antioxidant properties. Vitamin E is a collective term for a group of compounds called tocopherols
and tocotrienols. It exists in several forms, with alpha-tocopherol being the most biologically active
and commonly studied form. Vitamin E plays a crucial role in protecting cells from oxidative
damage caused by free radicals and reactive oxygen species. It helps maintain the integrity of cell
membranes and protects lipids from oxidation. Additionally, vitamin E is involved in immune
function, gene expression, and various enzymatic activities within the body. One notable natural
source of vitamin E is Capsicum, a genus of flowering plants commonly known as peppers.
Capsicum spp. are highly variable, such as bell peppers, chili peppers, and paprika. These vibrant
and flavorful vegetables are not only rich in vitamin E but also provide other essential nutrients, such
as vitamin C, vitamin A, and dietary fiber.
Here, we will explore the extraction of vitamin E from a selected Capsicum germplasm, specifically
focusing on the utilization of Capsicum. The extraction process and methodology will be discussed,
along with the potential applications and significance of the extracted vitamin E in various industries.

Protocol:
About 0.5gm of Capsicum fruit tissue was taken.

The tissues was finely chopped and homogenized with liq. N2.

Finely chopped sample was transferred into a 15 ml falcon tube.

The sample was mixed with 10ml of chilled acetone containing 0.1%BHT.

The samples were centrifuged at 5000g for 5min at 4℃

Pelleted samples were repeatedly extracted until the pellet became colourless.

Supernatant from all the extractions were kept in a vaccum drier for drying.

The extracts were recovered with 5ml of chilled acetone containing 0.1% BHT.

Then the liquid was filtered through a 0.45 µm membrane syringe filter.

Then the filtered sample was transferred to amber colored HPLC vial and sent for HPLC analysis.

Results:
Fig 2. Known concentration of vitamin E were used for estimation of retention time & peak area
through HPLC analysis.

FAT SOLUBLE VITAMINS NEW #190 41 UV_VIS_1

5,500
mAU WVL:290 nm
3 - VITAMIN E - 1.560

4,000

3,000

2,000

1,000

4 - 2.320
1 - 1.153 2 - 1.373

min
-500
0.00 0.50 1.00 1.50 2.00 2.50 3.00

Fig 3. The characteristics peak for vitamin E obtained for unknown sample in HPLC analysis.
Table 2. Retention time, height and peak area for vitamin E sample in HPLC analysis

No. Ret.Time Peak Name Height Area Rel.Area Amount Type

min mAU mAU*min %
1 1.15 n.a. 9.982 2.035 0.35 n.a. BM
2 1.37 n.a. 39.745 4.930 0.85 n.a. M
3 1.56 VITAMIN E 4959.161 539.493 93.34 187.509 Mb
4 2.32 n.a. 335.899 31.544 5.46 n.a. bMB
Total: 5344.787 578.002 100.00 187.509

Discussion:
The standard curve obtained for 3 different concentration of Vitamin E was plotted against the peak
area obtained from sample through HPLC analysis. The Vitamin E peak was obtained at 1.56 min
retention time with an area coverage of 539.49 milli O.D @440nm (mAU*U). The equation for
straight line was deduced and used to calculate and estimate the concentration/amount of vitamin E
content in the unknown Capsicum sample.
The final concentration of Vitamin E was found to be 47.462 mg/gm. The vitamin E concentration in
capsicum fruit was satisfactorily high.
Objective 3
Media preparation, primary culture of E. coli (DH5ɑ) and plasmid isolation.
Requirements:
Chemicals required:
1. LB agar Media
2. LB broth
3. Re-suspension solution
4. Lysis solution
5. Neutralization solution
6. Washing solution/ Autoclaved Water
Apparatus required:
1. Petri-dishes
2. Micro centrifuge tubes
3. Incubator shaker
4. Culture tubes
5. GeneJET spin column
6. Table-top Centrifuge
Principle:
Luria Broth is a lysogenic broth for the culture of bacteria in laboratories first created by Guiseppe
Bertani. The media is routinely used in molecular biology to grow bacterial cultures.
Plasmids are circular DNA that are present in prokaryotes. They can replicate independently without
the use of chromosomal machineries. The term plasmid initially introduced by Joshua Lederberg in
1952. Plasmids usually carry genes that confer antibiotic resistance to the bacteria. Plasmids are
efficiently used in genetic engineering to transform desired gene of interest in a heterogeneous
system.
Plasmid isolation involved the lysis of bacterial cells using alkaline solutions. Bacterial plasmids
have low molecular weight compared to the genomic DNA and is easily separated during isolation in
the through phase separation. The process of plasmid isolation involves repeated lysis using alkaline
solution such as NaOH incubated in a buffer solution or osmoticum in order to prevent unwanted
lysis of cell debris. The plasmid isolated is then precipitated with a precipitation reagent such as
ammonium acetate. The plasmid is then washed using 70-75% ethanol and dissolved in sterile water.
Protocol:
Pick a single colony from LB agar plate. Incubate 12-16 hrs at 37℃ while shaking at 200- 250 rpm.
Use a tube or flask with a volume of at least 4 times the culture volume.

Centrifuge at 8000 rpm for 2 min at room temperature. Decant the supernatant and remove all the
remaining medium.

The pelleted cells were re-suspended in 250 µL of resuspension solution. The cell suspention was
transferred in MCTs. The bacteria were resuspended completely by vortexing or pipetting up and
down until no cell clumps remaining.

250 µL of the lysis Solution was added and mixed thoroughly by inverting the tube 4-6 times until
the solution becomes viscous and slightly clear.

350 µL of the neutralization Solution was added and mixed immediately and thoroughly by inverting
the tube 4-6 times. The neutralized bacterial lysate became cloudy.

Centrifugation was done at 13800 rpm for 5 min at room temperature to pellet cell debris and
chromosomal DNA.

The supernatant was transferred to the supplied GeneJET spin column by decanting or pipetting
without disturbing or transferring the white precipitate.

Centrifugation was done at 13,800 rpm for 1 min at room temperature The flow-through was
discarded and the column placed back into the same collection tube.
500 µL of the autoclave water was added to the GeneJET spin column. Centrifugation is done for 30-
60 seconds. The flow-through was discarded. The column was placed back into the same collection
tube.

The previous step was repeated using 500 µL of the autoclave water.

The flow-through was discarded and centrifuged for an additional 1 min to remove residual Wash
Solution. This step is essential to avoid residual ethanol in plasmid preps.

The GeneJET spin column was transferred into a fresh 1.5 mL microcentrifuge tube. Then 50 µL of
the water was added to the center of GeneJET spin column membrane to elute the plasmid DNA.
Then it was incubated for 2 min at room temperature and centrifuged for 2 min.

The column was discarded and the purified plasmid DNA was stored at -20°C.

Results and Discussion

The quantity of plasmid DNA was checked using Nano drop spectrophotometer & quality was
checked on 1% agarose gel using gel electrophoresis.

Table 3. Concentration of isolated plasmid DNA.

Sample No. Abs (260/280) nm Abs (260/230) nm Concentration (ng/ul)

1 1.81 1.53 104.4
2 1.88 2.06 132.8
3 1.98 1.89 57.7
 Sample-1 Sample-2 Sample-3
Fig 4. LB agar plate showing E. coli growth after overnight incubation.

Supercoiled plasmid
Linear plasmid

Fig 5. Gel image showing the bands

Sample 1 and 2 showed characteristics ratio of (260/280) close to 1.8 and the concentration was also
significantly high, while sample 3 showed the ratio of (260/280) higher than 1.8 which means it may
have RNA contamination.
The agarose gel electrophoresis (fig 5) showed 2 characteristics bands in all three samples which
indicated good quality of plasmids in the sample.
Objective 4
Estimation of total anthocyanin and carotenoid content from Capsicum
Requirements:
Chemicals Required:
1. 70% Ethanol
2. Acetone
Apparatus Required:
1. Mortar and pestle
2. Table-top Centrifuge
3. Micro centrifuge Tubes
4. UV-spectrophotometer
Principle
Carotenoids are triterpenoid pigments majorly present in plants. They exhibit color (mainly red,
yellow, orange, purple) in the fruits and leaves of lower and higher plants. They are classified into
two main groups: carotenes and xanthophylls. Carotenes constitute α, β, γ-carotenes and lycopenes
which are long chain hydrocarbons. Xanthophylls are pigments that contain oxygen atoms in the
form of carboxy, hydroxy and carbonyl groups. They include lutein, zeaxanthin, fucoxanthin,
astaxanthin, β-cryptoxanthin, and peridinin. The health benefits of carotenoids exceed the range of
properties exhibited through antioxidant properties. Due to their lipophilic properties, carotenoids
have been used to treat hypoglycemia in diabetic patients in a dose dependent. Relatively, these
compounds also exhibit anti-obesity activity by activating lipases or leading to excessive ATP
production that resulted in weight loss.
Anthocyanins represent glycosylated polyphenolic compounds that impart color such as orange,
purple, red, and blue in vegetative tissues of plants. They are water soluble pigments localized
mostly in the vacuole. Up to ~600 anthocyanins have been reported in plants. Major anthocyanins
include the pelargonidin, cyanidin, delphinidin, malvidin, petunidin, peonidin etc. Anthocyanins
provide rich color to fruits which is preferred by humans, and they also protect the plants against
biotic and abiotic stresses. Anthocyanins impart color to plants which serves as an attractant for
pollinators. They have strong antioxidant properties and have defensive role against abiotic and
biotic stresses. They also protect the plants from photoinhibition, photobleaching and scavenge free
radicals. They usually accumulate at the vegetative tissues where UV exposure is maximum
suggesting their photoprotective role. Anthocyanins have also been reported to improve crops post-
harvest by preventing lipid peroxidation and ensuring membrane stability by decelerating senescence.
Protocol:
500 mg of fruits was crushed after drying in Liq. N2

The powdered sample was added in acetone and ethanol for carotenoid and anthocyanin respectively.

The extract was kept in dark for shaking & overnignt incubation.

Then the samples were centrifuged at 2000rpm for 10 mins at room temperature.

Collect the supernatant and quantify using UV-spectrophotometer.

Results & Discussion

C. annuum (Kosom Moso) C. chinense (Chocolate) C. chinense (BJCF)

Fig 6. Different Capsicum genotypes used for carotenoid and anthocyanin estimation

Table 4. Amount of carotenoid and anthocyanin of 3 different genotypes

Pigment Carotenoid (mg/100g) Anthocyanin(mg/100g)
Genotype Early Mature Early Mature
Kosom Moso 15.35 24.97 26.40 11.20
Chocolate 14.50 70.70 1.16 17.47
BJCF 5.37 52.67 1.55 46.39
Fig 7. Carotenoids and anthocyanins profile in different Capsicum germplasm

In the Fig 7, we can see that in Kosom Moso, Chocolate and BJCF genotypes, mature stages have
higher carotenoid content than early stages. The highest genotype content was found in Chocolate
genotype. We also observe that, in Kosom Moso the Anthocyanin content was higher in early stages
than mature stages. But in Chocolate and BJCF genotypes the anthocyanin content of mature stages
is higher than early stages.

Conclusion:
For Objective 1, we isolated RNA using Trizol and quantified the same using nanodrop
spectrophotometer. The quality of the RNA was checked in 1% agarose gel (1X TAE) by
electrophoresis.
For Objective 2, We estimated the concentration of Vitamin E via HPLC and found relatively higher
concentration in capsicum.
For Objective 3, we successfully isolated plasmid DNA and quantified the same using nanodrop
spectrophotometer.
Finally for objective 4, We quantified and compared the carotenoid and anthocyanin content in
different capsicum genotypes by spectrophotometric analysis.
During 1 month Internship programme in SLS Life Sciences, JNU. I was able to learn many new
techniques and experimental procedure such as RNA isolation, plasmid isolation, Vitamin E
extraction, quantification of DNA and RNA, Streaking of transformed bacteria.
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