“EXTRACTION AND QUANTIFICATION OF CITRAL FROM

CYMBOPOGAN FLEXUOSUS LEAVES”

A
PROJECT WORK REPORT
SUBMITTED
AS A PARTIAL FULFILLMENT FOR THE DEGREE OF

BACHELOR OF PHARMACY
IN
THE FACULTY OF PHARMACY
TO
GANPAT UNIVERSITY

MAY – 2024

GUIDED BY: SUBMITTED BY:

Dr. Bhoomi M. Patel Milind H. Barot (20022411007)
M. PHARM., Ph.D. Harsh K. Patel (20022411050)
Sharad D. Patel (20022411087)
Pinal K. Solanki (20022411115)

SHREE S. K. PATEL COLLEGE OF PHARMACEUTICAL EDUCATION AND

RESEARCH, GANPAT UNIVERSITY, GANPAT VIDYANAGAR – 384012,
DIST. – MEHSANA (GUJARAT), INDIA
 CERTIFICATE
This is to certify that Milind H. Barot, Harsh K. Patel, Sharad D.
Patel, Pinal K. Solanki have completed their Project Work for the
degree of Bachelor of Pharmacy in the Faculty of Pharmacy on the
topic “EXTRACTION AND QUANTIFICATION OF CITRAL
FROM CYMBOPOGAN FLEXUOSUS LEAVES”. I further certify
that work was carried out under my supervision and guidance at
Department of Pharmaceutical Chemistry & Quality Assurance,
Shree S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University during the academic year 2023-24. This work is up
to my satisfaction.
GUIDED BY: HEAD OF DEPARTMENT:

_________________ _________________
Dr. Bhoomi M. Patel Dr. Paresh U. Patel
M.Pharm, Ph.D., M.Pharm, Ph.D.,
Assistant Professor, Professor and HOD.,
Dept. Of Pharm. Chemistry Dept. Of Pharm. Chemistry
GUNI-SKPCPER GUNI-SKPCPER

Forwarded Through:

______________
Dr. P. D. Bharadia
M. Pharm., Ph.D.
Principal,
Shree S. K. Patel College of Pharmaceutical
Education and Research, Ganpat University

DATE:
PLACE: Ganpat University
 DECLARATION
We, Milind, Harsh, Sharad, Pinal hereby declare that the
Project Work entitled “EXTRACTION AND QUANTIFICATION
OF CITRAL FROM CYMBOPOGAN FLEXUOSUS LEAVES ”
which is submitted to the Ganpat University, in the partial
fulfilment for the award for degree of Bachelor of Pharmacy at
Shree S. K. Patel College of Pharmaceutical Education &
Research, is Our own project work carried out under the
supervision and guidance of Dr. Bhoomi M. Patel,
M.Pharm,Ph.D, Department of Pharmaceutical Chemistry &
Quality Assurance, Shree S. K. Patel College of Pharmaceutical
Education and Research, Ganpat University. The work
presented in this report has not been submitted for the award of
any degree in this or any other University. The work was
carried out by us during academic year 2023-24.

Milind Barot
Harsh Patel
Sharad Patel
Pinal Solanki
B. Pharm. - VIII

Date:
 ACKNOWLEDGMENT
We express our gratitude to my guide Dr.Bhoomi M. Patel,
Assistant professor of the Department of Pharmaceutical
Chemistry & Quality Assurance, of Shree S.K. Patel College Of
Pharmaceutical Education And Research, Ganpat University, for
their genuine guidance and constent encouragement throughout
this Project Work. We are highly obliged as our honorable guides
have devoted their valuable time and shared their experience and
knowledge.

We extend our sincere thanks to Dr.Praful D. Bharadia, principal.,

Dr. Paresh U. Patel, Head Of Department., Dr. Satish A. Patel,
professor., Janvi S. Patel, Roshani A. Patel, Riddhi J. Jani, Mansi
R. Patel, Ami P. Patel, assistant professors., Praharsh B. Raj, Nisha
J. Patel, Laboratory Assistant., of Shree S.K. Patel College Of
Pharmaceutical Education And Research for providing us such an
opportunity to do our work in the subject practice school. We also
sincerely thanks Shree S.K. Patel College Of Pharmaceutical
Education And Research for providing us with knowledge about
the Project Work.
I also wish to express a heartfelt appreciation to Dr.Bhoomi M.
Patel for providing us a good training and support for the
successful completion of the work.
 INDEX

Table of Contents
1
.INTRODUCTION OF LEMON GRASS ..........................1
2. INTRODUCTION OF CITRAL....................................4
3. METHOD OF EXTRACTION.....................................5
4.HIGH PERFORMANCE THIN LAYER CHROMATO-
GRAPHY (HPTLC):.......................................................7
5.METHODOLOGY :....................................................9
6.CONCLUSION :......................................................15
7.REFERENCES..........................................................15
INTRODUCTION OF LEMON GRASS :

Lemongrass is a tropical perennial and aromatic plant which belongs to the family
Graminae (Poaceae) and the genus Cymbopogon. The name Cymbopogon came from
Greek words ‘kymbe’ (boat) and ‘pogon’ (beard) meaning arrangement of flower spike.
The herb is cultivated and distributed in sub regions of Africa, Asia, North America,
South America, Australia, Oceania . There are about 55-80 species of lemongrass already
identified which includes Cymbopogon citratus (West Indian lemongrass), Cymbopogon
flexuosus (East Indian lemongrass), and Cymbopogon pendulus (Jammu lemongrass).
Out of all the species identified, only two have economic importance as cultivated plants
namely C. citratus and C. Flexuosus.

A. Dried pieces of leaves B. Plant

Fig.1.1 Cymbopogon flexuosus

1
Cymbopogon flexuosus which is the second most important lemongrass specie is a tall,
fast-growing, lemon-scented, perennial grass reaching an average height of about 1.5 m.
It has distinct, dark-green foliage and also produces seed. Lemongrass plants persist for
many growing season and it produces essential oil.
It contains many phytochemical compounds like phenol, citral, geranial, terpenoids,
benzenoids and other nitrogenous compounds which help in metabolic process of plant.
The leaves of lemongrass and its oil possess lemon-like odour characteristic aromatic
flavours due to its citral contents which is the most dominant constitutes.
Dry leaves of lemongrass contain approximately 1-2 percent essential oil.
Lemongrass extracts has found industrial application in pharmaceutical, cosmetics, food
processing and perfumery industries.
Scientific studies have showed that Cymbopogon citratus oil possesses various
pharmacological properties such as antibacterial, anti-amoebic, anti-filarial, antidiarrheal,
anti-inflammatory, antifungal, antimalarial, anti-mycobacterial, anti-mutagenicity,
hypoglycemic, antioxidants and neurobehavioral. antiprotozoal, anticarcinogenic,
antioxidant, anti-rheumatic and cardio-protective.
For instance, in India, China, Thailand among other countries, lemongrass is generally
used as food flavoring agent, ingredient in beverages because its ability to aid digestion,
boost body immunity and blood circulation.
Also, Okpo and Edeh (2022) has reported that in some parts of Nigeria, lemongrass is
use for the treatment of fever, convulsion in children, throat inflammations, stomach
upset, skin diseases, and ears/eyes infections. Particularly, in Isoko part of Nigeria,
lemongrass is used as ingredient in pepper soup, curries, and local drink. Lemongrass
extract have been reportedly used for treatment of diarrhea, skin infections and painful
irregular menstruation in females. In sub-regions of the world, lemongrass is reportedly
used to reduce blood pressure Devi, et al., (2011) affirmed the use of lemongrass extracts
for local treatment of gastrointestinal discomfort. In Guatemala, an extract of lemongrass
leaves have been reportedly applied for various purposes. For other application of
lemongrass especially in the manufacturing industries, it had been reportedly used as a
major ingredient of perfumery, cosmetics, soaps, tea, cleansing agent and confectionary
products.

2
It is also said that lemongrass oil can help accelerate the healing of scratches and cuts, but
Karma (2006) confirmed a burning sensation from direct application of lemongrass oil on
the skin. The essential oil of lemongrass is in high demand due to its commercial value.
Extracting and characterizing bioactive molecules from medicinal plant is very important
for drugs with high therapeutic value. (1-4).

Chemical constituents :
Major constituents :
Citral, cymbopogone, cymbopogonol.

Citral (2:1 mixture of E & Z isomer)

3
 Cymbopogone Cymbopogonol

Others constituents :
Linalool, citronellol, linalyl acetate, gerany lacetate, elemol, neral, geranial, myrcene,
borneol, camphor, ∆³-carene, fenchone, nerol, ocimene, α- pinene, terpinolene, α-
oxobisabolene, menthone, menthol, geraniol, limonene, terpineol, luteolin, luteolin 6-C-
gluvoside luteolin 7-O- neohesperoside, homoorientin, 2"-O- rhamnosyl-homoorientin,
chlorogenic, caffeic and p-coumaric acids, 2-undecanone, methylheptenone,
methylheptenol, octacosanol, doteiacontanol,triacontanol, hexacosanol, β-sitosterol,
fucosterol glucoside, fructose, sucrose.geranial. (5).

2. INTRODUCTION OF CITRAL
Citral contains a strong aroma, and is useful in treating gastrointestinal issues. Citral is
widely used for its scent in perfumes.
POTENTIAL BENEFITS :
 Antioxidants
 Antimicrobials
 Relaxant

4
 Anti-inflammatory
 Pain relief
 Gastrointestinal health
 Balance hormones
 Aids in vitamin a synthesis (6-7)

3. METHOD OF EXTRACTION
Soxhlet Extractor :
The original Soxhlet extractor was developed by Franz Von Soxhlet, a German
agricultural chemist, in the early part of this century. In Soxhlet extractor, within an
enclosed flask there is an inverted condenser pointing down into the flask from the top.
Just below that condenser will be suspended either what's called a soxhlet basket or a
recovery vessel depending on whether you are extracting or recovering solvent. The
condenser will have cold liquid circulating through it to keep the condenser cold. In the
bottom of the main flask, solvent is placed. To do an extraction, the powered plant
material is placed in the soxhlet basket which is a vessel with perforated sides and bottom
so that liquid can fall through it. When gentle heat is applied to the main flask, the
solvent begins to evaporate and the solvent vapors reach the cold condenser at the top of
the flask and begin to liquefy on the sides of the condenser (much the same way that a
cold glass of water becomes wet on the outside of itself on a hot day). The re-condensed
solvent on the sides of the condenser begins flowing down the sides of the condenser and
begins dripping off of drip points on the end of the condenser. This solvent drips into the
top of the soxhlet basket where it saturates the herb being extracted. The solvent flows
through the basket and out of the holes in the bottom of the basket carrying the extract
with it into the bottom of the flask. The extract laden solvent falling from the soxhlet
basket is dark in color and as it becomes clearer, one can know that the plant material is
leached out and the process is finished.
At this point one can do one of 3 things:
1. Stop the operation and pour the extract infused solvent out of the main flask.

5
2. Hook up the recovery vessel and remove the solvent from your extract which generally
leaves a paste behind.
3. Dump and squeeze out the spent plant material in the soxhlet basket, then start a fresh
basket of herb in the extractor using the same solvent which continually redistills and
extracts regardless of how much extract is infused into it in the bottom of the main flask.
The recovery vessel is simply a cup which is suspended below the condenser. As solvent
vapors re-condense and fall off the tip of the condenser, they fall into the cup and are thus
separated from the extract itself.
There are several advantages of Soxhlet extraction. The most important are that the
sample is repeatedly brought into contact with fresh portions of the solvent. This
procedure prevents the possibility of the solvent becoming saturated with extractable
material and enhances the removal of the analyte from the matrix. Moreover, the
temperature of the system is close to the boiling point of the solvent. This excess energy
in the form of heat helps to increase the extraction kinetics of the system. Soxhlet
extraction has several disadvantages, including it requires several hours or days to
perform the sample is diluted in large volumes of solvent, and due to the heating of the
distillation flask losses due to thermal degradation and volatilization have been observed.
(8).

Fig.3.1 Soxhlet extractor

6
HIGH PERFORMANCE THIN LAYER CHROMATO-
GRAPHY (HPTLC):

Principle and working

It is a powerful analytical method equally suitable for qualitative and quantitative
analytical tasks. Separation may result due to adsorption or partition or by both
phenomenon, depending upon the nature of adsorbents used on plates and solvents
system used for development. The mobile phase solvent flows through because of
capillary action. The components move according to their affinities towards the
adsorbent.

(fig. 4.1 HPTLC method development)

7
 The component with more affinity towards the stationary phase travels slower. The
component with lesser affinity towards the stationary phase travels faster. Thus the
components are separated on a chromatographic plate. Difference between TLC and
HPTLC .

INSTRUMENTATION :
 Steps involved
1. Selection of the HPTLC plates & sorbents
2. Sample preparation including prewashing & prechromatographic
derivatization
3. Application of sample
4. Pre-conditioning
5. Development techniques(separation)
6. Detection including post chromatographic derivatization
7. Quantitation
8. Documentation

SOLVENTS USED FOR PRE-WASHING:

 Methanol
 Chloroform: methanol ( 1:1 )
 Chloroform: Methanol: Ammonia (90:10:1 )
 Methylene chloride: Methanol ( 1:1 )
 Ammonia solution (1%)

SAMPLE PREPARATION:
 For normal chromatography: Solvent should be non-polar and
volatile.
 For reversed chromatography: Polar solvent is used for dissolving the
sample

8
 Sample and reference substances should be dissolved in the same
solvent to ensure comparable distribution at starting zones.

APPLICATION OF SAMPLE:
1. Sample application is the most critical step for obtaining
good resolution for quantification by HPTLC.
2. Some applicators used for spotting are:
a) Capillary tubes
b) Micro bulb pipettes
c) Micro syringes,
d)Automatic sample applicator.
The major criteria is that they shouldn’t damage the surface while
applying sample. (9-12).

METHODOLOGY :
Preparation of mobile phase
AR grade solvents toluene : Ethylacetate in the ratio of 9:10, v/v was used as mobile
phase. Properly mixed solvents were poured in TLC chamber and kept for saturation for
30 min at 25 ± 3ºC.
Preparation of standard solutions of citral:
Preparation of standard stock solutions
Accurately weighed 1ml of pure citral were transferred to 50 ml volumetric flasks
separately, dissolved and diluted up to mark with n-hexane.
Preparation of sample solution
Freshly cut the leaves of lemongrass and then stored it in dark storage for 5-6 days. Then,
transfer it into powder form. Take 25gm powder of lemongrass in thimble and 350ml of
n-hexane in flask. Once, the soxhlet process is complete, let it vaporized on room
temperature and then filtered through whatman filter paper No. 41 in 50 ml volumetric
flask. The residue was washed with n-hexane and final volume was adjusted up to mark
to obtain solution of citral.
9
Chromatographic conditions
The chromatographic separation was performed using Silica Gel G60F 254 HPTLC plates
previously pre-washed with methanol and activated at 110ºC for 5 min. Solution of both
drugs was applied to plate (10×10 cm) by means of a Linomat V semiautomatic spotter
equipped with 100 μl syringe and operated with settings of band length, 6mm; distance
from the plate edge, 10 mm; and distance from the bottom of the plate, 10 mm. The
distance between the spots was automatically adjusted by using win-CATS software. The
plate was developed in a twin trough TLC chamber previously saturated for 30 min with
mobile phase, toluene:ethyl aceatate (9:1 v/v) to 85 mm distance at temperature 25 ± 3ºC.
Densitometric scanning was performed using a Camag TLC scanner III in absorbance
mode at 273 nm with slit dimension of 6×0.45 mm and scanning speed of 1 mm/second.
The scanned data were analyzed using win-CATS software.
Linearity and range
Analysis was performed on 10×10 cm HPTLC Silica Gel G60 F 254 aluminium based plate.
A calibration curve was plotted over a concentration range of 22-120 ng/bond for citral.
For the calibration curve, accurately measured mixed standard working solution 1, 2, 3, 4,
5, 6 of standard citral and 7 of test citral was spotted on precoated TLC plate under
nitrogen stream using Linomat 5 semiautomatic spotter. The plate was dried in air,
developed and scanned as described. The calibration curves were constructed by plotting
peak areas versus concentration corresponding to each spot and the regression equation
was calculated. Each reading was an average of five determinations.
Limit of detection and quantification
The limit of detection (LOD) was determined by visual method on basis of trial and error.
The lowest concentration of linearity range which is estimated with accuracy and
precision is considered as limit of quantitation (LOQ).
Specificity
The specificity of the method was ascertained by analyzing standard drug and
sample.The spots of citral in samples were confirmed by comparing the R f and spectra of
the spots with that of standard. The peak purity of citral was assessed by comparing the
spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E)
positions of the spot.

10
Figure 5.1 Spectrum comparison of standard and sample spot of citral at 273 nm

Analysis of citral in extract.

From the above prepared sample solution, 2µl was applied to the HPTLC plate and
followed by development as described in section 7.1.1.4. The amount of citral present in
formulation was determined by fitting the area response into the regression equation of
citral.

y = 412585x - 5867.4 (where, y=8326.4)

8326.4 = 412585x - 5867.4
8326.4 + 5867.4 = 412585x
14193.8 = 412585x
x = 14193.8
412585
x = 0.034.
11
Optimization of chromatographic conditions
HPTLC method has been not reported so far for simultaneous estimation of citral in
Cymbopogon flexuosus. So the aim of the present study was to develop HPTLC method
for simultaneous analysis of citral in Cymbopogon flexuosus. Several mobile phases were
tried to accomplish better separation and good peak symmetry citral. Initially neat
solvents such as toluene, ethylacetate in different ratio were evaluated as mobile phase.
Using the mobile phase toluene: ethylacetate (9:1,v/v) and 10×10 cm HPTLC Silica Gel
G60F254, better separation was attained where R f values were found to be 0.37 for citral.
Both the spots appeared to be more compact with a more symmetrical peak shape when
TLC plates were pre-treated with n-hexane and activated at 110ºC for 5 min. Well
defined spots were obtained when the chamber was saturated with mobile phase for 30
min at room temperature. 273nm wavelength was selected for quantification of citral. A
representative densitogram and 3-D chromatogram showing peaks of citral in different
concentrations at 273 nm are shown in Figure 7.1.1 and Figure 7.1.2, respectively.

Figure 5.2 Densitogram of standard spot of citral with corresponding Rf at 273 nm by

HPTLC method

12
Figure 5.3 Densitogram of spot of citral with corresponding Rf at 273 nm by HPTLC
metshod

Figure 5.4 (3-D chromatogram of citral by HPTLC method)

13
Caliberation And Curve :
Linearity of the method was determined at seven concentration levels ranging from 22-
120ng/bond for citral. The calibration plot was linear over this concentration range of and
the drug obeyed Lambert Beer’s law in this range (Figure 7.1.3 and 7.1.4). The linearity
of the calibration curve was validated by the high value of correlation coefficient
(r2=0.9975) for citral over this concentration range.

Citral at 273nm
45000
40000 f(x) = 412585.285714286 x − 5867.42000000001
R² = 0.997533321992803
35000
30000
25000
Area

20000
15000
10000
5000
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
Conc in μL/μL

Figure 5.5 Calibration curve of citral at 273 nm by HPTLC method

Linear regression data of tamsulosin hydrochloride and finasteride by HPTLC

method

Concentratio Slope ± Intercept ± Correlation

Drug n range SDa, % CVb SDa, % CVb coefficient ± SDa,
(ng/spot) (nc=5) (nc=5) % CVb (nc=5)
Citral 200-1200 0.728 ± 0.006, 168.61 ± 11.56, 0.9971 ± 0.0012,
0.863 8.055 0.127
14
6.CONCLUSION :
A HPTLC method has been developed and validated for simultaneous estimation of
Citral in formulation. The results obtained from the analysis of formulation using
proposed method are highly reproducible, reliable. The method has linear response in the
range of 22-120 ng/band for citral. The Soxhlet extraction method is used to extract the
essential oil from cymbopogan fleuxosus.
The proposed HPTLC method is simple, accurate, precise, specific and sensitive and has
ability to separate the chemical constituents from essential oils. It can be used for routine
simultaneous analysis of citral from their Cymbopogon flexuosus. The proposed HPTLC
method is less expensive, simple, rapid and more flexible.

7.REFERENCES

1. Abd-El Fattah, S.M., Hassan, A.Y., Bayoum, H.M., and Eissa, H.A. (2010). The use of lemongrass
extracts as antimicrobial and food additive potential in yoghurt. J. Am. Sci. 6 (11), 582–594.

2. Abderrahmane, D., Lynda, B. and Brahim, Y.M. (2013). Effect of extraction method on chemical
composition, antioxidant and anti-inflammatory activities of essential oil from the leaves of Algerian
Tetraclinis Articulata (Vahl) Masters. Industrial Crops and Products, 44: 32-36.

3. Abera, A. (2020). Extraction and physicochemical analysis of essential oils in lemongrass leaves
grown in Arbaminch, Ethiopia. International Journal of Engineering Research & Technology (IJERT).
Vol. 9 Issue 10, 638-641.

4. Aftab, K., Ali, M. D., Aijaz, P., Beena, N., Gulzar, H. J., Sheikh, K., Sofia, Q. and Tahir Abbas, S.
(2011). Determination of different trace and essential element in lemon grass samples by x-ray
flouresence spectroscopy technique. Int Food Res J, 18:265-270.

5. V. Ramalingaswami Bhavan, Quality Standard Of Indian Medicinal Plants, Volume 10 Indian Council
Of Medical Research, Published By Neeraj Tandon, Parul Sharma, New Delhi 110029, India, Page No.
145.

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6. Okpo, S. O. and Edeh, I. (2022). Determination of the Metals Contents of Essential Oil from
Lemongrass. Engineering Advances, 2(2), /141-146. DOI: 10.26855/ea.2022.12.001.

7. Onawunmi, Grace O. (September 1989). "Evaluation of the antimicrobial activity of citral". Letters
in Applied Microbiology. 9 (3): 105–108.

8. Prof. N. Raaman, Phytochemical Techniques, New India Publishing Agency. Pitam Pura, New Delhi
– 110088, Page No. !2-14.

9 . Attimarad M, Ahmed MKK, Aldhubaib BE, Harsha S. High-performance thin layer chromatography:
A powerful analytical technique in pharmaceutical drug discovery Symposium – HPTLC, 2014.

10. Bairy PS. A comparison study of hplc and hptlc: principles, instrumentations and applications.
ASIO Journal of Analytical Chemistry (ASIO-JAC). 2015;1(1):20-28.

11. Boudesocque L, Dorat J, Pothier J, Gueiffier A, Enguehard Gueiffier C. High-performance thin-

layer chromatography-densitometry: A step further for quality control of cranberry extracts. Food
Chemistry. 2013; 136:866-871.

12. Bukanski BW, Degroodt JM, Beernaert H. A two dimensional high-performance thin-layer
chromatographic screening method for sulphonamides in animal tissues. Zeitschrift fu¨r Lebensmittel
Untersuchung und Forschung. 1988; 187:242.

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