phytochemical screening and gc-ms analysis of leaf …

www.wjpps.com Vol 8, Issue 7, 2019.

1061

Ashyam et al. World Journal of Pharmacy and Pharmaceutical Sciences

PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS OF LEAF

EXTRACT OF CINNAMOMUM VERUM

Prasanth S. S., Deepthi Bhaskar P. and Ashyam K.*

Department of Pharmaceutical Analysis, Alshifa College of Pharmacy, Perinthalmanna,

Kerala, India.

ABSTRACT

The presence of phytochemical constitutes has been reported from

species of the Lauraceae. This study was implement to actuate the

chemical components of Cinnamomum Verum leaves using Perkin

Elmer Gas Chromatography–Mass Spectrometry, our results of GC-

MS was done using the database of Agilent network GC of Uwin

Lifesciences, Kerala. Phytochemical screening of the entire herb of

Cinnamomum Verum revealed the presence of some bio-active

components. The phytochemical tests showed the presence of tannins,

flavanoids, terpenes, carbohydrates, saponins, and phenols in higher

amount in Chloroform, Ethylacetate, Ethanol & Water extracts of

Cinnamomum verum leaves. The GC-MS analysis has shown the

presence of different phytochemical compounds in the Chloroform, Ethylacetate, Ethanol &

Water extracts of Cinnamomum verum A total of 118 compounds were identified by

Chloroform, Ethylacetate, Ethanol & Water extract composition. This study result will make

a way for the production of herbal medicines for various ailments by using Cinnamomum

Verum leaves.

KEYWORDS: GC-MS analysis, Cinnamomum Verum, phytochemical screening, whole

plant chloroform, ethylacetate, ethanol and water extracts.

INTRODUCTION

India has a rich culture of medicinal herbs and spices, which includes about more than 2000

species and has a vast geographical area with high potential abilities for Ayurvedic, Unani,

Siddha traditional medicines but only very few have been studied chemically and

pharmacologically for their potential medicinal value.[1]

The plants are indispensable to man

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.421

Volume 8, Issue 7, 1061-1074 Research Article ISSN 2278 – 4357

Article Received on

08 May 2019,

Revised on 28 May 2019,

Accepted on 18 June 2019

DOI: 10.20959/wjpps20197-14159

*Corresponding Author

Ashyam K.

Department of

Pharmaceutical Analysis,

Alshifa College of

Pharmacy, Perinthalmanna,

Kerala, India.


1062


for his life. Nature has provided a complete storehouse of remedies to cure all ailments of

mankind. The knowledge of drugs has accumulated over thousands of years as a result of

mans inquisitive nature, so that today we possess many effective means of ensuring health

care. The human being appeared to be affiliated with more diseases than any other species.

There can be little doubt that he very early sought to alleviate sufferings from injury and

diseases by taking advantage on the plants growing around him. In the past almost all the

medicines used were from plants, being man‟s only chemist for ages. The history of herbal

medicine is as that as old as human civilization.[2]

Cinnamomum verum belongs to the family Lauraceae and possesses significant antiallergic,

antiulcerogenic, antipyretic and anaesthetic activities. The bark yields an essential oil

containing cinnamaldehyde and eugenol. Several biological activities such as peripheral

vasodilatory, antitumor, antifungal, cytotoxic and antimutagenic activities has been attributed

to cinnamaldehyde.[3]

Plant produces these chemicals to protect themselves but the research

have shown that they have the capacity to treat human diseases in an effective way.[4]

There

are thousands of phytochemicals, each have their pharmacological properties of their own.[5]

The plant possesses various medicinal properties; the aim of this study was to identify the

phytocompounds in the chloroform, ethylacetate, ethanol and water leaf extract of

Cinnamomum verum by qualitative screening of phytochemicals and to identify each specific

compound with their concentrations by Gas Chromatography – Mass Spectrum (GCMS)

analysis.[6]

MATERIALS AND METHODS

COLLECTION OF THE PLANT LEAVES

Places within the Malappuram district were selected for the collection of the leaves of

Cinnamomum verum. [Figure 1] Collection was done during the month of May-June. Healthy

and mature plants were selected for the collection of plant part. Then, the leaves were

thoroughly washed with water to remove the dirts, if any present. Identified and authenticated

by Mr.A.K Pradeep, from Calicut University Herbarium which was certified that the given

specimen no-88430 belonged to Cinnamomum verum Presl [Lauraceae]. A voucher specimen

was deposited in the Dept of Pharmacognosy of Al Shifa College of Pharmacy.


1063


Figure 1: Cinnamomum verum.

PREPARATION OF EXTRACT

Petroleum Ether Extraction

The air-dried and coarsely powdered leaves were successively extracted with petroleum ether

in the soxhlet apparatus. The soxhlation process was carried out until the solvent found to be

colourless. Then the solvent was filtered and concentrated under reduced pressure and the

percentage yield was calculated.

Chloroform Extraction

The marc left after the extraction with petroleum ether was air-dried and it was again charged

in the soxhlet apparatus with chloroform. The soxhlation process was carried out until the

solvent found to be colourless. Then the solvent was filtered and concentrated under reduced

pressure and the percentage yield was calculated.

Ethyl Acetate Extraction

The marc left after the extraction with chloroform was air-dried and it was again charged in

the soxhlet apparatus with ethyl acetate. The soxhlation process was carried out until the

solvent found to be colourless. Then the solvent was filtered and concentrated under reduced

pressure and the percentage yield was calculated.


1064


Ethanol Extraction

The marc left after the extraction with ethyl acetate was air-dried and it was again charged in

the soxhlet apparatus with ethanol. The soxhlation process was carried out until the solvent

found to be colourless. Then the solvent was filtered and concentrated under reduced pressure

and the percentage yield was calculated.

Water Extraction

The marc left after the extraction with ethanol was air-dried and it was again charged in the

soxhlet apparatus with distilled water. (Chloroform is added in the ratio 1:99 as preservative).

The soxhlation process was carried out until the solvent found to be colourless.

Then the solvent was filtered and concentrated under reduced pressure.

PRELIMINARY PHYTOCHEMICAL SCREENING

The Phytochemical screening of Cinnamomum verum Leaf revealed that the petroleum ether

extract contains tannins. Chloroform & Ethyl acetate extract contain flavanoids. Water

extract contain saponins, terpenes[7]

, flavanoids. Whereas, the ethanolic extract contains

carbohydrates, saponins[8]

, flavanoids, terpenes, and phenols in higher amount.

Table 1: Preliminary phytochemical evaluation of chloroform, ethylacetate, ethanol and

water extracts of Cinnamomum verum.

SL.NO PHYTOCHEMICALS PEE CHE EAE ETL WTR

1 Alkaloids

2 Glycosides

3 Carbohydrates +

4 Saponins ++ ++

5 Flavones and Flavonoids + + ++ +

6 Terpenes ++ ++

7 Tannins + +

8 Phenols ++ +

9 Proteins and aminoacids

PEE-Petroleum ether extract, CHE-Chloroformextract, EAE-Ethylacetate, extract, ETL-

Ethanolic extract, WTE –Water extract.

(++) indicate active constituents in high amount, (+) absence of active indicate active

constituents in lesser amount, (-) indicates the constituents.


1065


GC-MS (Gas Chromatography-Mass Spectrometry) analysis

GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a

AOC-20i autosampler and gas chromatograph interfaced to a mass spectrometer (GC-MS)

instrument employing the following conditions: column HP-5 fused silica capillary

column(30 ×320µm×0.25µm composed of 5% Phenyl Methyl Siloxane), operating in

electron impact mode at 70 eV; Nitrogen was used as carrier gas at a constant flow of

2.5ml/min and an injection volume of 10µL was employed (split ratio of 25:1) injector

temperature 280°C; ion-source temperature 280°C. The oven temperature was programmed

from 110°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C/min, then

5°C/min to 250°C/min, ending with a 13 min isothermal at 250°C. Mass spectra were taken at

70 eV; a scan interval of 0.5 s and fragments from 40 to 550 Da.

Interpretation on mass spectrum of GC-MS was done using the database of Agilent network

GC of Uwin Lifesciences, Kerala. The results are tabulated in the tables(6,7,8,9) and

figures(9,10,11,12).

RESULTS AND DISCUSSION

After the successful conventional hot soxhlet extraction of the whole part of the plant in

investigation, the preliminary phytochemical study revealed that the petroleum ether extract

contains tannins. Chloroform & Ethyl acetate extract contain flavanoids. Water extract

contain saponins, terpenes, flavanoids. Whereas, the ethanolic extract contains carbohydrates,

saponins, flavanoids, terpenes, and phenols in higher amount.


1066


Table 2: Compounds identified in the chloroform extract of Cinnamomum verum In

GC-MS.

SL NO COMPOUND NAME

RETENTION

TIME IN

MIN

PERCENTAGE

COMPOSITION OF

THEVOLATILE

MATTER

(W/W)

PERCENTAGE

PROBABILITY

1 TERPINOLENE 1.25 1.25% 92%

2 HOTRIENOL 1.55 0.25% 95%

3 LINAYL ACETATE 2.03 3.12% 96%

4 BORNEOL 2.45 3.25% 95%

5 ALPHA-CITRAL 3.25 0.45% 99%

6 L-CARVONE 3.56 1.25% 93%

7 NEROL 3.19 1.62% 85%

8 HEXANAL 4.25 2.51% 86%

9 2-HEXANAL 5.02 1.02% 98%

10 2,4 HEPTADIENAL 6.40 2.15% 96%

11 HEXENYL ACETATE 7.15 3.05% 95%

12 HEXANOL 7.58 4.52% 98%

13 HYDROXYBUTNONE 7.29 0.52% 99%

14 METHYL HEPTENONE 8.25 0.65% 96%

15 HEPTADIENE 8.55 2.35% 95%

16 IONONE 10.24 1.25% 98%

17 JASMONE 12.35 0.21% 96%

18 OCTANONE 13.26 0.95% 95%

19 ALPHA-PINENE 13.55 1.25% 97%

20 SABINENE 14.02 0.25% 96%

21 BETA-PINENE 15.02 2.35% 96%

22 MYRCENE 15.32 7.25% 92%

23 OCTANAL 15.55 0.54% 99%

24 CARENE 16.24 2.01% 95%

25 LIMONENE 16.54 0.32% 98%

26 LINALOOL OXIDE 16.59 2.14% 94%

27 TERPIN-4 OL 17.22 1.25% 99%

28 TERPENEOL 17.35 1.24% 98%

29 GERANIOL 18.25 0.21% 95%

30 METHYL ANTHRANYLATE 18.55 0.30% 96%

31 ELEMENE 20.35 0.25% 98%

32 NERYL ACETATE 35.40 0.45% 95%

33 GERANYL ACETATE 36.20 5.01% 98%

34 CARYOPHELLENE 38.30 0.24% 96%

35 HUMULENE 39.41 0.32% 99%

36 CUBEBENE 40.25 3.25% 98%

37 CADINENE 41.35 3.01% 95%

38 VALENCENE 42.25 0.23% 98%

39 AMORPHENE 42.35 0.11% 97%


1067


GCMS Analysis shows that about 39 volatile constituents were present in the chloroform

extract of Cinnamomum verum leaves. The most abundant peak was found to be myrcene,

geranyl acetate, hexanal per reference and corresponds to retention time of 15.32,36.20,4.52

minutes respectively.

Table 3: Compounds identified in the Ethyl acetate extract of Cinnamomum verum In

GC-MS.

SL NO COMPOUND NAME

RETENTION

TIME IN

MIN

PERCENTAGE

COMPOSITION OF

THEVOLATILE

MATTER

(w/w)

PERCENTAGE

PROBABILITY

1 EUDESMOL 3.25 0.25% 98%

2 PENTENOL 3.54 1.24% 95%

3 HEXENOL 4.02 1.35% 96%

4 BENZALDEHYDE 5.12 2.52% 99%

5 METHYL BENZOATE 5.32 3.25% 95%

6 PHENYL ACETALDEHYDE 5.45 3.65% 96%

7 PINOCARVEOL 8.25 4.25% 99%

8 HEPTADIENAL 8.54 4.65% 95%


10 CUMINYL ALCOHOL 9.45 1.02% 99%

11 GERANIOL 12.02 3.02% 95%

12 NEROL 12.42 4.25% 99%

13 LEDOL 12.45 3.02% 96%

14 CITRAL 14.54 0.21% 98%

15 TERPENIOL 15.24 0.54% 94%

16 TERPENE-1 OL 16.25 9.25% 97%

17 BORNYL ACETATE 17.45 2.04% 99%

18 OCIMENE OXIDE 32.02 6.02% 98%

19 TERPINOLENE 32.21 6.25% 96%

20 BISABOOL 33.54 5.26% 95%

21 METHYL EUGENOL 36.25 5.24% 94%

22 METHYL CINNAMATE 37.25 1.25% 98%

23 PHENETHYL ALCOHOL 38.21 11.02% 99%

24 ANISALDEHYDE 38.35 3.04% 95%

25 SAFROLE 38.39 7.05% 96%

26 METHOXYCINNEMAL DEHYDE 40.02 0.54% 95%

27 ALPHA-CRESOL 41.23 8.02% 95%

28 CITRONELLAL 41.25 2.30% 96%

29 PIPERITOL 45.21 4.02% 98%

30 MYRTENOL 45.35 2.30% 95%

The GC-MS Analysis reports that about 30 volatile constituents are present in the

ethylacetate extract of Cinnamomum verum leaves. The most abundant peak was found to be


1068


phenethyl alcohol, terpene-1-ol, and alpha-cresol at corresponding retention time 38.21,

16.25, 41.23 minutes respectively etc.

Table 4: Compounds identified in the Ethanolic extract of Cinnamomum verum In GC-

MS.

SL NO COMPOUND NAME

RETENTION

TIME IN

MIN

PERCENTAGE

COMPOSITION OF

THEVOLATILE

MATTER

(w/w)

PERCENTAGE

PROBABILITY

1 CARVONE 1.23 2.52% 98%

2 BUTANOIC ACID 2.32 1.02% 95%

3 OCTANOIC ACID 3.26 2.35% 96%

4 DECANOIC ACID 3.55 4.25% 98%

5 ETHYL CINNAMAE 4.02 1.25% 99%

6 METHYL CINNAME 4.05 3.02% 97%

7 METHOXY CINNEMALDEHYDE 4.55 5.36% 96%

8 3-METHYL PROPANOL 5.26 2.01% 98%

9 2-METHYL PROPANOL 10.24 2.03% 99%

10 5-METHYLPROPANOL 11.23 2.45% 95%

11 CRESOLPHENETHYL ALCOHOL 15.02 1.62% 92%

12 BENZYL ALCOHOL 15.32 1.98% 89%

13 ANETHOL 16.45 2.89% 89%

14 METHYL ACETOPHENONE 25.44 1.85% 88%

15 CUMINALDEHYDE 26.02 1.74% 95%

16 SAFROLE 27.31 3.25% 96%

17 BORNEOL 28.26 2.01% 98%

18 GERANIOL 29.22 3.05% 99%


20 HYDROXY-CINEOLE 31.24 1.62% 95%

21 CUMINYL ALCOHOL 31.45 5.25% 99%

22 CARVEOL 41.26 2.65% 98%

23 NEROL 41.35 3.02% 96%

24 TERPENEOL 43.22 0.21% 95%

25 LINALYL ACETATE 44.44 0.52% 96%

26 BORNEOL 45.01 0.65% 98%

27 VERBENONE 45.44 2.35% 95%

The GC-MS Analysis reports that about 27 volatile constituents are present in the ethanolic

extract of Cinnamomum verum leaves. The most abundant peak was found to be

methoxycinnamaldehyde, cuminyl alcohol, Geranyl acetate at a retention time of 4.55,31.45

and 30.25 minutes.


1069


Table 5: Compounds identified in the Water extract of Cinnamomum verum In GC-MS.

SL NO COMPOUND NAME

RETENTION

TIME IN

MIN

PERCENTAGE

COMPOSITION OF

THEVOLATILE

MATTER

(w/w)

PERCENTAGE

PROBABILITY

1 MENTHOL 2.25 1.29% 98%

2 LAVANDULOL 3.56 0.25% 95%

3 TERPENYL FORMATE 3.58 0.45% 96%

4 CITRONELLAL 4.02 0.23% 99%

5 MYRTENOL 4.35 0.51% 98%

6 EUDESMOL 9.56 0.24% 98%

7 CARYOPHELLENE 10.12 1.23% 95%

8 CADINOL 11.23 2.52% 95%

9 SPATHULENOL 12.02 6.21% 96%

10 ELEMOL 12.54 0.45% 94%

11 BISABOOL 24.55 0.62% 98%

12 CYCLO-HEXANOL 25.02 0.52% 95%

13 OCTANOL 25.35 0.33% 95%

14 1,8 CINEOLE 27.48 1.25% 95%

15 HOTRIENOL 32.12 2.45% 96%

16 GERANIOL 35.25 2.03% 98%

17 ANETHOL 38.45 2.21% 99%

18 CRESOL 42.56 3.02% 95%

19 EUGENOL 44.02 2.30% 99%

20 METHYL CINNAMATE 44.35 2.03% 94%

21 DILLAIOLE 44.55 1.02% 95%

22 DIMETHYLPYRAZINE 46.58 2.30% 98%

The GC-MS Analysis reports that about 22 volatile constituents are present in water extract

of Cinnamomum verum leaves. Among them, the most abundant peak was found to be

spathulenol, cardinal and hotrienol at a retention time of 12.02,2.52,2.45 minutes.


1070


Figure 2: GCMS Data report of Chloroform extract of C.verum leaves.


1071


Figure 3: GCMS Data report of Ethylacetate extract of C.verum leaves.


1072


Figure 4: GCMS Data report of Ethanolic extract of C.verum leaves.


1073


Figure 5: GCMS Data report of Water extract of Cinnamomum verum leaves.

CONCLUSION

The phytochemicals present in the extracts were identified by qualitative phytochemical

screening, which revealed the presence of fats in petroleum ether extract; tannins, terpenes in

chloroform extract; terpenes, flavanoids in ethyl acetate extract; saponin, flavanoids in water

extract; and terpenes, phenolics, tannins and flavanoids in the alcoholic extract of

Cinnamomum verum leaves respectively. Various volatile constituents of the different

extracts were obtained from GC-MS Analysis. Ethanolic extract of Cinnamomum verum leaf

showed terpenes, tannins, flavonoid contents in higher amount.

REFERENCES

1. Benzie.F.F, Galor.S 2011; Herbal Medicine: Biomolecular and Clinical Aspects; Page no:

1-2.

2. C.K. Kokate, A. Purohit, S.B. Gokhale., (2002) Pharmacognosy 13th

edition, Nirali

Prakashan, Page. 1-14.


1074


3. Bullerman, Liew, & Seier, 1977; Koh et al., 1998; Kwon et al., 1998; Shaughnessy,

Setzer, & DeMarini, 2001.

4. Dutta A, Ghosh S. Chemical examination of Daemia extensa. J Am Pharm Assoc, 1947a;

36: 250-252.

5. Anjaneyulu ASN, Raju DVSN, Srinivasa Rao S. Chemical evaluation of Pergularia

daemia. Indian J Chem, 1998; 37: 318-320.

6. Dhar ML, Dhar MM, Dhawan BN, Mehrota BN, Ray C. Screening of Indian plants for

biological activity. Indian J Exp Biol, 1968; 11: 43-54.

7. Kumar GS, Jayaveera KN, Kumar CK, Sanjay UP, Swamy BM, Kumar DV.

Antimicrobial effects of Indian medicinal plantsagainst acne-inducing bacteria. Trop. J.

Pharm. Res., 2007; 6: 71723.

8. Parekh J, Chanda SV. In vitro antimicrobial activity and phytochemical analysis of some

Indian medicinal plants. Turk. J. Biol., 2007; 31: 53-8.

phytochemical screening and gc-ms analysis of leaf …

Documents