gc-ms analysis of bioactive compounds of … · pure phyto-pharmaceuticals need to be studied...

www.wjpr.net Vol 7, Issue 1, 2018.

1045

Mary and Giri. World Journal of Pharmaceutical Research

GC-MS ANALYSIS OF BIOACTIVE COMPOUNDS OF

ACHYRANTHES ASPERA

A. Pauline Fatima Mary1*

and Dr. R. Sagaya Giri1

*1

Department of Botany, K. N. Govt. Arts for Women (Autonomous),

Thanjavur-613 007, Tamilnadu, India.

ABSTRACT

Achyranthes aspera L. (Amaranthaceae) is an important medicinal

herb found as a weed throughout India. It has long been used in

different systems of medicine in the treatment of cancer, leprosy,

asthma, wound, insect and snake bite, dandruff, hepatitis, renal

disorders, dermatological disorders, gynecological disorders, malaria,

fever, cough, diabetes, dysentery, toothache etc.,. The powdered leaves

were subjected to phytochemical screening and were found to contain

alkaloids, flavonoids, Phenols, Saponins, Tannins, Phytosterols and

Terpenoids. To determine the bioactive components of ethanolic leaf

extracts of A. aspera by GC-MS analysis were carried out. Twenty five

compounds were identified. Some important compounds are; 9-Octadecenamide, (Z)

(21.15%), Squalene (19.04%), Phytol (7.06%), Phenol, 4,6-di (1,1dimethylethyl)

2-methyl- (6.50%), à–Amyrin (4.33%), Cyclopropane, nonyl- (3.37%), Dibutyl phthalate

(3.19%) etc.,. Some of the identified compounds have been reported to possess various

biological activities such as Antimicrobial, Antioxidant, Antiseptic, Pesticide, Fungicide,

Diuretic, Anti-inflammatory and Anticancer.

KEYWORDS: Achyranthes aspera, Antifungal activity, Bioactive compoounds,

Phytochemical.

INTRODUCTION

Nature has provided a complete store-house of remedies to cure all ailments of mankind. For

a long period of time, plants have been a valuable source of natural products for maintaining

human health, especially in the last decade, with more intensive studies for natural therapies.

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 7, Issue 1, 1045-1056. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. A. Pauline Fatima Mary

Department of Botany, K. N.

Govt. Arts for Women

(Autonomous), Thanjavur-

613 007. Tamilnadu, India.

Article Received on

13 November 2017,

Revised on 3 Dec. 2017,

Accepted on 24 Dec. 2017

DOI: 10.20959/wjpr20181-10540

8533


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A number of plants have been documented for their medicinal potential, which is in use by

the traditional healers, herbal folklorists and in Indian system of medicine namely Siddha,

Ayurveda and Unani.[1]

The use of herbal medicine has become increasingly worldwide

popular and medicinal plants are believed to be an important source of new chemical

substances with potential therapeutic effects. WHO has considered phytotherapy in its health

programme; because these drugs are safe, cost effective and most importantly people have

faith in them. The demand for crude drugs has undergone a considerable change in recent

years due to aggressive marketing of the crude drugs. Standardized extracts from them or

pure phyto-pharmaceuticals need to be studied extensively for their quality, purity, potency,

safety and efficacy.[2]

The novel molecules from plant sources have been instrumental in development of

structurally modified compounds, which assist a lot in the development of modern

therapeutic system.[3]

Phytochemicals are responsible for medicinal activity of plants and

these biochemicals are naturally ccurring in the plants that have defence mechanism and

protect from various diseases[4]

The phytochemical are very important in medicine and

constitute most of the valuable drugs.[5]

This biochemicals are often referred to as secondary

metabolites which is useful to traditional medicine system are identified by GC-MS

technique.[6]

In recent years Gas Chromatography – Mass Spectrum (GC-MS) studies have

been increasingly applied for the analysis of medicinal plants as this technique has proved to

be a valuable method for the analysis of essential oil, alcohols, acids, esters, alkaloids,

steroids, amino and nitro compounds.[7]

Achyranthes aspera L. (Amaranthaceae) is an important medicinal herb found as a weed

throughout India, tropical Asia and other parts of the world. It has long been used in different

systems of medicine in the treatment of cancer, leprosy, asthma, fistula, piles, arthritis,

wound, insect and snake bite, diabetes, malaria, pneumonia fever, dandruff, hepatitis, renal

disorders, dermatological disorders, gynecological disorders, malaria, fever, cough, diabetes,

dysentery, toothache etc. The aim of this study was to investigate the Achyranthes aspera

leaves for its phytochemicals by qualitative test and to study the phytochemical components

by GCMS.


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MATERIAL AND METHODS

Sample Authentication

Achyranthes aspera was identified and authenticated by Botanist, Dr. Soosai Raj, M.Sc.,

Ph.D., Department of Botany, St. Joseph’s college, Trichy, Tamil Nadu, India. A Voucher

specimen has been deposited at the Rapinat Herbarium, St. Joseph’s College, Tiruchirappalli,

Tamil Nadu, India. (Figure-1).

Botanical Description

Achyranthes aspera Linn

Systematic position

Class : Dicotyledons

Sub Class : Monochlamydeae

Serious : Curvembryeae

Family : Amaranthaceae

Genus : Achyranthes

Species : aspera

Achyranthes aspera Plants are small, much branched, monoecious perennial sub shrub up to

0.8–1×0.8 m. Rootstock stout, woody. Stems somewhat succulent at first, ribbed, becoming

basally woody with age, densely covered in velutinous, appressed hairs. Leaves opposite,

densely clustered toward branch tips 40–50×25–30 mm, spreading to decurved, mostly

broadly ovate, ovate-orbicular or elliptic; apex blunt to abruptly sub acute, sometimes very

shortly apiculate; base attenuate; lamina somewhat fleshy, purple-grey, veins often purple,

abaxial and adaxial surfaces silkycanescent, margins crenulate to crenate. Petioles 5–10mm

long, pink, fleshy, velutinous, basal abscission zone present. Inflorescence a terminal erect

spike, 150– 200mm long; peduncle 15mm long, fleshy, white villous; spike rachis fleshy,

white-villous to purple villous; flowers bisexual, retrorse, sessile, 180–200 per spike, these

spaced initially at 10-mm intervals along rachis, diminishing rapidly to <1-mm intervals

toward inflorescence apex.

Collection of Plant materials

Fresh leaves of Achyranthes aspera were collected (Figure-2) from vaduganputhupatti,

Thanjavur Dt., Tamilnadu. The leaves were washed in clean water and air dried in room

temperature. The dried plant materials were milled to a fine powder using grinder and stored

in the dark at room temperature in airtight containers.


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Preparation of plant extracts

50 g of dried powder of plant was extracted with ethanol by continuous hot percolation, using

soxhlet apparatus (Figure-3) and concentrated up to 50 ml in desiccator under reduced

pressure. The concentrated extract were lyophilized and used for further studies.

Qualitative analysis of phytochemical compounds

Phytochemical analysis of the all plant extracts was undertaken using standard qualitative

methods as described by various authors. The plant extracts were screened for the presence of

biologically active compounds such as alkaloids, flavonoids, carbohydrates, phytosterols,

proteins, phenolics, tannins and saponins.

Quantitative analysis of Phytochemical compounds by using GCMS

The ethanolic extracts of the selected plant samples were subjected to GC-MS studies for

identification of phytochemical compounds. The GC-MS analysis was carried out using a

Clarus 500 Perkin- Elmer (Auto System XL) Gas Chromatograph equipped and coupled to a

mass detector Turbo mass gold – Perking Elmer Turbomas 5.2 spectrometer with an Elite-1

(100% Dimethyl ply siloxane), 300 m x 0.25 mm x 1 μm df capillary column. The instrument

was set to an initial temperature of 110°C and maintained at this temperature for 2 min. At

the end of this period, the oven temperature was raised upto 280°C, at the rate of an increase

of 5°C/min and maintained for 9 min. Injection port temperature was ensured as 250°C and

Helium flow rate as 1 ml/min. The ionization voltage was 70 eV. The samples were injected

in split mode as 10:1. Mass Spectral scan range was set at 45-450 (mhz). The chemical

constituents were identified by GC-MS. The fragmentation patterns of mass spectra were

compared with those stored in the spectrometer database using National Institute of Standards

and Technology Mass Spectral database (NIST-MS). The percentage of each component was

calculated from relative peak area of each component in the chromatogram.

Identification of Compounds

Interpretation of mass spectrum of GC-MS was conducted using the database of National

Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum

of the known component was compared with the spectrum of the unknown components

stored in the NIST library. The Compound name, Retention time, Molecular formula and

Nature of the compounds and Area % of the test materials were ascertained.


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RESULTS AND DISCUSSION

Ethanolic extracts of leaves of A. aspera showed the presence of alkaloids, flavonoids,

carbohydrates, proteins, phenols, saponins and terpenoids whereas phytosterol and tannins

were absent (Table-1and Figure-4). Priya[8]

screened the phytochemical of A. aspera,

methanol extract showed the presence of carbohydrates, phenolic compounds, oil and fats,

saponins, flavonoids, alkaloids and tannins, whereas, aqueous extract contained phenolic

compounds, saponins, flavonoids and tannins as major phytochemical groups.

Danial[9]

reported the presence of polysaccharides, ecdysterone (harmone), achyranthine,

betaine (Alkaliods), vanillic acid, syringic acid, p-coumaric acid (phenolic acids), saponin A,

saponin B (saponins), protein and carbohydrates in A. aspera. Presence of phenolic

compounds in the plant suggests the potential use of A. aspera as a source of antioxidant

compounds[10]

.

The GC-MS spectral studies revealed the presence of 25 compounds from the leaf ethanol

extract of A. aspera. The GCMS chromatogram of ethanolic leaf extract of Achyranthes

aspera are shown in the figure-5. The compound name, retention time (RT), molecular

formula, compound nature and area % were presented in Table 2. The major compounds were

cyclopropane, nonyl- (3.37) 1-Hexadecanol (3.11%), Phenol, 4, 6-di (1,1-dimethylethyl)-2-

methyl- (6.5%), Squalene (19.04%), Dibutyl phthalate (3.19%), Phytol (7.06%), 9-

Octadecenamide, (Z)- (21.15%) and á-Amyrin (4.33%). The details of the identified

phytoconstituents and its therapeutic activity were given in the Table 3. The structure of the

identified compounds among were given in the Figure-6. Saurabh Srivastav et al., (2011)11

revealed that wide numbers of phytochemical constituents have been isolated from

Achyranthes aspera which possesses activities like antiperiodic, diuretic, purgative, laxative,

antiasthmatic, hepatoprotective, anti-allergic and various other important medicinal

properties.


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Figure 1: Sample Authentication.

Figure-2: Collection of Achyranthes aspera leaves.

Figure-3: Preparation of Plant Extract.


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Figure-4: Preliminary phytochemical analysis.

Table 1: Analysis the phytochemicals of Achyranthes aspera by qualitative test.

Figure 5: GC-MS Chromatogram of Ethanolic leaf extracts of Achyranthes aspera.

Phytochemicals Result

Alkaloids +

Flavonoids +

Carbohydrates +

Protein +

Phenols +

Saponins +

Tannins -

Phytosterols -

Terpenoids +


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Table 2: Components detected in Ethanolic leaf extracts of Achyranthes aspera.

S. No Compound Name RT Molecular

Formula Compound Nature

Area

%

1 Cyclotetrasiloxane, octamethyl- 5.99 C8H24O4Si4 OrganoSilicone

compound 2.15

2 3-Carene 6.29 C10H16 Monoterpene 1.14

3 Cyclopropane, nonyl- 9.06 C12H24 Gaseous

Hydrocarbons 3.37

4 1-Hexadecanol 11.86 C16H34O Terpene alcohol 3.11

5 Caryophyllene 12.4 C15H24 Sesquiterpene 0.8

6 Precocene I 12.93 C12H14O2 Essential oils 1.99

7 Cycloheptasiloxane,

tetradecamethyl- 13.17 C14H42O7Si7

Organo Silicone

compound 0.83

8 1-Hexadecanol 14.37 C16H34O Terpene alcohol 2.42

9 Phenol, 4,6-di (1,1dimethylethyl)

2-methyl 15.28 C15H24O Phenolic compound 6.5

10 Trichloroacetic acid, tetradecyl

ester 16.61 C16H29Cl3O2 Acetic acid compound 0.97

11 3-Eicosyne 17.08 C20H38 Organic compound 2.15

12 Squalene 17.94 C30H50 Triterpene 19.0

4

13 Dibutyl phthalate 18.42 C16H22O4 Plastilizer compound 3.19

14 Hexadecanoic acid, ethyl ester 18.63 C18H36O2 Palmitic acid eater 1.72

15 Phytol 19.78 C20H40O Diterpene 7.06

16 9,12,15 Octadecatrienoic acid,

ethyl ester,(Z,Z,Z) 20.28 C20H34O2 Linolenic acid ester 1.59

17 9-Octadecenamide,(Z) 22.07 C18H35NO Amide 21.1

5

18 Hexasiloxane,1,1,3,3,5,5,7,7,9,9,

11,11dodecamethyl 22.86 C12H38O5Si6

Organo Silicone

compound 0.55

19 Eicosane, 7-hexyl 23.09 C26H54 Aliphatic hydro

compounds 1.28

20 Diisooctyl phthalate 23.7 C24H38O4 Plastilizer compound 1.53

21 Docosanoic acid, 1,2,3-

propanetriyl ester 24.12 C69H134O6 Fatty acid 2.25

22 à –Amyrin 24.79 C30H50O Triterpene 4.33

23 9-Hexacosene 25.13 C26H52 Hydrocarbon 2.2

24 Heptacosane, 1-chloro 25.44 C27H55Cl Hydrocarbon 1.77

25

Glycine,N-[(3à,5á,7à,12à) 24-

oxo-3,7,12-tris [(trimethylsilyl

,methyl ester

25.95 C36H69NO6

Si3 Organic compound 0.77


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Table 3: Bioactive components and their activity in Ethanolic leaf extracts of A. aspera

by GCMS study.

S. No Compound Name ***Activity

1 Cyclotetrasiloxane, octamethyl-

Antimicrobial, Antiseptic, Hair

Conditioning Agent, Skin- Conditioning

Agent-Emollient; Solvent.

2 3-Carene Allergenic, Fungicide, Irritant,

Pesticide.

3 Cyclopropane, nonyl- Anethetic agent.

4 1-Hexadecanol Antimicrobial , Anti-inflammatory.

5 Caryophyllene

Anti-inflammatory, Antibiotic,

Antooxidant, Anticarcinogenic agent,

local anaesthetic activities.

6 Precocene I Antifeedant, juvabional, Pesticide.

7 Cycloheptasiloxane, tetradecamethyl-




8 1-Hexadecanol Antimicrobial, Anti-inflammatory.

9 Phenol, 4,6-di (1,1dimethylethyl) 2-

methyl

Antimicobial, Anesthtic, Antooxidant,

Antiseptic, Cancer preventive, Pesicide,

Fungicide.

10 Trichloroacetic acid, tetradecyl ester Precipatation agent, An Ingredient

agent, Herbicide.

11 3-Eicosyne Antimicrobial.

12 Squalene Antimicrobial, Antioxidant, Antitumor,

Cancer-Preventive, Pesticide.

13 Dibutyl phthalate Antimicrobial, Solvent, Plastilixer,

Pesticide, Repellent.

14 Hexadecanoic acid, ethyl ester Antioxidant, Nematicide, Pesicide.

15 Phytol Antimicrobial, Anticancer, Diuretic,

Anti -inflammatory.

16 9,12,15 Octadecatrienoic acid, ethyl

ester,(Z,Z,Z)

Analgesic, Antipyretic, Anticonvulsant,

Antiseptic.

17 9-Octadecenamide,(Z) Antimicrobial.

18 Hexasiloxane,1,1,3,3,5,5,7,7,9,9,11,11do

decamethyl




19 Eicosane, 7-hexyl No Activity reported.

20 Diisooctyl phthalate Antimicrobial, Solvent, Plastilixer,

Pesticide, Repellent.

21 Docosanoic acid, 1,2,3-propanetriyl ester Antimicrobial.

22 à-Amyrin Antimicrobial, Antitumor, antiviral,

Anti- inflammatory.

23 9-Hexacosene Antiinflammatory

24 Heptacosane, 1-chloro Anticancer drug

25 Glycine,N-[(3à,5á,7à,12à) 24-oxo-3,7,12-

tris [(trimethylsilyl ,methyl ester Antibacterial.

***Source: Dr. Duke’s phytochemical and ethno botanical databases (online database).


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1. 3-Carene 2. Cycloheptasiloxane, tetradecamethyl

3.1-Hexadecanol 4. Phenol,4,6-di(1,1-dimethylethymethyl

5. Squalene 6. Dibutyl phthalate

7. Phytol 8. 9-Octadecenamide,(Z)-

9. á-Amyrin

Figure-6: Structure of Bioactive compounds


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CONCLUSION

In the present study 25 compounds from the ethanol leaves extract of Achyranthes aspera

were identified by Gas Chromatography Mass Spectrometry (GC-MS) analysis. The

biological activities of each of the identified phytocomponents used for antimicrobial,

antioxidant, anti-inflammatory and pesticide activities. The research findings have shown that

the leaves extract of A. aspera is extensively rich in secondary metabolites. The plant leaves

has a high potential for a vast number of bioactive compounds which justified its use for

various ailments by traditional practitioners.

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gc-ms analysis of bioactive compounds of … · pure phyto-pharmaceuticals need to be studied...

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