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Year : 2018  |  Volume : 10  |  Issue : 2  |  Page : 230-235  

Gas chromatography-mass spectrometric determination of components of leaves of Aegle marmelos and Psidium guajava and seeds of Nigella sativa and correlation with In vitro antioxidant activity

1 Department of Pharmacology, Delhi Institute of Pharmaceutical Science and Research, New Delhi, India
2 Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bioactive Natural Product Laboratory, Jamia Hamdard, New Delhi, India

Date of Web Publication20-Apr-2018

Correspondence Address:
Dr. Rajani Mathur
Delhi Institute of Pharmaceutical Sciences and Research, Pushp Vihar, Sec III, MB Road, New Delhi - 110 017
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/pr.pr_93_17

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Objective: The aim of this study is to analyze the volatile components of the leaves of Aegle marmelos, Psidium guajava, and seeds of Nigella sativa, using gas chromatography-mass spectrometric (GC-MS) and correlate with their in vitro antioxidant activity. Materials and Methods: Plant material was analyzed for heavy metal content (HMC), pesticide residue (PR), and the presence of microbes/fungi. Following hydrodistillation, the volatile components (AM, PG, and NS) were subjected to GC-MS analysis and in vitro antioxidant assay (2,2-diphenyl-1-picrylhydrazyl). Results: The plant material passed the test for HMC, PR, microbial, and fungal contamination. Using GC-MS, the number of compounds identified in AM, PG, and NS were 62, 46, and 58, respectively. The antioxidant activity of AM, PG, and NS was 98.538%, 98.955%, and 97.755%, respectively. Conclusion: GC-MS-based methods can be successfully utilized for phytochemical profiling and standardization of plant material.
Abbreviations Used: AM: Volatile oil of Aegle marmelos Correa.; GC-MS: Gas chromatography-mass spectrometry; HMC: Heavy metal content; NS: Volatile oil of Nigella sativa Linn.; PG: Volatile oil of Psidium guajava Linn.; PR: Pesticide residue.

Keywords: Aegle marmelos, antioxidant activity, gas chromatography-mass spectrometric, Nigella sativa, Psidium guajava, volatile compounds

How to cite this article:
Jayachandran Nair C V, Ahamad S, Khan W, Anjum V, Mathur R. Gas chromatography-mass spectrometric determination of components of leaves of Aegle marmelos and Psidium guajava and seeds of Nigella sativa and correlation with In vitro antioxidant activity. Phcog Res 2018;10:230-5

How to cite this URL:
Jayachandran Nair C V, Ahamad S, Khan W, Anjum V, Mathur R. Gas chromatography-mass spectrometric determination of components of leaves of Aegle marmelos and Psidium guajava and seeds of Nigella sativa and correlation with In vitro antioxidant activity. Phcog Res [serial online] 2018 [cited 2021 Jun 25];10:230-5. Available from: http://www.phcogres.com/text.asp?2018/10/2/230/230759


The leaves of Aegle marmelos, Psidium guajava, and seeds of Nigella sativa, were analyzed for heavy metal content, pesticide residue, and the presence of microbes/fungi and their volatile oil content was profiled by gas chromatography-mass spectrometric and correlated with in vitro antioxidant activity.

   Introduction Top

Since time immemorial, plants have been an exemplary source of drugs. The traditional system of medicine like Ayurveda contains extensive mention of formulations that contain Nigella sativa Linn. (Ranunculaceae), Aegle marmelos (L.). Correa (Rutaceae), and Psidium guajava Linn. (Myrtaceae).[1],[2],[3] Recent pharmacological studies have also established the medicinal properties of these plants,[4],[5] that can be mainly attributed, to the presence of wide array of chemical moieties such as tannins, alkaloids, essential oil, quinones, sterols, triterpenoids, and flavonoids.

Thorough validation and standardization of the medicinal plant-derived drugs are essential but cumbersome and technically challenging. Thus, there is an urgent need to set-up simple methods that can be utilized for quality analysis of medicinal plant-based products. Here, we report a simple gas chromatography-mass spectroscopy-based method that was developed in accordance with ICH guidelines [6] to address this lacuna. In addition, the phytochemical constituents were pharmacodynamically assessed forin vitro antioxidant activity.

   Materials and Methods Top

Collection and identification of plant material

The fresh leaves of Aegle marmelos (L.) Correa. and Psidium guajava Linn. were locally collected (Herbal garden, DIPSAR) and the seeds of Nigella sativa Linn. were procured from the local market. Identification and authentication of the plant material were carried out by Principle Scientist, Resources (Indian Council of Agricultural Research), New Delhi, India, and vouchers were provided (NHCP/NBPGR/2014-5-7).

Extraction of plant material

The leaves of Aegle marmelos (L.) Correa. and Psidium guajava Linn. and seed of Nigella sativa Linn. were weighed (100 g), grounded (28 mesh size), and micronized separately. In Clevenger apparatus, each plant material was separately soaked in distilled water (1 L) at 90°C for 6 h. Using hydrodistillation, the volatile components were collected by the addition of equal volumes of n-hexane, dried over anhydrous sodium sulfate, stored in the amber colored bottle (−20°C) and clearly labeled as AM, PG, and NS, respectively. The extract yield was calculated as 0.50%, 0.39%, and 0.41% (w/v) for AM, PG, and NS, respectively.

Analysis for pesticide residue, heavy metal, and microbial content

The dried leaves and seeds were tested for residues of pesticide [2,2-bis (p-chlorophenyl)-1, 1, 1trichloroethane (DDT); 1, 2, 3, 4, 5, 6-hexachloro cyclohexane(HCH), a mixture of isomers; Endosulfan (α, β and sulphate isomers); Malathion and Parathion], aflatoxin (B1, B2, G1, G2), metallic elements (arsenic, lead, mercury, cadmium, iron, zinc, potassium, calcium) using liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS/MS). The plant material was also analyzed for the total aerobic microbial count, enterobacteria, fungal count, in accordance with British Pharmacopeia.[7]

Gas chromatography-mass spectrometric analysis and identification of volatile constituents

Gas chromatography-mass spectrometric analysis was carried out after extraction using hexane and filtered through 0.25 μM PTFE filter. A volume of 2 μL was injected at a split ratio of 1:5 into Agilent 7890 GC System, USA equipped with a CT-PAL autosampler attached to silica column (30 m × 0.25 mm, film thickness 0.25 μm) and interfaced with an electron ion spray mass detector (m/z 50–700 at 0.5 s/scan). The interface was set to 285°C and ion source was adjusted to 200°C with carrier gas (Helium @ 2 mL/min). After a 3 min solvent delay time at 70°C, the oven temperature was raised to 5°C/min–310°C. The volatile constituents were identified by matching the retention index and fragmentation pattern data with those of the standards using WILEY and NIST.[8]

In vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl Assay

The antioxidant activity of volatile constituents was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.[9] In brief, to different concentrations of the sample (1–50 μl/ml), DPPH· solution (2 ml, 0.1 mM) and Trolox (20–1000 μM) were added. The mixture was vortexed and kept undisturbed at room temperature for 30 min. The absorbance of the mixture was read at 517 nm against the blank using UV-Vis spectrophotometer (UV-1700 double beam, Shimadzu). The activity was calculated using the equation-DPPH scavenging effect (%) = [(A0–A1)/A0 × 100] where, A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of the sample or standard. The extract concentration versus DPPH scavenging effect (%) was calculated from the graph. The decrease in the absorbance indicates an increase in DPPH radical scavenging activity.

   Results and Discussion Top

The analysis of PG, AM, and NS did not detect presence of pesticide residues, microbial, and fungal contamination. The samples of AM, PG but not NS, tested positive for the presence of arsenic, lead, iron, zinc, potassium, and calcium. Cadmium was not detected in any of the samples [Table 1].
Table 1: Qualitative determination of the plant leaves of Psidium guajava Linn., Aegle marmelos (L) Correa and seeds of Nigella sativa Linn

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The chemical composition of the hydrodistilled volatile components of AM as analyzed using GC-MS showed the presence of 62 compounds [Figure 1]a. The major constituent was β-elemene (8.73%) [Table 2]. The other constituents were β-Farnesene (4.25%), β-caryophyllene (7.67%), myristinic acid, fumaric acid and hexadecanoic acid.
Figure 1: (a) Gas chromatography-mass spectrometric Spectra of volatile oil Aegle marmelos (L.) Correa. (b) Gas chromatography-mass spectrometric Spectra of volatile oil Psidium guava Linn. (c) Gas chromatography-mass spectrometric Spectra of volatile oil Nigella sativa Linn. (d) 2,2-diphenyl-1-picrylhydrazyl scavenging activity on volatile oils

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Table 2: Composition of essential oil from leaves of Aegle marmelos (L.) Correa (AM)

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Forty-six compounds were identified in PG by GC-MS analysis [Figure 1]b of which the major were β caryophyllene (23.4%), δ-cadinene, nerolidol (10.34%), torreyol (7.71%), followed by α-copaene, α-humlene, α-amorphene, cis-α-bisabolene [Table 3].
Table 3: Composition of essential oil from leaves of Psidium guava Linn (PG)

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The chromatogram of 58 compounds was obtained by GC-MS analysis of NS [Figure 1]c. The major compounds that could be identified were D-longifolene and β caryophyllene, followed by carvacrol (4.52%), α-cubebene (5.48%), n-heneicosane, farnesol isomer A, penta fluroproponicacid, and nor-vittalalactone [Table 4].
Table 4: Composition of essential oil from seed of Nigella sativa Linn (NS)

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Interestingly, the three samples (AM, PG, and NS) also detected positive for L naphthalene.

The antioxidant activity of AM, PG, and NS was calculated to be 98.538, 98.955 and 97.755%, respectively [Figure 1]d.

There is a deep-seated faith of all humans worldwide, in plant-derived medicines as generally safe, effective, and cheap means of healthcare. Thus, quality control and standardization of herbal medicines are of utmost importance and modern-day analytical techniques such as GC-MS help to achieve the same. The leaves of Aegle marmelos and Psidium guajava and seeds of Nigella sativa enjoy well-established pharmacological activity that mainly stems from scavenging property.[10],[11],[12] In conclusion, the volatile oil components of A. marmelos, P. guajava and N. sativa were analysed by GC-MS, and correlated with their antioxidant activity. Of the three, AM demonstrated the highest antioxidant activity. The GC-MS profile of volatile oils can be used for herbal standardization of the three plants.

Financial support and sponsorship

The authors would express their appreciation to department of biotechnology (DBT No. BT/PR9068/MED/97/139/2013) and All India Council of Technical Education for financial support.

Conflicts of interest

There are no conflicts of interest.

   References Top

The controller of publications, Ministry of AYUSH, Government of India. The ayurvedic pharmacopoeia of India. 2001;I (Ist ed):27-119.  Back to cited text no. 1
Maity P, Hansda D, Bandyopadhyay U, Mishra DK. Biological activities of crude extracts and chemical constituents of Bael, Aegle marmelos (L.) corr. Indian J Exp Biol 2009;47:849-61.  Back to cited text no. 2
Mahaseth RK, Kumar S, Dutta S, Sehgal R, Rajora P, Mathur R, et al. Pharmacodynamic study of interaction of aqueous leaf extract of Psidium guajava linn. (Myrtaceae) with receptor systems using isolated tissue preparations. Indian J Pharm Sci 2015;77:493-9.  Back to cited text no. 3
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Burkil HM. The Useful Plants of West Tropical Africa. London, UK: Royal Gardens Kew; 1994. p. 21-150.  Back to cited text no. 4
Gutiérrez RM, Mitchell S, Solis RV. Psidium guajava : A review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol 2008;117:1-27.  Back to cited text no. 5
www.ich.org. Current Step 4version Dated 4 June, 2008. Available from: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q10/Step4/Q10_Guideline.pdf. [Last accessed on 2018 Jan 25].  Back to cited text no. 6
The Stationery Office. British Pharmacopoeia. 2013;6:A475-5.  Back to cited text no. 7
Khan W, Chester K, Anjum V, Ahmad W, Ahmad S, Narwaria A, et al . Chromatographic profiling of pancharishta at different stages of its development using HPTLC, HPLC, GC-MS and UPLC-MS. Phytochem Lett 2017;20:391-400.  Back to cited text no. 8
Okawa M, Kinjo J, Nohara T, Ono M. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoids obtained from some medicinal plants. Biol Pharm Bull 2001;24:1202-5.  Back to cited text no. 9
Wang L, Wu Y, Huang T, Shi K, Wu Z. Chemical compositions, antioxidant and antimicrobial activities of essential oils of Psidium guajava L. Leaves from different geographic regions in China. Chem Biodivers 2017;14:e1700114.  Back to cited text no. 10
Ramadan MF, Kroh LW, Mörsel JT. Radical scavenging activity of Black cumin (Nigella sativa L.), Coriander (Coriandrum sativum L.), and Niger (Guizotia abyssinica Cass.) Crude Seed Oils and Oil Fractions. J Agric Food Chem 2003;51:6961-9.  Back to cited text no. 11
Baliga MS, Bhat HP, Joseph N, Farhan F. Phytochemistry and medicinal uses of the bael fruit (Aegle marmelos Correa): A concise review. Food Res Int 2011;44:1768-75.  Back to cited text no. 12


  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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