1. INTRODUCTION:

The evolution of the earth begun with a reductive environment and the earliest biotic forms on earth were used to gain energy via anaerobic respiration. Oxygen was produced as a toxic by-product.¹ Cyanobacteria was the first known living entity on the earth which was used to produce oxygen during respiration.² As time progresses, the evolution of the earth leads to an oxidative environment due to oxygen availability. Oxidative respiration had an edge over anaerobic respiration. Because aerobic respiration was associated with high energy production with the same unit of substrates.³

But the aerobic respiration also had some negative impacts on organisms. The aerobic respiration led to the production of oxidative free radicals. These free radicals were harmful to the structural machinery of the organism.⁴ Therefore, protecting the organism from oxidative stress has become of utmost importance to sustain and survive biotic life on earth. Antioxidants are compounds that prevent the oxidation of the substrates even at their low concentrations.⁵ The living entity acquires these compounds either from internal synthesis or environmental acquisition.⁶

The environment is a rich reservoir of structurally diverse free radical protectants. They either be of synthetic origin or naturally occurring. Synthetic antioxidants are a good alternative for free radical scavenging but are associated with several life-threatening effects. They are suspected to possess noxious effects on the living system. For instance; butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate can cause hormonal imbalance, delayed sexual maturation, cancer, epithelial damage, organelles (lung, liver, kidney, thyroid) dysfunction, and so on.⁷ Synthetic antioxidants also impair blood coagulation and neurobehavioral activity. These adverse effects limit the use of synthetic antioxidants while on the other side promotes the use of natural antioxidants. Natural antioxidants are more sustainable and environmentally friendly. They can be of animal origin or plant origin. The animal-originated antioxidants include some meats, poultry, and fish. However, they might be affiliated with certain subclinical diseases, metabolic disorders, stress, hormonal imbalances, and others. They also seem less sustainable and environmentally friendly as compared to plant-based antioxidants. Ultimately, plant-originated antioxidants have become the primary choice to combat oxidative stress.⁸

There are various sources of plant-based antioxidants including fruits, vegetables, beverages, cereals, herbs, and spices.⁹ There is a strong correlation between the intake of a plant-based diet and the prevention of chronic diseases.¹⁰ Although, all represent good antioxidants but are not affordable for everyone. Therefore, the category with a daily utilization chart is highly recommended. Spices are one of them that make an integral part of food.¹¹ They grace almost every culture's cuisine and, thus are preferred over other dietary commodities. Spices are majorly concentrated with antioxidant-rich secondary metabolites.¹² These secondary metabolites such as alkaloids, flavonoids, and polyphenols can be extracted in the form of essential oils (EOs). EOs are an affluent pool of structurally diverse volatile compounds. These compounds exhibit enormous free radical scavenging activity.¹³ Hence, EOs extracted from spices can be effectively used as free radical protectants.

The current research was focused on extracting and characterizing EOs from three renowned dietary spices viz. Cinnamomum verum (cinnamon), Cuminum cyminum (cumin), and Trigonella foenum-graecum (trigonella). Their EOs were screened against a selected panel of bacterial and fungal strains.

2. MATERIALS AND METHODS:

2.1. Chemicals and Reagents:

The analytical-grade chemicals were procured from Sigma-Aldrich and Himedia. Cinnamon, cumin, and trigonella powder were procured from Rohtak (Haryana) India-124001.

2.2. Plant Materials:

Three spices, viz. cinnamon, cumin, and trigonella, were chosen for the present study. The spices were selected based on their nutritional composition. Different commonly used dietary spices were analyzed for their dietary fiber, protein, and fat content. It was observed that cinnamon, trigonella, and cumin were rich sources of these components as compared to other spices (https://www.usda.gov/).

2.3. Extraction of Spices EOs:

2.3.1. Extraction by Clevenger Apparatus:

The bark and seeds of cinnamon and cumin, respectively were subjected to hydro distillation. The optimum operational conditions of the Clevenger apparatus were as follows: 50gm pulverized cinnamon and cumin; temperature 40-60˚C; 300mL distilled water; duration 4-5 hours. Anhydrous sodium sulfate was added to extracted EOs to remove water droplets and stored at 4 ˚C for further use.¹⁴ The cinnamon essential was coded E.O.A whereas cumin essential oil was coded E.O.B.

2.3.2. Extraction by Soxhlet Apparatus:

A total of 50gm of pulverized trigonella seeds were kept in a thimble made of Waltman filter paper. The thimble was placed in the Soxhlet apparatus. The extraction process was initiated by dispensing petroleum ether (300mL) in the round bottom flask. The cycle was carried out for 6 hours at 40-60˚C to complete the extraction. The completion of seed extraction was signified by the transparency of the thimble solution. The solvent was evaporated from the oil by placing it in a water bath. The high-viscosity yellowish trigonella seed oil (E.O.C) was collected and preserved in a refrigerator.¹⁵

2.4. Calculation of EO’s Yield:

The yield of EOs obtained from different apparatus was calculated using the following equation¹⁵:

Mass of extracted oil (gm)

Extraction yield (%) = --------------------------------- × 100

Mass of pulverized seeds (gm)

2.5. GC-MS Analysis:

GC-MS investigation was conducted on 394143602-GC Single Quadruple Mass Spectrometer SCION 436 GC; Fill Scan SIM, with CP-739651-CP8410 Liquid Auto Sample. Helium was used as carrier gas with 1mL/min flow rate. The samples were diluted as 10µL in 2mL dichloromethane (DCM). One µL was used as the sample injection volume. Column temperature was maintained at 100˚C. The chromatogram was obtained with several peaks. Each peak corresponds to a specific compound. The peak area signifies the relative quantity of a compound. The compounds were identified on the basis of commercial libraries and mass spectra documentation provided with GC-MS. GC retention indices were also used for the identification of the compounds.¹⁶

2.6. Antioxidant Assessment:

2.6.1. DPPH Assay:

The assay was performed according to Nanasombat and Wimuttigosol with minor modifications.¹⁷ The 0.1mM DPPH was prepared in methanol. One mL methanolic DPPH was mixed with 1 mL of different concentrations of essential oils. The solution was left in the dark for 30 minutes. The solution was stirred vigorously and then poured into quartz cuvettes. The fluctuations in the absorbance were monitored at 517nm. Pure methanol was used to zero down the spectrophotometer. Ascorbic acid was used as standard. The assay was done in triplicates. The radical inhibition by DPPH assay was calculated as follows:

% Inhibition = [(A_control-A_sample)/A_control] ×100

Where, A_control = Absorbance of the control reagents excluding test sample

A_{sample =}Absorbance of the test sample

2.6.2. ABTS Assay:

ABTS radical cation was prepared by mixing an aqueous ABTS solution (7mM) with an aqueous solution of K₂S₂O₈ (140mM). The solution was kept for 16hours in the dark followed by dilution in methanol up to an absorbance of 0.8 at 734nm. The one mL ABTS solution was dissolved with one mL concentrations of different essential oils. After vigorous stirring, the solution was poured into quartz cuvettes. The spectrophotometer was zeroed down with pure methanol. Ascorbic acid was used as the positive control. The decreased absorbance for each sample was registered at 734 nm and performed thrice.¹⁸

3. RESULTS:

3.1. EOs Yield:

The EOs extracted from different methods were quantified using the equation discussed above. The extraction yields of different EOs are as follows: cinnamon oil (2.75%), cumin oil (1.53%) and trigonella oil (4.35%). The maximum yield was obtained with trigonella EO. Therefore, it can be inferred that the Soxhlet apparatus gives a high yield of EO as compared to the Clevenger apparatus.

3.2. Composition Analysis of EOs:

GC-MS was employed for the composition analysis of different EOs. The compounds were identified by comparing the analytical libraries' retention indices and mass spectra. The GC-MS chromatograms of different EOs are depicted in Figures (1a, 1b and 1c). Compounds identified from different EOs are clubbed in Tables 1, 2 and 3.

Figure 1a: GC-MS chromatogram of cinnamon EO

Table 1. Chemical Composition of Cinnamon Eo

Retention time	Compound name	Area	Probability	Molecular formula	Molecular weight (g/mol)	Structure of compound
3.732	Benzaldehyde	1.035	34.52	C₇H₆O	106.12
6.905	Benzenepropanol	0.747	74.20	C₉H₁₂O	136.19
7.930	2-Propenol, 3-phenyl	1.244	22.25	C₉H₁₀O	134.18
8.987	Cinnamaldehyde	48.55	37.89	C₉H₈O	132.16
10.52	α-Copaene	6.479	51.71	C₁₅H₂₄	204.35
11.96	2-Propenoic acid, 3-phenyl	6.812	50.16	C₉H₈O₂	148.16
12.80	trans-Calamene	2.769	2.769	C₁₅H₂₂	202.33
14.64	ar-Tumerene	1.729	1.729	C₁₅H₂₀O	216.32

Figure 1b: GC-MS chromatogram of cumin EO

Table 2. Chemical Composition of Cumin Eo

Retention time	Compound name	Area	Probability	Molecular formula	Molecular weight (g/mol)	Structure of compound
3.893	Bicyclo-heptane 6,6-dimethyl	3.105	49.28	C₉H₁₆	124.22
4.551	o-Cymene	10.090	18.15	C₁₀H₁₄	134.22
5.074	γ-Terpinene	1.661	28.11	C₁₀H₁₆	136.23
7.471	1,3-cyclohexadiene 1-methanol	1.792	67.55	C₇H₈O	110.15
8.379	Benzaldehyde	56.268	35.10	C₇H₆O	106.12
9.105	1,3-cyclohexadiene 1-carboxyaldehyde	6.637	60.18	C₇H₈O	108.14
12.483	Caryophylleneoxide	15.004	31.58	C₁₅H₂₄O	220.35

Figure 1c: GC-MS chromatogram of trigonella EO

Table 3. Chemical Composition of Trigonella Eo

Retention time	Compound name	Area	Probability	Molecular formula	Molecular weight	Structure of compound
8.307	Benzaldehyde 4-(1-methylethyl)	9.074	35.39	NR	NR	NR
8.852	Cinnamaldehyde	13.518	42.87	C₉H₈O	132.16
10.644	n-Pentadecanol	2.345	4.62	C₁₅H₃₂O	228.41
12.566	Phenol, 2,4-bis (1,1-dimethyl ethyl)	6.785	46.70	C₁₄H₂₂O	206.32
13.707	Aspidospermidin-17-ol, 1-acethyl-19,21-epoxy-15,16-dimethoxy	7.417	25.85	NR	NR	NR
14.644	Dibutyl.pthalate	24.873	15.60	C₁₆H₂₀O₄^2-	276.33
16.653	Murolan-3,9 (11) diene-10-peroxy	7.407	10.33	NR	NR	NR
17.428	Pthalic acid butyl hexyl ester	8.315	8.23	C₁₈H₂₆O₄	306.4
17.556	i-Proxy 7,10,13,16,19-docosapent	10.203	9.29	NR	NR	NR

NR* = Not reported

4. DISCUSSION:

Spices are rich repositories of therapeutically active phytocompounds. In the present study, essential oils were extracted from cinnamon and cumin through the Clevenger apparatus, whereas essential oil from trigonella was extracted through the Soxhlet apparatus. Our results indicated that the Soxhlet apparatus produced a high yield of EO as compared to the Clevenger apparatus with an equal amount of substrate. The EO obtained with the Soxhlet apparatus was qualitatively low as compared to the Clevenger apparatus. The composition of volatile compounds was also low.¹⁹ GC-MS carried out the composition analysis of different essential oils. In a study, it was reported that cinnamon essential oil maximally constitutes cinnamaldehyde.²⁰ The present study also confirmed the maximum presence of cinnamaldehyde in cinnamon oil. The cumin essential oil also showed the presence of different compounds. In a study by Singh et al., it was stated that cumin essential oil maximally yields cumin aldehyde²¹, but it was not confirmed by the present study. However, the present study favored the existence of different aldehydes in cumin essential oil. Trigonella essential oil analysis also validated the presence of different phytoconstituents.²² These phytocompounds endow several nutritional and medicinal attributes.^23,24

EOs as food additives improve food quality and enhance food's antioxidant potential and self-life.²⁵ The radical scavenging potential of the compounds mainly renders antioxidant defense. In this regard, several methods have been designed for the antioxidant assessment of the compounds. The DPPH and ABTS are standard colorimetric assays.²⁶ These assays are easy to perform and are routinely used for antioxidant screening.¹⁸ Therefore, in the present study, the antioxidant activity of different EOs was estimated by DPPH and ABTS assays. The antioxidant results of the present study are presented in Figure 2. The results demonstrated that E.O.A with the maximum radical scavenging efficacy. Though, other essential oils were also noticed with significant antioxidant activity but were lower than E.O.A. E.OA was also noticed with maximum DPPH radical scavenging activity i.e., 0.29mg/mL among different spices (mace, prikhom, zedoary, anise, bitter zinger, bastard cardamom, and dill) essential oils.¹⁷ The maximum radical scavenging activity of E.O.A. was endowed by the synergistic effect of its various phytoconstituents. The phytoconstituents of spices essential oils were reported to exhibit significant antioxidant activity.²⁷ It was also reported that cinnamon essential oil possesses pronounced antioxidant activity. The cumin essential oil exhibited excellent antioxidant potential even better than BHT and BHA. The EO has shown dose-dependent antioxidant activity. The oil at a concentration of 5.4µg was sufficient to inhibit half of the DPPH free radicals.²⁸ It was worth mentioning that the antioxidant results obtained with ABTS assays were comparatively higher than the DPPH assay. The reason was that ABTS⁺ was highly reactive in nature as compared to DPPH. The reaction of antioxidant compounds with ABTS⁺involves very fast electron transfer followed by a proton transfer (ET-PT) mechanism.²⁹ The RSA was directly proportional to the number of active hydroxyl groups.³⁰ The antioxidant activity was noticed in a dose-dependent manner. Uttara and Mohini also reported the dose-dependent antioxidant activity of plant extracts.³¹

5. CONCLUSION:

The present study concluded that spices essential oils are an affluent pool of therapeutically active phytoconstituents, as confirmed by GC-MS analysis. These phytoconstituents are responsible for the multifaceted therapeutic effects of essential oils. The different antioxidant assays revealed cinnamon essential oil with maximum radical scavenging activity. Cumin and trigonella essential oils also showed significant antioxidant activity but were lower than cinnamon essential oils. The presence of cinnamaldehyde was the most probable reason behind the maximum radical scavenging potential of cinnamon EO. Therefore, spices' essential oils and their therapeutically active phytocompounds can be used as supplements in pharmaceutical industries for new and natural drug innovations. However, further studies are warranted to understand better these essential oils' in-vivo and clinical therapeutic effectiveness.

6. AUTHOR CONTRIBUTION:

Neetu Singh designed the paper, wrote the paper draft, and performed the experimentation; Surender Singh Yadav supervised the study, read the draft, and gave valuable suggestions; Balasubramanian Narasihman reviewed the study and analyzed the results.

7. FUNDING:

Financial assistance from the Council for Scientific and Industrial Research (CSIR), New Delhi, Haryana State Council for Science and Technology (HSCST), Panchkula, and Department of Science and Technology, Govt. of India, New Delhi is thankfully acknowledged.

8. DECLARATION OF INTEREST:

There is no conflict of interest.

9. REFERENCES:

1. Leisegang K. Life Under Aerobic Conditions. In: In Oxidants, Antioxidants and Impact of the Oxidative Status in Male Reproduction. 2019; 3–8. Academic Press. https://doi.org/10.1016/B978-0-12-812501-4.00001-8

2. Fischer WW. Hemp J. Valentine JS. How did life survive Earth’s great oxygenation? Current Opinion in Chemical Biology. 2016; 31: 166–178. https://doi.org/10.1016/j.cbpa.2016.03.013

3. Saltveit ME. Respiratory metabolism. In: In Postharvest physiology and biochemistry of fruits and vegetables. 2019; 73–91. Woodhead Publishing.https://doi.org/10.1016/B978-0-12-813278-4.00004-X

4. Menon R. Antioxidants and their therapeutic Potential-A review. Research Journal of Pharmacy and Technology. 2013; 6(12): 1426–1429.

5. Kirtawade R. Salve P. Kulkarni A.Dhabale P. Herbal antioxidant: Vitamin C. Research Journal of Pharmacy and Technology. 2010; 3(1): 58–61.

6. Gutteridge JM. Halliwell B. Mini-Review: Oxidative stress, redox stress or redox success? Biochemical And Biophysical Research Communications. 2018; 502(2): 183–186. https://doi.org/10.1016/j.bbrc.2018.05.045

7. Xu X. Liu A. Hu S. Ares I. Martínez-Larrañaga MR. Wang X. Martínez M.Anadón A. Martínez MA. Synthetic phenolic antioxidants: Metabolism, hazards and mechanism of action. Food Chemistry. 2021; 353: 129488. https://doi.org/10.1016/j.foodchem.2021.129488

8. Manju V. Revathi R. Murugesan M. In vitro antioxidant, antimicrobial, anti-inflammatory, anthelmintic activity and phytochemical analysis of Indian medicinal spices. Research Journal of Pharmacy and Technology. 2011; 4(4): 596–599.

9. Chakraborty P. Kumar S. Dutta D. Gupta V. Role of antioxidants in common health diseases. Research Journal of Pharmacy and Technology. 2009; 2(2): 238–244.

10. Jain N. Goyal S.Ramawat KG. Radical scavenging activity and total phenolic content in selected fruits and vegetables. Research Journal of Pharmacy and Technology. 2012; 5(1): 121–124.

11. Bhutkar MA. Bhise SB. Spices and condiments in the management of diabetes mellitus. Research Journal of Pharmacy and Technology. 2011; 4(1): 37–42.

12. Singh N. Yadav SS. Ethnomedicinal uses of Indian spices used for cancer treatment: A treatise on structure-activity relationship and signaling pathways. Current Research in Food Science. 2022a; 5: 1845–1872. https://doi.org/10.1016/j.crfs.2022.10.005

13. Singh N. Yadav SS. A review on health benefits of phenolics derived from dietary spices. Current Research in Food Science. 2022a; 5:1508–1523.https://doi.org/10.1016/j.crfs.2022.09.009

14. Pingret D. Fabiano-Tixier AS.Chemat F. An improved ultrasound Clevenger for extraction of essential oils. Food Analytical Methods. 2014; 7:9–12.https://doi.org/10.1007/s12161-013-9581-0

15. Akbari S. Abdurahman NH. Yunus RM.Alara OR. Abayomi OO. Extraction, characterization and antioxidant activity of fenugreek (Trigonella-Foenum Graecum) seed oil. Materials Science for Energy Technologies. 2019; 2(2):349–355.https://doi.org/10.1016/j.mset.2018.12.001

16. Caputo L. Amato G. de Bartolomeis P. De Martino L. Manna F. Nazzaro F. De Feo V. Barba AA. Impact of drying methods on the yield and chemistry of Origanum vulgare L. essential oil. Scientific Reports. 2022; 12(1): 3845. https://doi.org/10.1038/s41598-022-07841-w

17. Nanasombat S.Wimuttigosol P. Antimicrobial and antioxidant activity of spice essential oils. Food Science and Biotechnology. 2011; 20: 45–53. https://doi.org/10.1007/s10068-011-0007-8

18. Olszowy M. Dawidowicz AL. Essential oils as antioxidants: Their evaluation by DPPH, ABTS, FRAP, CUPRAC, and β-carotene bleaching methods. Monatshefte für Chemie-Chemical Monthly Monatshefte für Chemie-Chemical Monthly. 2016; 2083–2091.https://doi.org/10.1007/s00706-016-1837-0

19. Fagbemi KO. Aina DA.Olajuyigbe OO. Soxhlet extraction versus hydrodistillation using the clevenger apparatus: A comparative study on the extraction of a volatile compound from Tamarindus indica seeds. The Scientific World Journal. 2021; 1–8.https://doi.org/10.1155/2021/5961586

20. Singh N. Rao AS. Nandal A. Kumar S. Yadav SS.Ganaie SA. Narasimhan B. Phytochemical and pharmacological review of Cinnamomum verum J. Presl-a versatile spice used in food and nutrition. Food Chemistry. 2021a; 338: 127773. https://doi.org/10.1016/j.foodchem.2020.127773

21. Singh N. Yadav SS. Kumar S.Narashiman B. A review on traditional uses, phytochemistry, pharmacology, and clinical research of dietary spice Cuminum cyminum L. Phytotherapy Research. 2021b; 35: 5007–5030. https://doi.org/10.1002/ptr.7133

22. Singh N. Yadav SS. Kumar S. Narashiman B. Ethnopharmacological, phytochemical and clinical studies on Fenugreek (Trigonella foenum-graecum L.). Food Bioscience. 2022; 46: 101546. https://doi.org/10.1016/j.fbio.2022.101546

23. Balamurugan G. Arunkumar MP. Muthusamy P. Anbazhagan S. Preliminary Phytochemical Screening, Free radical Scavenging and Antimicrobial activities of Justicia tranquebariensis Linn. Research Journal of Pharmacy and Technology. 2008; 1(2): 116–118.

24. Roy A. Saraf S. Antioxidant and antiulcer activities of an Ethnomedicine: Alternanthera sessilis. Research Journal of Pharmacy and Technology. 2008; 1(2): 75–79.

25. Viuda‐Martos M. Ruiz Navajas Y. Sánchez Zapata E. Fernández‐López JA. Antioxidant activity of essential oils of five spice plants widely used in a Mediterranean diet. Flavour and Fragrance Journal. 2010; 25(1): 13–19. https://doi.org/10.1002/ffj.1951

26. Gunasekaran B. Muralidharan P. Pandiselvi A. Amutha P. Preliminary Phytochemical screening and Anti oxidant activities of Ethanolic extract of Caesalpinia sappan Linn. Research Journal of Pharmacy and Technology. 2008; 1(3): 179–181.

27. Tomaino A. Cimino F. Zimbalatti V. Venuti V. Sulfaro V. De Pasquale A. Saija A. Influence of heating on antioxidant activity and the chemical composition of some spice essential oils. Food Chemistry. 2005; 89(4): 549–554. https://doi.org/10.1016/j.foodchem.2004.03.011

28. Allahghadri T. Rasooli I.Owlia P. Nadooshan MJ. Ghazanfari T. Taghizadeh M.Astaneh SDA. Antimicrobial property, antioxidant capacity, and cytotoxicity of essential oil from cumin produced in Iran. Journal of Food Science. 2010; 75(2): 54–61. https://doi.org/10.1111/j.1750-3841.2009.01467.x

29. Kumar D. Rawat DS. Synthesis and antioxidant activity of thymol and carvacrol based Schiff bases. Bioorganic and Medicinal Chemistry Letters. 2013; 23(3): 641–645. https://doi.org/10.1016/j.bmcl.2012.12.001

30. Guo Z. Xing R. Liu S. Yu H. Wang P. Li C. Li P. The synthesis and antioxidant activity of the Schiff bases of chitosan and carboxymethyl chitosan. Bioorganic & Medicinal Chemistry Letters. 2005; 15(20): 4600–4603. https://doi.org/10.1016/j.bmcl.2005.06.095

31. Uttara J. Mohini U. Evaluation of antioxidant activity of aqueous extract bark of Ficus glomerata. Research Journal of Pharmacy and Technology. 2008; 1(4): 537–538.

Received on 22.03.2024 Modified on 09.05.2024

Research J. Pharm. and Tech 2024; 17(7):3367-3374.

DOI: 10.52711/0974-360X.2024.00526