Evaluation of Comparative Metabolomic Profile in Cardamom elettaria and Amomum subulatum Fruits

Introduction

Traditional medicine plays important role in curing several diseases. According to WHO, 88% of the world population depends on the plant-derived medications as home remedies and curation for the several ailments ^1,2. Essential oil (EOs) are extracted from leaves, fruits, stem and barks which have been natural aromatic, volatile liquids possessing the antibacterial, antiviral and insecticidal activities³. Many foods, including meat, peaches, sweet peppers, lemon, apple juice, and milk, use EOs as a natural preservative because they kill harmful bacteria like salmonella, E. coli, and Listeria monocytogenes and boost antioxidant activity and active shelf life by up to 5-6 days. It is now used as natural food preservatives from ancient times ⁴. Cardamom essential oils are most effective for treating asthma, oral mouth infections, gastrointestinal disorders, cardiovascular issues, nausea, diarrhoea, hepatotoxicity, gastric and ulcerogenic diseases ⁵.  Cardamom is the best curative medicine for severe cough, cold, wheezing and asthma, bronchitis, tuberculosis, antibacterial and sore throat ^6,7.

 C. elettaria and A. subulatum are major types and its capable to slow down the spoiling process and stop the spread of food-borne infections and it used as antimicrobials in the food industries⁸. After saffron and vanilla, little cardamom is considered the priciest spice in the world. Essential oil of C. elettaria  (L.) Maton used in perfume production, and it ranks third in cost, behind only vanilla and saffron, among spices. Large-scale production occurs mostly in the tropical countries of Guatemala, India, and Sri Lanka ^8,9 and it having higher economic values ¹³. In this study, we have compared the phytoconstituents profile of both C. elettaria and A. subulatum and their physico-chemical appearance that will be useful in further research.

Figure 1 : A) Cardomum eletteria and B) Amomum subulatum. Click here to View Figure

Materials and Methods

Sample Collection, oil extraction and physical characteristics observation

Fresh and healthy capsules of C. elettaria and A. subulatum has been procured from the local shop of Cochin, Kerala. Physical parameters such as size and shape, bulk density and volatile content were observed manually. Seeds with capsules were then made steam distillation in Clevenger apparatus for extraction of essential oils from both the seeds (C. elettaria and A. subulatum) separately and the physical parameters of both oils was observed manually.

Identification of Volatile phytoconstituent profile in C. elettaria and A. subulatum Oil by GC-MS analysis:

Gas chromatography (GC) coupled to a mass spectrometer detector (MSD) from an Agilent HP 6890/5977 was used to examine the oils from C. elettaria and A. subulatum. Specifically, the machine condition was Injector at 270°C; Detector 300°C;  oven 40°C @ 1min hold to 110 °C @ 1 mins hold up to 300 °C (5 mins hold), and 325 °C (10 mins hold) at a rate of 10°C per minute. The steady flow of helium was set at 3 mL/min. The scan mass/charge range was from 50 m/z to 550 m/z. NIST 05 and Wiley 275 intrinsic mass spectral libraries were used to determine the essential oil by comparing retention indices (RIs) to n-alkanes homologous series ¹⁴.

Results

15 g of both fruits were collected (A. subulatum and C. Elettaria) then observed the morphological characteristics and extracted oils for further studies.  Capsules of C. elettaria and A. subulatum shown greater difference in their physical morphological appearance. Where C. elettaria has green colour and having slighter wider area of 5-7 mm and A. subulatum has daker brown in colour and having the wider area of 9-11mm which is more comparable to the green cardamom. The length of A. subulatum largely differs from the C. elettaria having 22-30 mm larger were C. elettaria has 16-20 mm length which measured by the vernier calliper.  The Bulk density of the A. subulatum has the larger difference as 410gm/l whereas C. elettaria has only 360 gm/l. This shows the wild type of cardamom has larger volume and denser in quantity grade.  The volatile content of C. elettaria has greater 7.5% then A. subulatum having 5.5% shown in Table. 1. 15g of separate samples of both species yielded 3.4 µl ± 0.02µl (Cardamom elettaria) and 2.5 µl ± 0.31µl (Amomum subulatum) respectively.

Table 1: Raw material analysis of C. elettaria and A. subulatum

S.No.,	Parameters	Cardamom elettaria	Amomum subulatum
1.	Size & Shape	(16-20 mm) X (5-7 mm) More parabolic or curved	(22-30 mm) X (9-11 mm) More Spherical
2.	Bulk Density	360gm/lit Approx	410gm/lit Approx
3.	Volatile Oil Content	7.5% Approx	5.5% Approx

As mentioned before the respective oils were obtained and checked for the tentative values of Optical rotation, Refractive index, Specific gravity, Solubility, Odour and taste.  Optical rotation of C. eletteria has +29.11/ (25 to 35) which is higher and the  A. subulatum has the negative scale of rotation such as -9.59/ (-5 to-15). The Refractive index has the similar range of both of the species with difference of 0.0003 and with similar indexing of 1.460 to 1.464. Specific gravity of the oil of A. subulatum (0.915) has the slighter difference while C. elettaria has 0.927 with the standard deviated range of C. elettaria 0.917 to 0.937 and A. subulatum 0.905 to 0.925 respectively. Oil texture and quality of C. elettaria has been identified as Spicy, Sweet, Pleasant, Oily but not that rancid, Slight minty and A. subulatum has the Waxier, Camphoraceous, Oily Rancid, more minty texture.

Table 2: Physical parameter analyzation of C. eletteria oil and A. subulatum oil

S.No.,	Parameters	Cardamom eletteria Oil	Amomum subulatum Oil
1.	Optical Rotation (Value/Range)	29.1/ (25 to 35)	-9.59/ (-5 to -15)
2.	Refractive Index (Value/Range)	1.4620 / (1.460 to 1.464)	1.4623 (1.460 to 1.464)
3.	Specific Gravity (Value/Range)	0.927 (0.917 to 0.937)	0.915 (0.905 to 0.925)
4.	Solubility	Partially soluble in ethanol	Partially soluble ethanol
5.	Odor, Aroma & Taste	Spicy, Sweet, Pleasant, Oily but not that rancid, Slight minty	Waxier, Camphoraceous, Oily Rancid, more minty

A. subulatum

Hydro distillated oils of C.eletteria and A. subulatum has been carried out in the phytoconstituent analysis using the GC-MS analysis and compared (Table. 3). Within the detected Ester type phytoconstituents C.eletteria possess the greatest percentage area such asα-terpinyl acetate (46.316), δ-terpinyl acetate (0.24), Linalyl acetate (0.932), Geranyl acetate (1.111), Bornyl acetate (0.062). Where the major presence of Bormeal acetate plays the major role comprising the volatile ester group in C. eletteria. 1,8 Cineole is plays key role in the aromaticity of the cardamom species as already reported in many research. A. subulatum has the larger area percentile (70.061) while comparing to C. eletteria (30.148). Monoterpenes such as Sabinene (2.091) and β-myrcene (1.406) are greater in C. eletteriawhere in A.  subulatum has the greater percentile area such asβ-pinene (6.348), Limonene (d) (5.562) and δ-3-carene (1.159) of monoterpenes.  The monoterpenol, α-terpineol (5.205) is found abundant in A. subulatum showing they are strongest phytoconstituent associated to the monoterpenol shown in figure 2b. The aldehyde, Geranial is found major in the oil source of C. elattaria (0.69) having the greater difference while comparing to the amomum species shown in figure 2a.

Figure 2a: Chromatogram of Cardamomum elettaria Oil showing the major phytoconstituents. Click here to View Figure

Figure 2b: Chromatogram of Amomum subulatum Oil showing the major phytoconstituents. Click here to View Figure

Table 3: Comparison of oil phytoconstituents between the C. eletteria and A. subulatum.

CARDAMOM OIL
Sl. No.:	Compound	CAS No.	Molecular Formula	Cardamom Elettaria (%)	Amomum subulatum (%)
	ESTER
1	α-terpinyl acetate	80-26-2	C12H20O2	46.316	4.078
2	δ-terpinyl acetate	93836-50-1	C12H20O2	0.24	0.0231
3	Linalyl acetate	115-95-7	C12H20O2	0.932	0.022
4	Geranyl acetate	105-87-3	C12H20O2	1.111	0.15
5	Bornyl acetate	76-49-3	C12H20O2	0.062	Not detected
	OXIDE
6	1,8-cineole	470-82-6	C10H18O	30.148	70.061
	MONOTERPENE
7	Sabinene	3387-41-5	C10H16	2.091	1.31
8	β-myrcene	123-35-3	C10H16	1.406	0.736
9	β-pinene	127-91-3	C10H16	0.283	6.348
10	α-thujene	02-05-2867	C10H16	0.279	0.286
11	α-terpinene	99-86-5	C10H16	0.404	0.301
12	(E)-β-ocimene	13877-91-3	C10H16	0.056	0.052
13	Limonene (d)	5989-27-5	C10H16	3.095	5.562
14	α-pinene	80-56-8	C10H16	1.17	3.08
15	γ-terpinene	99-85-4	C10H16	0.749	0.493
16	p-cymene	99-87-6	C10H14	0.731	0.812
17	Terpinolene	586-62-9	C10H16	0.409	0.162
18	Camphene	79-92-5	C10H16	0.022	0.053
19	δ-3-carene	13466-78-9	C10H16	0.018	1.159
	MONOTERPENOL
20	Linalool	78-70-6	C10H18O	0.438	0.158
21	Terpinen-4-ol	562-74-3	C10H18O	1.689	1.741
22	α-terpineol	98-55-5	C10H18O	1.809	5.205
23	δ-terpineol	7299-42-5	C10H18O	0.106	0.73
24	P-cymen-8-ol	1197-01-9	C10H14O	0.368	0.032
	ALDEHYDE
25	Geranial	5392-40-5	C10H16O	0.69	0.0891
26	Neral	106-26-3	C10H16O	0.069	0.032
27	Decanal	112-31-2	C10H20O	0.072	0.046
	SEQUITERPENOL
28	(E)-nerolidol	40716-66-3	C15H26O	1.084	1.533
	SEQUITERPENE
29	β-selinene	17066-67-0	C15H24	1.199	0.743
30	β-caryophyllene	87-44-5	C15H24	0.0216	0.097

Discussion

C. eletteria grown in the regions of Kerala state and A. subulatum found in the regions of Karnataka (Mysore) which were adjoining states in the Western Ghats. Due to the increased export quality C. eletteria is preferred after the bleaching which is found in green elongated shape. The practice of employing Sulphur dioxide or compounds containing chlorine to brighten the hue was abolished by this system. In some cases, metanyl yellow solution was reportedly used to get a consistent, attractive bleached colour. Among the E. cardamomum species recognised by ISO 676:1995 are Thwaites Variety Major and Minuscule Thwaites Variety Burkill ¹⁵. Rich sandy loams soil is ideal for cardamom plants by enjoying the shade and need some rain. Capsules of cardamom are dried in artificial furnaces. At high temperatures and high humidity, the chlorophyll on the outside of the capsule can be degraded. Steam distillation was originally practiced in Western consumer countries. About 65% of the capsule is made up of the seeds when they are dry. The husk makes about 35% of the total. Grade level and geographical location can both cause for difference. C.eletteria Originally from South India, but now widespread around the world, wild maton is a long, perennial reed-like herb. The plant has been called “the queen of spices” because of its reputation for rarity and high price. The husk is valuable since it is employed in solvent extraction and its has many flavoring molecules. Essential oils have varying chemical compositions depending on the plant’s genus, species, chemotype, harvesting place, and period. The biological effects of C. elettaria essential oils are consistent with those predicted by their chemical composition. Monoterpenes such as 1,8-cineole, terpineol, linalool, and pinene, as well as the ester -terpinyl acetate, fall within this category. On a dry weight basis, cardamom capsules provide B vitamins, ascorbic acids, and minerals, along with 10% crude protein, 42% carbohydrates, and 20% crude fibre. Essential oil, found at concentrations of 5–11% depending on area and grade, is the most important component due to its primary usefulness as a flavouring substance. 1,8-Cineole is the primary component in eucalyptus oil. The component has a camphoraceous and refreshing aroma. Free and acetate forms of alcohols such linalool and terpineol are found. Thus, the volatile phytoconstituents of the C. eletteria and A. subulatum has the key differentiator and rich source that provide many medicinal properties such as anticancer, antidiabetic, antiasthmatic and anti-rheumatics.

Conclusion

Morphological characteristics of C.eletteria showed predominant than A. subulatum except the size and length. The principal phytoconstituents of A. subulatum oil contains 1,8-cineole, pinene, limonene (d), and 3-carene, while the phytoconstituents of C.eletteria oil are rich in terpinyl acetate, geranyl acetate, sabinene, myrcene, and selinene. Further, the volatile profile of these two essentials oils was revealed and showed differences. These findings will help to the natural flavors in food industries.  

Acknowledgement

Author express sincere thanks to Karpagam Academy of Higher Education for providing the lab facilities to carry out this research work.  

Conflict of Intertest

The authors do not have any competing interests, either financially or otherwise.

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