INTRODUCTION:

Medicinal plants play a key role in the treatment of many diseases in humans from ancient times and these are the vital part of the Indian traditional medical system, such as the Ayurveda and Siddha. Globally, an extensive activity of research is undergoing on diverse plant group and their remedial values. Scientifically, medicinal flora have proven to be a rich resource of biologically active compounds. India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world^[1]. Genus Alpinia is a large genus of the Zingiberaceae family, which is widely distributed in many tropical regions of Asia, including China, India and Indonesia.

This genus consists of almost 250 species and these plants have been utilized as traditional medicines, food and spices in many countries such as China, Japan and India. Alpinia galanga and Alpinia calcarata are two important plants of this genus.

Alpinia galanga L. is a rhizomatous plant which is cultivated in many countries like India, Indonesia, Malaysia, Thailand, and China. Its rhizomes are also used as spices and condiment and flavouring agent. The rhizome has enough importance to treat many diseases like rheumatism, ulcers, colds, whooping cough, vomiting, throat infections, stomach ache and indigestion. The plant is reported to have antimicrobial, antioxidant, anti-inflammatory, antiulcer, antitumour, anticalculi and anti-HIV properties^[2-6]. Alpinia calcarata is a rhizomatous perennial herb, commonly used in the traditional medicinal systems. Rhizome is the economically important part, which is a major constituent of many formulations of the indigenous system of medicine. A. calcarata possesses antibacterial, anthelmintic, antifungal, antinociceptive, antioxidant, gastroprotective, aphrodisiac and antidiabetic^[7,8]. This medicinal plant has enormous significance to treat throat inflammation, purifying blood, stimulating digestion, rheumatism, bronchitis and asthma .

In the last few years, gas chromatography mass spectrometry (GC-MS) has become one of the best technique for profiling of plant secondary metabolites, which are the key factor in therapeutics^[9,10]. GC-MS studies are very much useful in identifying the phytochemical constitutes present in the plant parts. Knowledge of the chemical constituents of plants is desirable because such information will be valuable for the synthesis of complex chemical substances.

To the best of our knowledge, though the rhizome extracts of A.galanga and A. calcarata have been investigated, leaf extracts of A.calcarata have not studied through GC-MS analysis till date. Considering the importance of the plants and existing lacuna, the present study was aimed for phytochemical screening, evaluation of total phenolic and flavonoid content and GC-MS analysis of methanolic leaf and rhizome extracts of A.galanga and A. calcarata.

MATERIALS AND METHOD:

Plant Material:

The rhizomes of Alpinia galanga were collected from Cilviculture, Bhubaneswar, Odisha whereas Alpinia calcarata plants were collected from Phulbani district, Odisha. The samples were authenticated by Dr. P.C. Panda, Principal Scientist, taxonomy and conservation division, Regional Plant Resource Centre, Bhubaneswar and was grown in the medicinal plant garden of Centre for Biotechnology, Siksha O Anusandhan University, Bhubaneswar.

Preparation of extracts:

The fresh leaves and rhizomes were washed and cut into small pieces followed by drying for 15-20 days under shade separately. Then the dried materials were grounded into coarse powder and were extracted with methanol for 12h in soxhlet apparatus. The extracts were filtered with Whatman 40 filter paper and then concentrated by using a rotary evaporator to give rise to a semi-solid mass. Each extraction method was repeated thrice for the purpose of accuracy. The residues obtained were stored in the refrigerator for further analysis.

Preliminary phytochemical screening

The leaf and rhizome extracts of A.galanga and A.calcarata were subjected to different chemical tests for the identification of different phytoconstituents^[11,12].

Evaluation of Total phenolic content (TPC) and Total flavonoid content (TFC):

Total phenolic content of the leaf and rhizome extracts of both A. galanga and A. calcarata were determined by Folin-Ciocalteu method as described by Sahoo et al, 2013^[13] using Gallic acid as a standard. The result was shown as mg Gallic acid equivalents (GAE)/g of extract. Likewise, total flavonoid content of leaf and rhizome extracts of both A.galanga and A.calcarata were estimated using aluminium chloride colorimetric method ^[13]. TFC was analyzed from the calibration curve obtained through Quercetin, which was represented as mg Quercetin equivalent/g of the extract. All the determinations were performed in triplicate for ensuring the result.

GC-MS analysis of plant extracts:

GC-MS (gas chromatography coupled with mass spectrometry) analysis was carried out on a 6890 series instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with mass selective detector with quadrupole analyzer, MSD 5973. The electron ionization energy was 70 ev, ion source temperature 230°C and the interface temperature 280°C. A fused silica column HP-5 was used as the stationary phase. The oven was programmed as 50°C to 240°C at 4°C/min; then 240°C to 270°C at 15°C/min; held isothermal at 50°C for 1 min and at 270°C for 15 min. A sample of 1μl was injected through the mobile phase as helium with flow rate 1.0ml/min. The identification of compounds was performed by comparing their mass spectra with data from the US National Institutes of Standards and Technology (NIST, USA).

RESULTS AND DISCUSSION:

Phytochemical screening:

Preliminary phytochemical screening of the leaf and rhizome extract of A.galanga and A.calcarata revealed the presence of different phytoconstituents like alkaloids, flavonoids, steroids, triterpenoids, tanins and saponins which are shown in table 1.

Table 1: Preliminary phytochemical screening of leaf and rhizome extract of Alpinia galanga and Alpinia calcarata.

Phytoconstituents	Test performed	AG leaf	AG rhizome	AC leaf	AC rhizome
Alkaloids	Dragendroff’s test	-ve	-ve	-ve	+ve
Alkaloids	Mayer’s test, Wagner’s test, Hager’s test	+ve	+ve	+ve	+ve
Steroids	Libermann Burchard test, Salkowski test	+ve	+ve	+ve	+ve
Flavonoids	Alkaline reagent test, Shinoda test	+ve	+ve	+ve	+ve
Triterpenoids	Libermann Burchard test, Salkowski test	+ve	+ve	+ve	-ve
Carbohydrates	Molisch’s test, Fehling’s test	-ve	-ve	-ve	-ve
Carbohydrates	Barfoed’s test, Benedict’s test	-ve	-ve	-ve	-ve
Tanins	FeCl₂ test	+ve	+ve	-ve	-ve
Saponins	Foam test	+ve	-ve	-ve	-ve
Aminoacids	Millon’s test, Ninhydrin test	-ve	+ve	+ve	+ve
Glycosides	Killer-Kiliani test	-ve	-ve	-ve	+ve
Glycosides	Brontrager’s test	-ve	-ve	-ve	+ve

(+): Indicates the presence of chemical constituents, (-): Indicates the absence of chemical constituents

Evaluation of total phenolic and total flavonoid contents:

In our study, Total Phenolic Content (TPC) of leaf and rhizome extracts of A. galanga and A. calcarata was estimated by using modified Folin-Ciocalteu calorimetric method and represented in terms of GAE/g of the extract. It was calculated using the standard curve of Gallic acid (Standard curve equation: Y=0.004x+0.063, R²= 0.998). TPC of the leaf and rhizome extract of A.galanga was found to be 77.25±1.56 and 32.44±1.35 mg GAE/g of the extract respectively (Table 2). Similarly, in case of A. calcarata, TPC of the leaf extract was found to be higher (59.25±0.92mg GAE/g) than that of rhizome extract (37.75±0.95mg GAE/g) (Fig. 1).

Fig. 1: TPC and TFC value of both Alpinia galanga and Alpinia calcarata

Total Flavonoid Content (TFC) of leaf and rhizome extract of both the plants was calculated using the standard curve of Quercetin (standard curve equation: Y= 0.013x+0.487, R²=0.886) and represented in terms of Quercetin equivalent/g of the extract. Alpinia galanga leaf extract possessed high flavonoid contents (64.69±1.12 mg Quercetin equivalent/ g of extract) in compared to A.calcarata extracts (Table 2).

Table 2 : Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of leaf and rhizome extract of Alpinia galanga and Alpinia calcarata.

Sl. No	Name of Plant extracts	TPC	TFC
1	A.galanga leaf	77.25±1.56	64.69±1.12
2	A.galanga rhizome	32.44±1.35	39.46±1.05
3	A.calcarata leaf	59.25±0.92	38.38±0.56
4	A.calcarata rhizome	37.75±0.95	36.92±0.24

The plant secondary metabolites like phenolics and flavonoids are responsible for the plant antioxidant activity and consequently, it is pretty important to evaluate the total phenolic and total flavonoid content of plant extracts^[14]. The phenolics, one of the major and most abundant groups of plant secondary metabolites, possess bioactivities like antiapoptosis, antiaging, anticarcinogen, antiinflammation, antiatherosclerosis, cardiovascular protection and cell proliferation activities ^[15]. These diverse groups of compounds serve as potential natural antioxidants in terms of their capability to work as radical scavengers.

GC-MS analysis of extracts:

The result concerning to Gas chromatography and mass spectrometry analysis of the leaf and rhizome extracts of A. galanga lead to the identification of many chemical constituents. The various components present in the A.galanga extracts that were detected by the GC-MS are shown in (Table 3). Benzenepropanal (37.35±0.5%) and 3-phenyl-2-butanone (20.49±0.6%) were found to be the constituents of the leaf extract, whereas rhizome extract was found to contain carotol (17.44±0.3%), eucalyptol (13.89±0.2%), 5-hydroxymethylfurfural (11.28±0.3%) as major constituents (Table 3). The leaf and rhizome extract were rich in benzenepropanal and carotol respectively. Eucalyptol (13.89±0.2%) was also found in the rhizome extract. Carotol was reported in other Alpinia species like Alpinia nigra and Alpinia calcarata ^[16,17]. Antifungal activity of carotol has been reported by ^[18].

GC-MS analysis of A. calcarata leaf and rhizome extracts revealed the presence of 6 and 3 identified components accounting for 99.97% and 50.7% of the total peak area respectively. The major constituents of the leaf extract were olealdehyde (32.41±0.95%), hexadecanal (31.84±0.34%), phytol (11±0.52%), 9-hexadecenal (10.06±0.12%) whereas rhizome extract was found to contain hydroquinone (44.17±0.36%) and pyranone (6.19±0.35%) as major constituents. Detailed list of all the detected compounds with their chemical names, area percentages and retention time of each was given in (Table 3). The details including molecular formula, molecular weight and the structure of identified active principles were given in the table 4.

Table 3: Chemical constituents identified in leaf and rhizome extracts of Alpinia galanga and Alpinia calcarata.

S. No	Compound name	Peak Area (%) Mean±SD				Retention time (RT)
S. No	Compound name	AG leaf	AG Rhizomes	AC Leaf	AC Rhizomes	Retention time (RT)
1	Benzenepropanal	37.35±0.5	Nd	Nd	Nd	9.051
2	3-phenyl-2-butanone	20.49±0.6	Nd	Nd	Nd	11.880
3	Eucalyptol	Nd	13.89±0.2	Nd	Nd	5.470
4	Pyranone	Nd	7.74±0.3	Nd	Nd	9.174
5	α-Terpineol	Nd	9.09±0.3	Nd	Nd	10.519
6	Fenchyl acetate	Nd	5.44±0.1	Nd	Nd	11.508
7	5-Hydroxymethylfurfural	Nd	11.28±0.3	Nd	Nd	12.840
8	Cinnamic acid	Nd	3.82±0.1	Nd	Nd	17.957
9	Carotol	Nd	17.44±0.3	Nd	Nd	25.894
10	Palmitic acid	Nd	4.26±0.1	Nd	Nd	39.062
11	9-Hexadecenal	Nd	Nd	10.06±0.12	Nd	31.899
12	Hexadecanal	Nd	Nd	31.84±0.34	Nd	32.694
13	9,12-Octadecadienal	Nd	Nd	6.67±0.42	Nd	38.089
14	Olealdehyde	Nd	Nd	32.41±0.95	Nd	38.487
15	Octadecanal	Nd	Nd	7.99±0.63	Nd	39.155
16	Phytol	Nd	Nd	11.00±0.52	Nd	41.904
17	Pyranone	Nd	Nd	Nd	6.19±0.35	9.876
18	Hydroquinone	Nd	Nd	Nd	44.17±0.36	15.001
19	2-Deoxy-D-ribose	Nd	Nd	Nd	0.34±0.18	31.471

nd: not detected

Table 4: Details of identified phytoconstituents

S. No	Compound name	Molecular Formula	Molecular Weight	Structure
1	Benzenepropanal	C₉H₁₀O	134.1751
2	3-phenyl-2-utanone	C₁₀H₁₂O	148.2017
3	Eucalyptol	C₁₀H₁₈O	154.2493
4	Pyranone	C₆H₈O₄	144.1253
5	α-Terpineol	C₁₀H₁₈O	154.2493
6	Fenchyl acetate	C₁₂H₂₀O₂	196.2860
7	5-Hydroxymethylfurfural	C₆H₆O₃	126.1100
8	Cinnamic acid	C₉H₈O₂	148.1586
9	Carotol	C₁₅H₂₆O	222.3663
10	Palmitic acid	C₁₆H₃₂O₂	256.4241
11	9-Hexadecenal	C₁₆H₃₀O	238.415
12	Hexadecanal	C₁₆H₃₂O	240.4247
13	9,12-Octadecadienal	C₁₈H₃₂O	264.4461
14	Olealdehyde	C₁₈H₃₄O	266.4620
15	Octadecanal	C₁₈H₃₆O	268.4778
16	Phytol	C₂₀H₄₀O	296.5310
17	Pyranone	C₆H₈O₄	144.1253
18	Hydroquinone	C₆H₆O₂	110.1106
19	2-Deoxy-D-ribose	C₅H₁₀O₄	134.1305

GC-MS analysis has been done for determining the presence of possible bioactive chemical constituents present in the leaf and rhizome methanolic extracts. The presence of various bioactive compounds justifies the use of plant as medicine. The phytochemical profiling of the A. calcarata leaf still remains to be unexplored and to the best of our knowledge, this is the first report on the GC/MS profiling of the leaf extract of A. calcarata.

CONCLUSION:

The results of GC–MS analysis of the methanol extract of A. galanga and A. calcarata have furnished an array of compounds which could be responsible for a wide range of bioactivities. The presence of various bioactive compounds in both of the plants validates their use by traditional practitioners. Further investigation may lead to isolation of bio-active compounds and their structural elucidation and screening of pharmacological activity will be helpful for further drug development.

ACKNOWLEDGEMENT:

The authors are grateful to the Dean, Centre for Biotechnology, Siksha O Anusandhan University for providing facilities to carry out this research work.

CONFLICT OF INTEREST:

There is no conflict of interest.

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Received on 28.10.2019 Modified on 02.12.2020

Research J. Pharm. and Tech. 2020; 13(10):4735-4739.

DOI: 10.5958/0974-360X.2020.00834.3