Phytochemical, proximate analysis and antioxidant activity of the rhizome of Alpinia nigra (Gaertn.) B.L. Burtt (Zingiberaceae) in Tamulpur district, Assam

The present research examines the chemical composition, proximate characteristics and antioxidant analysis of Alpinia nigra ’ s rhizome extract. Re-sults of GC - MS analysis revealed a total of 22 volatile compounds of which Tris (tert - butyldimethylsilyloxy) arsane (2.89%), 2 - ethylthiolane, S, S - dioxide (3.47%), Ambrial (6.68%) and Silicic acid, diethyl bis (trimethylsilyl) ester (4.59%) were the major compounds. The rhizome was found to have variable quantities of proximate compositions such as moisture content (55.5%), ash content (4.72%), crude fat (52%), crude fibre (41.25%), crude protein (28.95 µg/g), carbohydrate (46.27%) and dry matter (33.4%). The crude extraction of the rhizome was done by Soxhlet apparatus. The phyto-chemical screening showed the presence of alkaloid, tannin, terpenoid, gly-coside, steroid, carbohydrate and a considerable amount of phenol and flavonoid. Phenolic and flavonoid content in the extracts was determined by the Folin - Ciocalteu and aluminium chloride assays as gallic acid equivalent and quercetin equivalent, respectively. To evaluate the antioxidant activity of rhizome extract, phosphomolybdenum assay, ferric - reducing anti-oxidant power (FRAP), and 1,1 - diphenyl - 2 - picryl - hydrazil (DPPH) were used. The findings indicate that the rhizome extract is rich in phenol and flavonoid content and would serve as a promising source of natural antioxidants in the food, cosmetics, and pharmaceutical industries.


Introduction
Medicinal plants represent the most ancient form of medication, used for thousands of years in traditional medicine in many countries around the world.The empirical knowledge about their beneficial effects was transmitted over the centuries within human communities (1).About 80 % of people around the globe still rely primarily on traditional medicines for their primary healthcare, demonstrating the vital role played by the plant-based traditional healing system in healthcare (2).Nutrition and medicines obtained from plant resources have been extensively used by evaluating their health benefits because of its richness in antioxidant properties and other chemical constituents.Plants naturally synthesize chemical compounds known as secondary metabolites, which are distributed throughout in various parts of the plant.These secondary metabolites play a crucial role in the plant's de-fence mechanisms and serve as a means to protect the plant from oxidative damage caused by free radicals (3).The ability of free radicals to induce oxidative stress plays an important role in human pathophysiological diseases (4,5).Phytochemicals are able to neutralize the free radicals in the human body and hence alleviates many chronic diseases (6).In pursuit of compounds that promote natural defence, special attention is focused on secondary metabolites, generated by plants with known nutritional or therapeutic effects.Polyphenols, which include flavonoids, isoflavones, anthocyanins and catechins, are believed to possess the strongest antioxidant capabilities.Since humans cannot produce these substances, consuming them through food is crucial for maintaining good health, particularly in fighting against free radicals (7).
A recent study shows that 1403 wild species of plants under 184 families are consumed throughout India (8).Amongst these, Alpinia nigra (Zingiberaceae) is one of the wild herbaceous plants which have been used for therapeutic and dietary purposes.In one of the most common herbal treatments, A. nigra is used in Thai traditional medicine to treat stomach ailments and gastrointestinal disorders, as it has antibacterial and antifungal properties (9).It is found mostly in Yunnan and Hainan Province of China, Thailand, Bhutan, Sri Lanka and India (10).North-east India is one of the richest and most diversified regions for Zingiberaceae, where 88 different species of plants under 19 genera were documented (11). A. nigra occurs in marshy slope between low hillocks in different parts of Assam and Tripura.Different tribal communities of north-east India have been using A. nigra as a part of their diet with rice.Shoot and rhizome parts of the plant are also well known for its use in medicinal and culinary item.Indigenous people of Tripura drink the aqueous juice of A. nigra shoots to treat intestinal parasite infections.Although the plant is a beloved vegetable in the diet, the native people of Tripura in some areas also utilise it as a food flavouring agent.Similarly, in Assam, the aqueous extract of A. nigra's shoot and rhizome has been used to treat conditions like gastric ulcers, irregular menstruation, bone weakness and jaundice (12).Leaf extract from A. nigra exhibits cytotoxic, antimicrobial and analgesic properties (13,14).Experiments conducted using rhizome extract validated it as antibacterial and cytotoxic drugs (15).But there is no scientific justification on the use of rhizome of A. nigra as nutritional and antioxidant source.Despite this, this medicinal plant still has a bright future because more research is still needed to fully understand the phytochemical makeup and potential health benefits of many species.Therefore, this study has been devoted to work on phytochemical, proximate and antioxidant analysis of the rhizome of A. nigra.

Collection of plant material
The specimen, Alpinia nigra was collected from a village, Kekerikuchi under Tamulpur district, Assam (Fig. 1) and authenticated in Department of Botany, Bodoland University, Kokrajhar, Assam.

Preparation of rhizome
The rhizomes of A. nigra were separated and washed with running water for 3-5 times and further washed with distilled water.The washed specimens were cut into slices and shade dried on blotting paper at room temperature for 3-4 days (Fig. 2).

Preparation of rhizome extract
The dried rhizomes were grinded using an electronic grinder.About 10 g powdered rhizome of A. nigra was extracted with Soxhlet apparatus for 7-8 h using methanol and hexane as solvents separately.Filtrate obtained was concentrated in rotary evaporator and crude extracts were kept at −4 °C for further analysis.The extracts were dissolved in 5 % dimethyl sulfoxide (DMSO) at the concentration of 10 mg/mL and subjected to analysis of antioxidant activity.

Preliminary phytochemical screening of A. nigra
Methanolic extracts of rhizome was used to detect the presence of different secondary metabolites using standard laboratory procedures.The different tests, Wagner's test for alkaloid, Ferric chloride test for tannin, foam test for saponin, aluminium and ammonium test for flavonoids, Salkowski's test for terpenoid and steroid, Folin-Ciocalteu test for phenol, Keller-kiliani test for glycoside, Molisch test for carbohydrate were performed (16)(17)(18)(19)(20)(21)(22).Alkaloid 1-2 mL of the extract was warmed with 2 % sulphuric acid (H2SO4) for 2 min.Appearance of reddish-brown precipitation upon the addition of Wagner's reagent indicates the presence of alkaloid.Carbohydrate 1 mL of extract was treated with 0.5 mL of Molisch reagent and 1 mL of conc.H2SO4 was added to it.Appearance of purple to violet colour ring at the junction indicates the presence of carbohydrates.

Flavonoid
Ammonium test 1 mL of the extract was heated with 5 mL of ethyl acetate in boiling water for 3 min.The mixture was filtered and the filtrates were shaken with 1 mL of dilute ammonium solution (1 %).The layers were allowed to separate.A yellow colouration at ammonium layer indicates the presence of flavonoid.

Aluminium test
1 mL of the extract was heated with 5 mL of ethyl acetate in boiling water for 3 min.The mixture was filtered and the filtrates were shaken with 1 mL of 1 % aluminium chloride solution.Light yellow colouration indicates the presence of flavonoid.

Glycosides
1 mL of glacial acetic acid was mixed with 2 mL of rhizome extract and 5 % FeCl3 was added to it.Then conc.H2SO4 was added by the side wall of the test tube.Reddish brown appearance at the junction of 2 liquids and bluish green in the upper layer confirms the presence of glycosides.Phenol 1-2 mL of the extract was treated with few drops of 5 % ferric chloride solution.Appearance of bluish black colour indicates the presence of phenol.

Saponin
A small amount of the extract was shaked with 5 mL of distilled water in a test tube.Appearance of foamy layer on the top indicates the presence of saponin.

Steroid
1-2 mL of the extract was treated with 2 mL of chloroform and 3 mL of conc.H2SO4 was added carefully by the side wall of test tube.Formation of greenish yellow fluorescent in the chloroform layer with acid confirms the presence of steroid.

Tannin
A small amount of the extract was diluted with distilled water and 2-3 drops of 5 % ferric chloride solution was added.If the solution turns transient green to black colour, it indicates the presence of tannin.Terpenoid 1-2 mL of extract was treated with 2 mL of chloroform and 3 mL of conc.H2SO4 was added carefully.Formation of reddish-brown colouration in the interface indicates the presence of terpenoids.

Ash content
For ash content, 2 g of the powdered sample was placed in an incubated tared crucible and weighed.Then it was placed under muffle furnace at 550 °C for 5-6 h until the sample became white colour (ash) (23) and its content determined as follows:

Determi-nation of crude fat content
A cleaned round bottom flask was dried in oven at 105 °C for 2 h to ensure the weight of the bottle is stable.About 3 g of sample was weighed and wrap with filter paper.It was

Dry matter of the plant
It was calculated using the formula given below (14).

Determination of carbohydrate
It was estimated by the difference method based on traditional carbohydrate determination (25).

Protein estimation
Lowry's method using Bovine serum albumin (BSA) as the working standard was performed for protein estimation (26).Reagents A (2 % of Na₂CO₃ in 0.1 NaOH), B (0.5 % CuSO4 in 1 % NaK tartarate solution) and C (100 mL of reagent A + 2 mL of reagent B) was prepared.To 0.2 mL of the methanolic extract (2 mg/mL) taken in 3 test tubes, the volume was made up to 1 mL with distilled water and 5 mL of reagent C was added to each test tubes and incubated for 15 min at room temperature.Further 0.5 mL of FC reagent was added to the mixture and incubated for 30 min.
The absorbance was measured at 660 nm against distilled water as blank in UV-VIS spectrophotometer (UV-1900i, Shimadzu).The protein in the extract was determined as micrograms of BSA equivalent (BSAE) per gram of dry weight (µg BSAE g -1 DW) using standard BSA graph.

Antioxidant activity of rhizome of A. nigra
Total phenolic content (TPC) The total phenolic contents (TPC) were determined by Folin-Ciocalteu reagent after Slinkard and Singleton, 1977 using gallic acid as a standard phenolic compound (27).A total of 50 µL of the extract was taken in a test tube and diluted with distilled water (1.8 µL).Then 1 mL of a 10 % Na2CO3 and 150 µL of Folin-Ciocalteu was added and thoroughly mixed.The mixture was then allowed to stand undisturbed for a duration of 10-20 min.The absorbance was measured at 765 nm in UV-VIS spectrophotometer (UV-1900i, Shimadzu) against blank consisting of all the reaction agents except the extract.The total concentration of phenolic compounds in the extract was expressed as milligrams of gallic acid equivalent (GAE) per gram of dry weight (mg GAE g -1 DW) using standard graph.

Total flavonoid content (TFC)
Total flavonoid content (TFC) was determined following Dowd method (28).To 50 µL of extract solution, diluted using 1.95 mL distilled water, 1 mL of 2 % aluminium trichloride (AlCl3) in methanol was added.The mixture was further incubated at ambient room temperature for 10 min.Absorbance was measured at 415 nm in UV-VIS spectrophotometer (UV-1900i, Shimadzu) against blank that lack extracts.The total flavonoid content was expressed as mg of quercetin equivalent (QE) per g of dry weight (mg QE g -1 DW) using standard quercetin graph.

Phosphomolybdenum assay
Phosphomolybdenum assay was done to evaluate total antioxidant compounds (29).The assay is used for the reduction of Mo(VI)-Mo(V) by antioxidant compound and subsequent formation of a green phosphate Mo(V) complex at low pH.Briefly, in a 3 mL vial, a 0.1 mL aliquot solution was mixed with 1 mL of a reagent solution comprising 0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate and diluted upto 3 mL with distilled water.The mixture was incubated for 90 min in water bath at 95 °C.After cooling to room temperature, the absorbance was measured at 765 nm in UV-VIS spectrophotometer (UV-1900i, Shimadzu) against blank (0.1 mL of methanol in place of extract).The results were estimated from the standard ascorbic acid graph as milligrams of ascorbic acid equivalent (AAE) per g of dry weight (mg AAEg -1 DW).

Ferric-reducing antioxidant power (FRAP) assay
Reducing power of the extracts was determined (30), using ascorbic acid as standard.Various concentrations of the extracts (10-50 µL) were mixed with 1 mL of phosphate buffer (0.2 M, pH 6.6) and 1 mL of 1 % potassium ferricyanide [K3Fe (CN)6].After 30 min of incubation at 50 °C in water bath, 1 mL of 10 % trichloroacetic acid was added to the mixture to stabilise the reaction and it was centrifuged (3000 rpm) for 10 min.Then, 1 mL of supernatant was mixed with 1.5 mL distilled water and FeCl3 (0.5 mL, 0.1 %) and absorbance measured at 700 nm in UV-VIS spectrophotometer (UV-1900i, Shimadzu) against blank (all the reagents without extract).The reducing power is increased when the reaction mixture shows higher absorbance.

Scavenging DPPH radicals
The free radical scavenging activity of plant extracts was https://plantsciencetoday.online determined by 1,1-diphenyl-2-picryl-hydrazil (DPPH) method (31,32).Different concentrations of methanolic extract and standard in the range 10-50 µL were prepared in test tubes and added 2 mL of methanolic solution of DPPH at the concentration of 0.05 mg/mL and then the total volume in each test tube was adjusted to 3 mL by adding distilled water.The mixture was vigorously shaken and left to stand still at room temperature for a period of 30 min.Then the absorbance at 517 nm was determined using a UV-VIS spectrophotometer (UV-1900i, Shimadzu), with methanol serving as the blank.The DPPH scavenging effect was computed using the following equation: DPPH scavenging effect (%) = (A0 -A1 /A0) × 100….. (Eqn.8) Where, A0 is the absorbance of the negative control (2 mL of methanolic solution of DPPH + 1 mL of 5 % DMSO) and A1 is the absorbance of reaction mixture or standards.Ascorbic acid was used as a standard.

GC-MS analysis
For the GC-MS analysis, the methanol extract of the sample was run in GC-MS instrument (Perkin Elmer (USA), Model Clarus 680 d amp, CalruMS, 600C MS) comprising a liquid auto-sampler.TurboMass Ver 6.1.2.software was used and the peaks were analysed by data analysis NIST-2014 software.The capillary column used was 'Elite-5MS' having dimensions-length-60 m, ID-0.25 mm and film thickness-0.25 µm.The stationary phase constituted 5 % diphenyl, 95 % dimethyl polysiloxane.In the GC-Protocol, helium gas (99.99 %) was used as a carrier gas at a flow rate of 1 mL/min.An injection volume of 2 µL was employed in splitless mode.Injector temperature was 280 °C, and ion-source temperature was 180 °C.The oven temperature was programmed at 60 °C (for 1 min), with an increase at the rate of 7 °C/min to 200 °C (hold for 3 min), then again increased at a rate of 10 °C/min to 300 °C (hold for 5 min).The total run time was ~39 min.Solvent delay was kept for 8 min.A MS protocol mass spectral measurement was taken in electron impact positive (EI+) mode at 70 eV.A solvent delay of 8 min was there for MS scan.Mass range i.e., m/z was 50-600 amu.Interpretation of the peaks obtained from GC chromatogram were done by comparing with the mass spectrum library of corresponding peaks using the database software of National Institute Standard and Technology (NIST-2014).The mass spectra of unknown components were compared with the known components of the NIST library and compounds were identified by name, molecular weight and empirical formula.

Statistical analysis
Statistical analyses were performed in excel by doing Kruskal-Wallis test in OriginPro 9.0, as the data were not normally distributed.All the values were represented as mean ± SE of three parallel measurements.Differences were considered significant at p < 0.05 and p < 0.01.The IC50 value (µg/mL) was calculated using the AAT Bioquest IC50 value calculator (AAT Bioquest, Inc., Sunnyvale, CA, USA).

Qualitative phytochemical screening of A. nigra
Phytochemical investigation is of utmost significance in identifying new source of medicinally and scientifically valuable compounds.Different colours observed after performing with different tests and the inference of phytochemical screening is tabulated in Table 1.

Proximate analysis of rhizome of A. nigra
The assessment of the proximate composition of edible plant parts is necessary to determine their nutritional relevance and their byproducts are dependent on the digestion of digestible fibre in it.This will enable higher enzyme activity by increasing the food's surface area.The results of the proximate composition of rhizome of A. nigra are given in Table 2.

Total phenolic content (TPC)
The total phenolic compounds in rhizome extracts of A. nigra were calculated as gallic acid equivalent (GAE) using the equation obtained from a standard gallic acid graph (y = 0.0047x + 0.0082, R² = 0.9985) (Fig. 3).The phenolic content was higher in the methanol extract (11.97 ± 0.38 mg GAE/g DW) than the hexane extract (3.25 ± 0.23 mg GAE/g DW).

Total flavonoid content (TFC)
TFC were determined as quercetin equivalent using the equation obtained from the standard quercetin graph (y = 0.0121x + 0.0059, R 2 = 0.9984).Hexane extract had higher flavonoid content compared to methanol extract (Fig. 4).

Ferric-reducing antioxidant power (FRAP) assay
The FRAP activity was expressed as ascorbic acid equivalent using the equation obtained from the standard ascorbic acid graph (y = 0.0119x -0.053, R² = 0.9978).Methanol extract showed higher reducing power than hexane extract (Fig. 6).

Scavenging DPPH radicals
The percentage of scavenging DPPH radicals of the extracts are shown in Table 3. From the result it is clear that the % of free radical scavenging properties of both the methanol and ethanol extract increased linearly with the increase in concentration and was lower than the standard.However, the inhibitory effect was at equilibrium with the standard at high concentration (40 μg/mL) for methanol extract.At p < 0.05 and p < 0.01, there was a statistically significant difference among the concentration and extracts (Table 4 and 5).

GC-MS analysis
From phytochemical analysis, methanolic extracts showed better result so it was considered for further GC-MS analysis.Fig. 7 represents the chromatogram of GC-MS analysis of the extracts.Results of GC-MS analysis of the extracts revealed 22 compounds (Table 6).

Discussion
Phytochemical research is essential when looking for novel compounds with medicinal and scientific significance.
The data obtained from the study demonstrated that the rhizome of Alpinia nigra has chemicals of medicinal importance.The rhizome contained alkaloid, carbohydrate, flavonoid, glycoside, phenol, steroid, tannin and terpenoid.The information regarding the identification and standardisation of secondary metabolites is validated by the current study.It necessitates additional research on notable sources of distinctive bioactive compounds for its therapeutic and pharmacological applications.
Proximate composition of rhizome extract showed moisture, ash, fat, fibre, protein, carbohydrate and dry matter.Since there was a fair amount of moisture content, the chances of developing microorganisms are high which shortens the sample's shelf life.Ash content was found to be low (4.72 %).It is a diagnostic purity index and represents physiological and non-physiological ash.Its determination is crucial as it helps to understand the chemical composition and potential use of the sample.The adequate carbohydrate content present in the sample signifies it as a good source of energy and facilitates digestion and absorption of other nutrients.Significant proportion of crude protein in A. nigra suggests that the rhizome can be exploited as a possible source of protein.Examining the advantages of consuming more plant protein instead of animal protein is particularly relevant in terms of lowering the risk of cancer.High crude fibre content helps the gut to absorb trace elements, avoids diverticulosis, and promotes the removal of undigested food through the intestine (20,33,34).
Of all the classes of plant metabolites, phenolic compounds are one of the biggest and most prevalent (35).Phenolic compounds contain hydroxyl groups, which are significantly responsible for antioxidant activity and free radical terminators.These substances are the main sources of hydrogen for free radicals to stop the initial chain reaction of lipid oxidation.The hydroxyl groups in phenolic substances can inhibit the formation of free radicals in the human body (36).They also exhibit biological attributes including prevention of ageing, reducing inflammation, preventing atherosclerosis, protecting the cardiovascular system, improving endothelial function and suppressing angiogenesis and cancerous cells (37).
Flavonoid is a very significant natural phenol with low molecular weight known to have a wide range of chemical and biological activities, including the ability to scavenge free radicals.Flavonoids have major function in how a plant interacts with its surroundings and shields it from a variety of biotic and abiotic stresses.It is understood to be a class of polyphenolic compounds with two phenyl rings connected by a propane bridge to form the distinctive 15-carbon (C6-C3-C6) flavan skeleton (38).The ability to counteract free radicals depends on the number and position of the OH group, which are linked to the ability of these compounds to donate electrons (39)(40)(41).As indicated in Fig. 5 the methanol extract contained the highest concentration of antioxidant compounds, whereas hexane had the lowest antioxidant compound.The antioxidant compounds can stabilize the highly reactive free radicals (42).
The potential antioxidant activity of substances is determined by their reducing power.The reducing potential is typically associated with the presence of its reductons, generated on breaking of free radical chain by donating hydrogen atom.The reduction of ferrous ions (Fe 2+ ) to ferric ions (Fe 3+ ) is observed by the intensity of the bluegreen resultant solution, which absorbs at 700 nm.The present result shows the ferric reducing activity of extracts, and is due to the presence of polyphenol compounds, which may act in a similar way to reductons by donating electrons and reacting with free radicals to convert them into more stable products and terminate chain reactions of free radicals.In this assay, there is an electron transfer mechanism present that could affect the activity of phenolic compounds present in the extracts (43).
DPPH method has been employed for the evaluation of the free radical scavenging activity of the extracts of several natural products (44).This assay is used for the preliminary test which indicates the reactivity of the test extract compounds with a stable free radical.It gives purple-coloured strong absorption maximum band at 517 nm, which on reduction by the extract compound decreases in absorbance and discolouration from purple to yellow.It has been reported that the presence of the hydroxyl functional group of phenolic compounds having redox properties attributes the antioxidant activity (45).A statistically significant difference was observed within the concentrations and between the extracts and control (Tables 4 and  5).
Rhizome extracts showed a reasonably high level of antioxidant activity that is connected to flavonoid and phenolic components and are able to neutralise free radicals including superoxide, singlet oxygen and hydroxyl radicals, which makes them considerably more effective at scavenging radicals.The discovery of this work provides strong evidence that rhizome extracts of A. nigra are potent radical scavengers and contain a variety of naturally occurring antioxidants and active bioactive chemicals with desired therapeutic potential.

Conclusion
The nutritional value, phytochemical screening and antioxidant activity formed the central focus of this study.The findings of the present study indicate that the rhizome of Alpinia nigra can be considered as good sources of nutrition.The consumption of this species as food can be nutritionally advantageous.The presence of phytoconstituents may have potential antimicrobial, anti-diabetic and antioxidant activity which are being considered as an important aspect in pharmacognostic evaluation of the medicinal plant.The outcomes are really promising, and in fact, compound analysis is required to implement them for pharmaceutical applications.

Table 1 .
Preliminary phytochemical screening of methanolic extract of the rhizome of A. nigra.

Table 2 .
Proximate composition of rhizome of A. nigra.

Table 3 .
Percentage scavenging rate of DPPH free radicals by the rhizome of A. nigra.

Table 4 .
Kruskal-Wallis Anova of DPPH between the extracts and ascorbic acid in each concentration.(NS = not significant).

Table 5 .
Kruskal -Wallis Anova of DPPH between the concentration in each extract.

Table 6 .
GC-MS chromatogram of methanol extract of the rhizome of A. nigra.Compounds detected and identified in the methanol extract of the rhizome of A. nigra by GC-MS.