INTRODUCTION:

Allium tuberosum is a species of onion, native to south western parts of the Chinese province of Shanxi, and cultivated and naturalized elsewhere in Asia and around the world. Allium tuberosum is commonly known as Garlic chives or Chinese chives. In India, it is called as Maroi Nakuppi.In cooking, it is used as a substitute for onions and garlic^1,2. It is commonly found in South-east Asian countries like China, Japan, Korea, India, Thailand, Nepal, etc. In India, it is commonly found in Northeast state specially Manipur. The whole plant of Allium tuberosum is antibacterial, cardiac, depurative, stimulant, stomachic and tonic³. It is an anti-emetic herb that improves kidney function. It is used internally to treat urinary incontinence, kidney and bladder weakness, etc. The seed of the plant is carminative and stomachic.

They are used in India for the treatment of spermatorrhoea. The leaves and the bulbs are applied to bites, cuts and wounds. In traditional folk medicine, Allium tuberosum was used for treating parasites in the intestines, reinforce the immune system, promote digestion as well as to cure anaemia^4,5. The spicy smell of Allium tuberosum have the role of eliminating stasis to activate blood circulation and invigorate the circulation of blood, suitable for traumatic injury, nausea, vomiting, enteritis, chest pain and other symptoms⁶. Allium tuberosum contain a lot of vitamins, fiber, iron, calcium, sulfur and magnesium⁷. While keeping in mind the extensive use of this herb in medicines, the current study was designed to determine the various phytochemical compounds present in Allium tuberosum and to determine its antioxidant and α-amylase activity from its hexane and methanolic extracts.

MATERIALS AND METHODS:

Collection of plant materials⁸

The fresh plant sample (Allium tuberosum)was collected from Imphal, Manipur. The plant materials were washed properly with water and shade dried for evaporation of water molecules. After drying, the plant materials were grinded well into fine powder and transferred into airtight containers.

Preparation of leaf extracts:

The plant extracts were prepared by Soxhlet extraction method. Two different solvents of varied polarity (methanol and hexane) were used. Extraction was continued for 24 hours for each solvent. Further, the extract was poured in porcelain dish and using hot plate, heated at 30-40ºC for solvent evaporation.

Phytochemical screening:

Phytochemical screening of hexane and methanolic extracts wereperformed using standard protocols^9-12.

Test for Saponin:

2ml of extract was added in 5ml distilled water. And 3 drops of olive oil was added. Then the test tube was shaken vigorously for about 30 seconds.Appearance of froth indicates presence of saponin.

Test for Carbohydrates:

Benedict's test – 2ml of extract was added in 2ml of Benedict’s reagent and was gently boiled in hot water bath for 5 minutes.

Fehling's test – 2ml of extract was added in 2ml of Fehling's A and 2ml Fehling's B and was gently boiled in hot water bath for 5 minutes.

Test for Tannins:

2ml of extract was added in 2ml of 10% solution of ferric chloride.A bluish-black or brownish-green precipitate indicated the presence of tannins.

Test for Glycosides:

Liebermann's test – 2ml of extract was added in 2ml of chloroform and 2ml of acetic acid. Then the mixture was cooled well in ice for 30 minutes. Concentrated sulphuric acid was then added.

Salkowski' test – 2ml of extract was added in 2ml of chloroform and 2ml of concentrated sulphuric acid.

Test for protein:

2ml of extract was added in 2ml of concentrated nitric acid.

Test for Flavonoids:

Alkaline reagent test – 2ml of extract was added in 2ml of 2% of sodium hydroxide. Appearance of green colour indicates the presence of flavonoids.

Sulphuric acid test – 2ml of extract was added in 2ml of 15% ferric chloride. Appearance of orange colour indicates the presence of flavonoids.

Test for Alkaloid:

1ml of Mayer’s reagent was added in 2ml of extract. Appearance of yellow colour indicates the presence of alkaloids.

Antioxidant Assay (DPPH Method):

In-vitro antioxidant activity of the plant extracts were assayed by DPPH method^13-15. DPPH is a commercially available stable free radical which is purple in colour.

The preparation of the test solution with varying concentration of the plant sample was prepared for stock solution of 1000µg/ml by making the total volume up to 1ml with distilled water. 2ml of aqueous DPPH solution was added and the final volume was made up to 3ml. Solutions were incubated for about 30mins. Further, absorbance was taken at 517nm against a blank aqueous solution without DPPH.

The DPPH radical-scavenging assay of the plant sample was calculated by the formula:

(Acontrol – Asample)

% of inhibition = ----------------------------------- x 100x

Acontrol

In vitro α-AMYLASE ASSAY:

In vitro α-amylase inhibition assay was performed to evaluate the anti-diabetic properties of the extract^16-18,.Different concentration of sample- 50, 100, 200, 300 and 400 µg/ml was prepared. 1% starch was prepared by dissolving1gm of starch in 100ml distilled water. The starch solution was boiled and then filtered for use. DNS was prepared by mixing 1gm DNS, 200 gm crystalline phenol and 50 gm sodium sulphite in 100ml of 1% NaOH. Amylase prepared by taking 1ml saliva in 19ml 0.2M phosphate buffer (7.4). Amylase was mixed with the sample at different concentrations to which 1% of starch solution and phosphate was added. The reaction was allowed to occur at 37ºC for 5 min and terminated by addition of 2ml of DNS reagent. The reaction mixture was heated for 15 min at 100ºC. α-amylase activity was determined by measuring colour intensity at 540 nm in spectrophotometer.

RESULTS AND DISCUSSION:

Phytochemical Screening:

Preliminary phytochemical screening of hexane and methanolic extracts were done and the results are summarized in Table 1. Phytochemicals such as flavonoids, alkaloids, glycosides, saponins, etc. were found to be present.

Table 2 :Phytochemical analysis of A. tuberosum

Phytochemicals	Results
Phytochemicals	Hexane extract	Methanol extract
Carbohydrates
a. Fehling test	-ve	-ve
b. Benedict test	-ve	-ve
Proteins	+ve	-ve
Glycosides
a. Liebermann's test	+ve	+ve
b. Salkowski'stest	-ve	+ve
Flavanoids
a. Alkaline reagent test	+ve	+ve
b. Sulphuric acid test	-ve	-ve
Alkaloids	+ve	+ve
Saponins	+ve	+ve
Tanins	-ve	-ve

Antioxidant ASSAY:

The results of in-vitro antioxidant activity for hexane and methanolic extracts (10µg/ml, 40µg/ml, 70µg/ml, 100µg/ml and 130µg/ml) are summarized in Table 2and 3.Upon using standard formula of % Inhibition (absorbance) for calculation, it is found that antioxidant activity increases along with the increase in the concentration of the extracts (figure1 and 2). The increase in in-vitro antioxidant activity along with the increase in concentration of sample is plotted in bar graphs for their pictorial presentation. Methanolic extracts showed more activity compared to hexane extract against the same concentrations (10µg/ml, 40µg/ml, 70µg/ml, 100µg/ml and 130µg/ml).

Hexane extract:

Table 2: Absorbance for antioxidant assay of hexane extract

Concentration of sample	Absorbance
10 µg/ml	0.394
40 µg/ml	0.325
70 µg/ml	0.289
100 µg/ml	0.210
130 µg/ml	0.188

Positive control: 0.510

% Inhibition:

Absorbance of positive control – Absorbance of sample

= --------------------------------------------------------- × 100

Absorbance of positive control

10µg/ml: I % = 22.7%, 40µg/ml: I % = 36.2%, 70 µg/ml: I % = 43.3%, 100µg/ml: I % = 58.8%, 130 µg/ml: I % = 63.1%

Fig 1: Graph representing inhibition %of hexane extract

Methanol extract:

Table 3: Absorbance for antioxidant assay of Methanol extract

Concentration of sample	Absorbance
10µg/ml	0.271
40µg/ml	0.240
70µg/ml	0.190
100µg/ml	0.181
130µg/ml	0.166

Positive control: 0.510

% Inhibition

Absorbance of positive control – Absorbance of sample

= --------------------------------------------------------- × 100

Absorbance of positive control

10µg/ml: I % = 46.8%, 40µg/ml: I % = 52.9%, 70 µg/ml: I % = 62.7%, 100µg/ml: I % = 64.5%, 130 µg/ml: I % = 67.4 %

Fig 2: Graph representing inhibition % of methanol extract

In-vitro α-amylase assay (Anti-diabetic assay):

The results of in-vitro α-amylase assayof hexane and methanol extract are summarized in Table 4 and 5. Upon standard calculation of % Inhibition, it was found that the more the concentration of the sample the more was the inhibition activity in both the extracts (hexane and methanol).

Hexane extract:

Table 4: Absorbance for α-amylase inhibition assay of hexane extract

Concentration of sample	Absorbance
50 µg/ml	0.319
100 µg/ml	0.294
200 µg/ml	0.223
300 µg/ml	0.188
400 µg/ml	0.151

Positive control: 0.405

% Inhibition

Absorbance of positive control – Absorbance of sample

= --------------------------------------------------------- × 100

Absorbance of positive control

50µg/ml: I % = 21.2 %, 100µg/ml: I % = 27.4 %, 200 µg/ml: I % = 44.9 %, 300µg/ml: I % = 53.5 %, 400 µg/ml: I % = 62.7 %

Methanol extract:

Table 5: Absorbance for α-amylase inhibition assay of Methanol extract

Concentration of sample	Absorbance
50 µg/ml	0.300
100 µg/ml	0.274
200 µg/ml	0.212
300 µg/ml	0.184
400 µg/ml	0.145

Positive control: 0.405

% Inhibition

Absorbance of positive control – Absorbance of sample

= --------------------------------------------------------- × 100

Absorbance of positive control

50µg/ml: I % = 25.9%, 100µg/ml: I % = 32.3%, 200 µg/ml: I % = 47.6%, 300µg/ml: I % = 54.5%, 400 µg/ml: I % = 64.19%

The present work was conducted to study the qualitative phytochemical nature, antioxidant activity and α-amylase activity of the extract of Allium tuberosum which were collected from Manipur. The results of the qualitative phytochemical nature, antioxidant activity and α-amylase activity of the extract of Allium tuberosum were confirmed by using standard protocols. The phytochemical analysis of hexane and methanol extract shows better results for protein, glycosides, flavonoids, alkaloids and saponins (Table 1). Carbohydrates and tannins are absent in both the extracts. Presence of alkaloids supports the use of these extracts as anti-inflammatory, antibacterial and anti-malarial agents^19,20. Moreover, presence of flavonoids indicates the health promoting properties such as anti-allergy, antibacterial, anti-cancer, etc. of these extracts^{21, 22}. The results showed that the plant extract under study showed good antioxidant properties. The maximum percentage of inhibition in hexane extract is 63.1% at 130µg/ml concentration. And the maximum percentage of inhibition in methanol extract is 67.4% at 130µg/ml concentration (Table 2 and 3).Oxidative potential of plants have found effective towards oxidative stress which otherwise leads to the development of life threatening disease²³. The study also reveals that the extracts possess considerable α-amylase inhibiting properties. The maximum percentage of inhibition in hexane extract is 62.7% at 400µg/ml concentration. And the maximum percentage of inhibition in methanol extract is 64.19% at 400µg/ml concentration (Table 4 and 5)

CONCLUSION:

In the present study, phytochemical screening and evaluation of antioxidant and α-amylase activities of the extract of Allium tuberosumwere performed by using hexane and methanol as a solvent. Presence of secondary metabolites (phytochemicals) can be attributed to the pharmacological property of the plant leaves. The results indicate the potentiality of the plant as a source of natural antioxidant and anti-diabetic molecules. It also illuminates the consideration of these extracts for further clinical trials in various pharmaceutical purposes.

CONFLICT OF INTEREST:

None

ACKNOWLEDGEMENT:

The authors would like to acknowledge Assam down town University for providing the lab facilities.

REFERENCES:

1. Saini N, Wadhwa S, Singh GK, Rawal S, Singhal S. Comparative study between cultivated garlic (Allium sativum) and wild garlic (Allium tuberosum). Asian Journal of Pharmaceutical Research and Development. 2013 Jul 1:108-25. https://ajprd.com/index.php/journal/article/view/96

2. Panda S, Mandal M, Satpathy M. Evaluation of the extract of Allium cepa Linn. for biochemical and antibacterial activities. Asian Journal of Research in Chemistry. 2016;9(3):113-5.10.5958/0974-4150.2016.00020.1

3. Khalid N, Ahmed I, Latif MS, Rafique T, Fawad SA. Comparison of antimicrobial activity, phytochemical profile and minerals composition of garlic Allium sativum and Allium tuberosum. Journal of the Korean Society for Applied Biological Chemistry. 2014 Jun;57(3):311-7. https://doi.org/10.1007/s13765-014-4021-4

4. Roshan S, Khan A, Ali S, Tazneem B. Effect of Allium sativum in Immoblilization Stress Induced Albino Rats. Liver. 2010 Aug 28;5(62.5):6-818.

5. Choudhary R. Benificial effect of Allium sativum and Allium tuberosum on experimental hyperlipidemia and atherosclerosis. Pakistan Journal of Physiology. 2008 Dec 31;4(2):7-10.https://pjp.pps.org.pk/index.php/PJP/article/view/826

6. Sultana F, Mohsin M, Sah AN. In-vitro Antioxidant and Antimicrobial Activity of Allium tuberosum Rottler. ex Spreng. Int. J. Adv. Res. Biol. Sci. 2015;2(12):178-87.

7. Savithramma N, Rao ML, Suhrulatha D. Screening of medicinal plants for secondary metabolites. Middle-East Journal of Scientific Research. 2011;8(3):579-84.

8. Karthik VP, Suresh P, David DC. In vitro hydrogen peroxide scavenging activity and alpha amylase inhibitory activity of Croton tiglium extract. Research Journal of Pharmacy and Technology. 2019 Jun 1;12(6):3045-7. DOI: 10.5958/0974-360X.2019.00515.8

9. Gogoi P, Islam M. Phytochemical screening of Solanum nigrum L and S. myriacanthus Dunal from districts of upper Assam, India. IOSR J Pharm. 2012;2(3):455-9. 10.9790/3013-0230455459

10. Banu KS, Cathrine L. General techniques involved in phytochemical analysis. International journal of advanced research in chemical science. 2015 Apr;2(4):25-32.

11. Jose BE, Panneerselvam P. In vitro Antidiabetic activity of Ethanol and Aqueous leaf extracts of Azima tetracantha Lam. Research Journal of Science and Technology. 2019 Mar 31;11(1):64-8. 10.5958/2349-2988.2019.00009.3

12. Singh MP, Jaiswal R, Mandal S. Evaluation of Immunomodulatory Activity of Aqueous Extract of the Bulb of Allium sativam In Wistar Rats. Research Journal of Pharmacology and Pharmacodynamics. 2010;2(6):401-6.

13. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. sci. technol. 2004 Dec;26(2):211-9.

14. SLDV RM, Das MC, Vijayaraghavan R, Shanmukha I. In vitro evaluation of antidiabetic activity of aqueous and ethanolic leaves extracts of Chloroxylon swietenia. National Journal of Physiology, Pharmacy and Pharmacology. 2017;7(5):486.

15. Bimal KG, Eun SS, Eun HK, Amal KG, Chang YY, Bal KG. A comparative evaluation of the antioxidant activity of some medicinal plants popularly used in Nepal. Journal of Medicinal Plants Research. 2011 May 18;5(10):1884-91.https://doi.org/10.5897/JMPR.9000006

16. Nhut PT, An TN, Minh LV, Truc TT, Anh NH. Phytochemical screening of Allium Tuberosum Rottler. ex Spreng as food spice. InIOP Conference Series: Materials Science and Engineering 2020 Dec 1 (Vol. 991, No. 1, p. 012021). IOP Publishing.

17. Manimaran M, Kannabiran K. Alpha amylase activity of Streptomyces sp. VITMK1 isolated from marine soil sample of Pichavaram, Tamil Nadu, India. Research Journal of Pharmacy and Technology. 2018 Jun 30;11(6):2180-2. 10.5958/0974-360X.2018.00403.1

18. Helen PA, Bency BJ. Inhibitory potential of Amaranthus viridis on α-amylase and glucose entrapment efficacy In vitro. Research Journal of Pharmacy and Technology. 2019;12(5):2089-92.

19. Khanam Z, Wen CS, Bhat IU. Phytochemical screening and antimicrobial activity of root and stem extracts of wild Eurycoma longifolia Jack (Tongkat Ali). Journal of King Saud University-Science. 2015 Jan 1;27(1):23-30. 10.1016/j.jksus.2014.04.006

20. Rangasamy P, Hansiya VS, Maheswari PU, Suman T, Geetha N. Phytochemical analysis and evaluation of In vitro antioxidant and anti-urolithiatic potential of various fractions of Clitoria ternatea L. Blue flowered leaves. Asian Journal of Pharmaceutical Analysis. 2019 Apr;9(2):67-76. 10.5958/2231-5675.2019.00014.0

21. Talukdar N, Dutta AM, Chakraborty R, Das K. Screening of phytochemicals, antioxidant and inhibitory effect on alpha-amylase by ethanolic extract of Elaeocarpus Ganitrus (Bark). International Journal of Pharmaceutical Sciences and Research. 2017 Dec 1;8(12):5270-5.10.13040/IJPSR.0975-8232.8(12).5270-75

22. Kasote DM, Katyare SS, Hegde MV, Bae H. Significance of antioxidant potential of plants and its relevance to therapeutic applications. International journal of biological sciences. 2015;11(8):982. 0.7150/ijbs.12096

23. Narasimhan R. Phytochemical screening and antioxidant studies in the Pulp extracts of Cucurbita maxima. Asian Journal of Pharmaceutical Research. 2016 Mar 1;6(1). 10.5958/2231-5691.2016.00001.0

Received on 17.12.2021 Modified on 04.05.2022

Research J. Pharm. and Tech 2023; 16(4):1987-1991.

DOI: 10.52711/0974-360X.2023.00326