Indian Journal of Traditional Knowledge Vol. 15(4), October 2016, pp. 669-674

Nutritional and phytochemical evaluation of A. lividus L. syn. subsp. oleraceus (L.) Costea

Nazish Nehal, Sonia Mann & Rajinder K Gupta* University School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, Delhi - 110078, India E-mail: [email protected]

Received 05 November 2015, revised 04 January 2016, updated 14 June 2016

The determination of the proximate composition of A. lividus showed the extract to be rich in protein (17.28 ± 0.42 gm/100 gm and dietary fiber (8.35 ± 0.16 gm/100 gm) but very low in fat content (0.69 ± 0.05 gm/100 gm). The phytochemical screening of the various extracts revealed the presence of phenolics ranging between 1.38 to 9.07 µg GAE/mg sample, flavonoid in the range 0.88-5.04 µg RE/mg sample, alkaloids (8.16 %) saponins (29.44 %) and tannins (4.27 %). The bioactive compounds were explored through GC-MS which showed presence of compounds like β-sitosterol, phytol and tetradecane in high amounts. Methanolic soxhlet extract showed highest radical scavenging activity against ABTS (IC50 - 1.61±0.01 mg/ml), DPPH (IC50 - 3.18±0.04 mg/ml), FRAP (161.39±0.64 TE/g extract) and reducing power assays. The extracts screened against various bacterial strains showed good to moderate antibacterial activity. Findings of the study justify the traditional usage of A. lividus and provide evidence of its potential use as antioxidants and antimicrobials.

Keywords: A. lividus, Proximate, Phytochemical, Antioxidant, GC-MS, Antibacterial activity IPC Int. Cl.8: A61K 36/00, C09K 15/00, C07

FAO’s (Food and Agriculture Organization) recent received less research attention4. Vegetable estimates of population suffering from chronic Amaranthus has been rated equal to or superior in undernourishment across the world (842 million; taste to spinach and is considerably higher in calcium, 12 % of the global population) and in developing iron, and phosphorous5. A. lividus has two synonyms regions (827 million)1 indicates towards finding Amaranthus blitum L. (1753), Amaranthus oleraceus diet-based ways of combating such nutritional L. (1763). This species of Amaranthus is available in disorders. One such remedy can be the exploration of various parts of the world and has common names underutilized foods like indigenous leafy vegetables such as wild , pigweed and purple which are inexpensive sources of nutrition and can be Amaranthus. In India, amaranthus plant is used to eradicate micronutrient malnutrition and indigenously known as Chaulai or lalsaag. Leaves degenerative diseases owing to their allelopathic and stem are prepared with other vegetables as a di, properties2. Moreover, the growing interest in especially potato. In North and South India, lal bhaji exploration of dietary phytochemicals with rich is a favourite dish and eaten with rice6. The antioxidant activity is paving the way towards the Amaranthus being resistant to varied soil and commercial development of new, natural, cost- agroclimatic conditions, has low production cost and effective and safe sources of antioxidants3. is one of the cheapest vegetable in the tropical market Amaranthus grows in the wild of the Indian (esp. North India), therefore, often described as the subcontinent and is often considered a weed. The poor man’s vegetable7. Earlier studies have Amaranthus and its different genotypes are emphasized the use of Amaranthus as a vegetable and divided into vegetable Amaranthus (two major grain crop which can be a cheap alternative rich species, A. tricolor and A. lividus) and grain source of protein, carotenoid, vitamin C, dietary fiber8 Amaranthus. In contrast to grain Amaranthus which is and nutrient for poor people, particularly in a multipurpose high-potential crop for agriculture, developing countries. food, feed and forage uses; vegetable Amaranthus has In Nigeria, A. lividus is used as a medicine against —————— lung disorders. The fluid extract of the plant is used as *Corresponding author an astringent internally in the treatment of ulcerated 670 INDIAN J TRADIT KNOWLE, VOL. 15, NO. 4, OCTOBER 2016

mouths and throats; and externally as a wash for Phytochemical analysis ulcers and sores9. It is also recommended in diarrhea, The total phenolics content of the various extracts dysentery and hemorrhages from bowels. A. lividus of A. lividus were determined by Folin-Ciocalteu also finds application in Ayurveda (known as Marisa, method. Gallic acid (10-50 µg/ml) was used as the Vaspaka, etc., in Sanskrit) and is used to relieve vata, standard and the total phenolic content of the extracts cough and cold, and control excessive bile secretion. were expressed in terms of µg of gallic acid The whole plant acts as an excellent cooling agent for equivalents (GAE) per mg of dry weight. Aluminium urinary troubles, as a lotion for external use, in chloride colorimetric method was used to estimate relieving pain during pregnancy and as a remedy total flavonoids content of the sample. The results were for skin diseases. Its root extracts along with amla expressed in µg of Rutin equivalents (20-100 µg) per mg and bark of Ashok and Daru Haldi are used to of dry weight of the extracts based on the linear equation treat leucorrhea6. In the Unani system of medicine, using the calibration curve of Rutin as the standard. the juice of the Amaranthus root is considered a The crude alkaloid and saponin content in the extract remedy for gastric problems, leucorrhoea, was determined on dry weight basis. Assessment of menorrhagia, boils, burns and nausea. It is popularly crude tannin content was performed by determining used as antipyretic, appetizer, diuretic, febrifuge, the optical density measured at 605 nm using tannic galactogogue, haematinic, laxative and stomachic acid as the standard to plot the calibration curve12. effects and as treatment for hallucination, leprosy, eczema and piles10. Thus, the present study has been Determination of secondary metabolites composition carried out to explore the various aspects (nutritional, Secondary metabolites were identified by GC-MS phytochemical, antioxidant, antimicrobial) of (Agilent Technologies; Agilent 6890 GC and 5975B underutilized plant Amaranthus lividus. MSD) by split injection (1:20) at 280 C13. Screening of volatiles and semi-volatiles were performed using Material and methods NIST’05 library. Plant material The plant specimen, Amaranthus lividus was Free radical scavenging activity collected from a local market of Dwarka, New Delhi, The 1,2,2'-azino-bis-(3-ethylbenzthiazoline-6- India. The collected sample was identified (Ref. No. sulphonic acid) (ABTS); 2,2-diphenylpicryl- hydrazyl NISCAIR/RHMD/Consult/2013/2219/225) by Dr Sunita hydrate (DPPH); ferric reducing antioxidant power Garg at National Institute of Science Communication (FRAP) free radical-scavenging activity and reducing and Information Resources, Council of Scientific and power of various extracts were also determined12. Industrial Research, New Delhi. Amaranthus lividus, an annual plant species belonging to the plant family Anti-microbial assay is commonly called purple Agar Well-diffusion method Amaranthus. This plant is native to Mediterranean The various extracts of A. lividus were tested for region and is now available in other parts of the world. their antibacterial activity14. Antimicrobial activity

Sample preparation and extraction was determined by measuring the diameter of the The aerial portion of the plant specimen, zone of inhibition. Amaranthus lividus ( and stem) was chopped and MIC (Minimum inhibitory concentration) solvent extracted with methanol. Two methanolic Plant extracts were screened to determine MIC extracts [by soxhlet extraction (MSE) and modified against bacterial microorganisms, using broth methanolic extraction (ME)] and one aqueous extract microdilution susceptibility assay. For detecting (AE) were prepared, and stored at 4 C for further growth, 40 µl of 70 % alcoholic solution of INT analysis. (conc. 0.1 mg/ml) was added to each well where Proximate composition analysis microbial growth was detected by the change in the The nutritional constituents of A. lividus were color of INT dye from yellow to purple. MIC values determined for moisture, ash, protein, fat, were defined as the lowest concentration which carbohydrate and crude fiber content (AOAC, 2005). completely inhibited microbial growth. The results Mineral content was analyzed by inductively coupled were expressed in mg/ml. Tetracycline was used as plasma optical emission spectrometry (ICP-OES)11. the positive control. NEHAL et al.: NUTRITIONAL AND PHYTOCHEMICAL EVALUATION OF A. LIVIDUS LEAVES 671

Results and discussion for their biological functions such as protection Proximate analysis against allergies, inflammation, free radicals, platelet Moisture content and dry matter analysis during aggregation, microbes, ulcers, hepatoxins, viruses and 17,18 nutrition reporting is very important because it tumours . Secondary metabolites like alkaloids are directly affects its nutritional content, stability and known to control development in living system and 19 storage. The result of the proximate composition of have a protective role in animals . A. lividus was found A. lividus leaves show high moisture content (85.01 %). to be an abundant source of alkaloids (8.16 ± 0.41 %) Ash content, which is an index of mineral contents indicative of its therapeutic values. The results preserved in the food material, was low, 13.81 % for reveal the crude sample to have high levels of A. lividus leaves as compared to that of other green saponin content (29.44 ± 0.62 %) suggesting its leafy vegetables. The variability is dependent upon pharmacological and commercial use. Saponins are the species and also on parameters like environmental known for their medicinal uses, as they exhibit temperature, relative humidity during growth and antispasmodic, antidiabetic and anticancerous 20 relative amount of metabolic water produced in the activity . Tannins (often considered as antinutrients) plant. The crude protein content of A. lividus was was found under acceptable limits (4.27 ± 0.69 %) found to be 17.28 % . According to Pearson15, plant which can easily be detoxified by soaking, boiling or food that provides more than 12 % of its calorific frying. Tannins also have astringent properties, hastens 21 value from protein is considered good source of wound healing and inflamed mucous membranes . protein. A. lividus showed negligible crude fat content The above results of the various biologically significant (0.69 %), thus an ideal component in several diets and chemical constituents of A. lividus leaves demonstrate more palatable, as dietary fats function to increase its potential use in therapeutics. palatability by absorbing and retaining flavours. The carbohydrate content of A. lividus was found to be GC-MS analysis very low, i.e., 4.13 % as compared to other green GC-MS analysis showed presence of various leafy vegetables. The crude fiber content in A. lividus bioactive compounds in methanolic crude extract was recorded as 8.35 % which was significantly high. (ME). β-sitosterol was present in maximum amount Crude fiber helps in digestion, prevention of colon (27.44 %) followed by phytol (10.68 %) and cancer, controlling cholesterol metabolism and eicosamethyl-cyclodecasiloxane (8.63 %). Phytol regulation of blood sugar16. Low calorific value of possesses anticancer, antioxidant, anti-inflammatory, antitumor, antimicrobial, diuretic, chemopreventive any food makes it good for the diet of obese people. 22 The energy value of A. lividus was estimated to be properties and often used in vaccine formulations . 91.92 kcal/ 100 gm and found to contain macro- Sitosterol is reported to show antihyperlipoproteinaemic, antibacterial, antimicotic and antitumor activity minerals (K: 33410.10 ppm, Ca: 19295.00 ppm, Na: 23 5138.20 ppm, Mg: 19101.00 ppm, P: 3964.90 ppm), in vivo . Presence of tetradecane was also detected micro-minerals (Fe: 309.60 ppm, Mn: 24.30 ppm, Zn: which exhibits antibacterial activity. Earlier studies have reported heptadecane to possess antioxidant and 1.20 ppm, Cu: 6.30 ppm) and negligible amount of 24 toxic heavy metals (Pb: 1.40 ppm, Cd: 0.30 ppm, Cr: antimicrobial properties . Ergost-5-en-3-ol, (3, β) is a steroid and found to be biologically active against liver 0.00, As: 0.00, Hg: 0.00) 25 diseases, jaundice and artherosclerosis . Hexadecanoic Phytochemical analysis acid, methyl ester has been reported to show Phenols and phenolic compounds have been used bioactivities like antioxidant, hypocholesterolemic, as disinfectants and remain the standard when pesticide, 5-α reductase inhibitor and also imparts compared to other bactericides. The total phenolic flavor26. The presence of such prevailing bioactive content of the various extracts of A. lividus was found compounds shows that A. lividus can be a potential to be highest for MSE (9.07 ± 0.05), followed by ME source of pharmaceutical raw material (Table 1). (6.41 ± 0.11) and then, AE (1.38 ± 0.038). The content of flavonoid expressed in µg of Rutin Free radical scavenging activity equivalents (RE) per mg of dry weight of the extracts The antioxidant activity A. lividus extracts was was estimated as 5.04 ± 0.04µg RE/mg sample evaluated in accordance with the decolorization of the (MSE), 4.81 ± 0.05µg RE/mg sample (ME) and 0.88 ABTS to its radical cation ABTS+. The ABTS radical ± 0.03 µg RE/mg sample (AE). Flavonoids are known scavenging activity was found to be linearly 672 INDIAN J TRADIT KNOWLE, VOL. 15, NO. 4, OCTOBER 2016

Table 1—Secondary metabolites profiling of A. lividus MeOH extract Compound Name Molecular RT Cas# % formula Area

Tetradecane C14H30 17.532 000629-59-4 0.25

Valeric acid C5H10O2 18.631 109717-37-5 0.97

Phytol C20H40O 21.761 000150-86-7 10.68

Cyclodecene C10H18 19.506 066633-38-3 2.51

Nonadecane C19H40 19.663 000629-92-5 1.42

Hexadecanoic acid, C17H34O2 19.932 000112-39-0 1.40 methyl ester

Cyclodecasiloxane, C20H60O10Si 20.370 018772-36-6 8.63 eicosamethyl- 10

Eicosane C20H42 20.650 000112-95-8 0.91

Ergost-5-en-3-ol, C28H48O 21.334 004651-51-8 5.35 (3.β.)-

Heptadecane C17H36 21.581 000629-78-7 6.31

Cyclooctasiloxane, C16H48O8Si8 21.873 000556-68-3 7.96 hexadecamethyl-

Rolicyprine C14H16N2O2 22.030 002829-19-8 1.41

Cyclononasiloxane, C18H54O9Si9 23.174 000556-68-3 5.94 octadecamethyl-

1,1,1,5,7,7,7- C16H48O6Si7 24.374 038147-00-1 4.16 Heptamethyl-3,3- bis(trimethylsiloxy) Fig. 1—ABTS scavenging activity of various extracts of tetrasiloxane A. lividus, a) ME-methanolic solvent extract, MSE- methanolicsoxhlet extract, b) AE- aqueous extract 3,6-Dioxa-2,4,5,7- C10H30O2Si4 25.495 004342-25-0 2.31 tetrasilaoctane, 2,2,4,4,5,5,7,7- octamethyl-

β-Sitosterol C29H50O 25.753 000083-46-5 27.44

5-Methyl-2- C15H13N 29.421 036944-99-7 1.50 phenylindolizine

N-Methyl-1- C13H21NO 30.968 031897-93-5 1.77 adamantaneacetamide

1,5- C11H18O3 31.226 069225-59-8 7.67 Dioxaspiro[5.5]undecan- 9-one, 3,3-dimethyl increasing with the increasing concentration of the extracts (Fig. 1). Ascorbic acid and BHT were used as standards. The inhibitory concentration (IC50) of various extracts for ABTS radical scavenging ability can be ranked as MSE (1.61 ± 0.01) > ME (1.89 ± 0.02) > AE (83.91 ± 0.52). Among the various extracts of A. lividus, MSE exhibited the highest DPPH radical scavenging activity in a dose-dependent manner (Fig. 2). Present free radical scavenging results showed the IC 50 Fig. 2—Free radical scavenging activity of various extracts of (mg/ml) ranking order as MSE (3.18 ± 0.04) > ME A. lividus by DPPH antioxidant assay; a) ME-methanolic solvent (3.22 ± 0.03) > AE (51.91 ± 0.62). extract, MSE- methanolicsoxhlet extract, b) AE- aqueous extract NEHAL et al.: NUTRITIONAL AND PHYTOCHEMICAL EVALUATION OF A. LIVIDUS LEAVES 673

Table 2—Antibacterial activity of various extracts of A. lividus against bacterial strains Bacterial strains Diameter of Zone of inhibition (mm) Agar Well-Diffusion method MIC values ME MSE AE MSE Gram positive B. cereus 10.0 ± 0.00 11.3 ± 0.56 _ 7.0 B. subtilis 12.7 ± 0.49 11.5 ± 0.63 _ 12.5 M. luteus 10.7 ± 0.53 7.0 ± 0.0 10.0 ± 0.0 _ S. aureus _ _ _ 6.25 S. epidermis 8.0 ± 0.0 7.0 ± 0.0 8.0 ± 0.0 6.25

Gram negative Fig. 3—Reducing power estimation of A. lividus extracts; E. coli 8.3 ± 0.39 _ _ 1.25 ME- methanol solvent extract, MSE- methanol soxhlet extract P. aeruginosa _ _ _ _ current values with previously reported other S. typhi 9.3 ± 0.58 8.8 ± 0.56 _ 6.8 Amaranthus species (A. hybridus, A. spinosus and A. S. flexneri _ _ _ _ caudatus) reflect that A. lividus is more potent in antibacterial action than others27. A. viridis on the The ferric reducing activity of the various extracts other hand shows better inhibition against E. coli and were found to be 161.39 ± 0.64, 132.86 ± 0.80, 7.50 ± S. aureus than A. lividus28. In order to confirm the 0.04 µg TE/mg extract for MSE, ME and AE, validity of the results obtained in the study and its use respectively. In comparison to the aqueous extract, the infood preservation or medicinal purposes, in vivo methanolic extracts (MSE and ME) had shown better studies may be required to address issues of safety antioxidant activity, being highest for MSE. Increase and toxicity29. in absorbance was noticed in a concentration- dependent manner for standard and various extracts of Conclusion A. lividus (Fig. 3). The present study has shown the presence of phenolics, flavonoid, alkaloids and saponins in high Antimicrobial activity amount in A. lividus. The plant was found to be All the 6 bacterial strains used to demonstrate nutritionally adequate due to the high protein and low antibacterial activity by broth dilution method showed some degree of sensitivity against the 3 extracts fat content. Present findings show that this vegetable plant can be used as a functional food. The presence ranging from 1.25-12.5 mg/ml. The quantitative of several therapeutically important bioactive estimation for antimicrobial activity for A. lividus compounds makes A. lividus a potential source of extracts against food-borne and pathogenic food as well as biopharmaceuticals. Further, effective microorganisms are shown in Table 2. scientific study is needed for isolation of such Comparatively, MSE exhibited good inhibition compounds before being put to commercial use in the against the test microorganisms which can be drug industry. attributed to the phenolic content of the sample. MIC values reflected E. coli to be the most sensitive References (1.25 mg/ml) to the extracts while B. subtilis 1 The State of Food Insecurity in the World: The multiple exhibited least inhibition with MIC values of dimensions of food security, Food & Agriculture 12.5 mg/ml. The antibacterial activity demonstrated Organization of the United Nations, Rome, Italy, 2013 by well diffusion method was not in accordance with 2 Gupta S, Lakshmi AJ, Manjunath MN & Prakash J, Analysis of nutrient and antinutrient content of underutilized green MIC results. These problematic results can be leafy vegetables, LWT-Food Sci Technol, 38 (2005) 339-345. attributed to differences in microbial growth, 3 Selvamuthukumaran M & Khanum F, Processing exposure of micro-organisms extracts, non-uniform seabuckthorn fruit for antioxidant rich jam development and diffusion of extracts in the agar medium. shelf stability assessment, Indian J Tradit Knowle, 13(2) (2014) 335-346. Antibacterial activity of A. lividus extract has not 4 Lopez V, Akerreta S, Casanova E, Garcia-Mina JM, been reported yet. However, a comparison of the Cavero RY & Calvo MI, In vitro antioxidant & anti-rhizopus 674 INDIAN J TRADIT KNOWLE, VOL. 15, NO. 4, OCTOBER 2016

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