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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 11  |  Issue : 2  |  Page : 88-96

Phytochemical analysis and evaluation of antioxidant potential of ethanol extract of Allium cepa and ultra-high homoeopathic dilutions available in the market: A comparative study


1 Department of Chemistry, St. Stephen's College, University of Delhi, New Delhi, India
2 Central Council for Research in Homoeopathy, New Delhi, India

Date of Web Publication8-Jun-2017

Correspondence Address:
Ekta Kundra Arora
Department of Chemistry, St. Stephen's College, University of Delhi, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijrh.ijrh_13_17

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  Abstract 


Introduction: As per statistics available with the WHO, 80% of the earth's population rely on traditional medicine for their primary health-care needs and most of this therapy involves the use of plant extracts and their active components. Objectives: To confirm the presence of alkaloids, fats, steroids, tannins, flavonoids, sugars, amino acids and saponins in Allium cepa extract. Method: A qualitative phytochemical analysis has been performed to confirm the presence of these components in Allium cepa extract and its homoeopathic formulations (mother tincture [MT], Allium cepa 30C and Allium cepa 200C). The total phenolic content, flavonoid content and antioxidant potential of the prepared extract and the various homoeopathic formulations have also been studied. Results: Presence of various phytochemicals such as flavonoids, alkaloids, saponins, steroids, carbohydrates and amino acids have been confirmed in the prepared extract as well as the MT. According to calculations using Avogadro's limit, preparations above 12C dilution should have no source material present. It is interesting to note that even though the formulations Allium cepa 30C and 200C are considered ultra-high dilutions, they gave a positive result for many phytochemicals. The total phenolic content, flavonoid content and antioxidant potential of the prepared extract and various homoeopathic formulations have also been studied. Conclusion: The positive qualitative and quantitative results also reinforce the growing belief that Homoeopathy is not just a placebo effect but a 'Smart medicine' which may be working on the nanoscale. More research is required to understand various aspects.

Keywords: 2,2-diphenyl-1-picrylhydrazyl, Allium cepa extract, Homoeopathy, Mother tincture, Nanomedicine, Phytochemical analysis


How to cite this article:
Arora EK, Sharma V, Khurana A, Manchanda A, Sahani DK, Abraham S, Kundu D, Gupta H, Chiru LB, Sharma N, Garg N, Jomy S. Phytochemical analysis and evaluation of antioxidant potential of ethanol extract of Allium cepa and ultra-high homoeopathic dilutions available in the market: A comparative study. Indian J Res Homoeopathy 2017;11:88-96

How to cite this URL:
Arora EK, Sharma V, Khurana A, Manchanda A, Sahani DK, Abraham S, Kundu D, Gupta H, Chiru LB, Sharma N, Garg N, Jomy S. Phytochemical analysis and evaluation of antioxidant potential of ethanol extract of Allium cepa and ultra-high homoeopathic dilutions available in the market: A comparative study. Indian J Res Homoeopathy [serial online] 2017 [cited 2017 Jul 27];11:88-96. Available from: http://www.ijrh.org/text.asp?2017/11/2/88/207660




  Introduction Top


Traditional medicine is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, treatment or improvement of physical and mental illness.[1] The WHO has developed and launched the traditional medicine strategy 2014–2023 in response to the World Health Assembly resolution on traditional medicine (62.13). The strategy aims to support member states in developing proactive policies and implementing action plans that will strengthen the role traditional medicine plays in keeping populations healthy. Allium cepa (Onion) is one of the most important commercial condiment vegetables grown and consumed not only in India but also worldwide. It is known that onions may help guard against many chronic diseases and are highly valued for their therapeutic properties. It has been reported to be very high in quercetin content. It is said to be particularly useful in the treatment of patients suffering from running eyes and nose. The onions ability to relieve congestions, especially in the lungs and bronchial tract is well documented.[2] Onions contain large amounts of flavonoids such as quercetin which is abundant in onions and protects against cataracts, cardiovascular diseases and cancer.[3],[4] Organosulfur compounds present in it have been linked to lowering blood pressure and cholesterol levels.[5] It is known to possess anthelmintic, anti-inflammatory, antiseptic, antispasmodic, carminative, diuretic, expectorant, febrifuge, hypoglycaemic and hypotensive properties.[6],[7] Onion relieves stomach upset and other gastrointestinal disorders and also strengthens the appetite.

Homoeopathy is a time-tested two-century-old empirical system of healing. Homoeopathic medicines are prepared through a characteristic process known as potentization, where serial dilutions are performed with strong strokes at each step of dilution.[8] Homoeopathic pharmacists prepare medicines from various biologically active substances that belong to two main groups: organic materials (plants, animal products and nosodes) and inorganic substances (synthetic chemicals, metal alloys and natural and synthetic ceramic materials).[9] These bioactive materials are subjected to specific manufacturing procedures whereby they are dissolved in water and alcohol. To become homoeopathic remedies, medicinal compounds are diluted hundreds, even thousands of times to reduce their toxicity and to ensure that they are biologically active and compatible with the processes of human physiology. The major objection to homoeopathic medicine is that the doses of medicine prescribed in some cases are too dilute (beyond Avogadro limit) for any active ingredient to be present. The medicines, therefore, receive criticism on their activity and are termed as having a placebo effect. A further examination of dilution to establish that homoeopathic medicines may not be as dilute as a simplistic application of Avogadro's Principle but retains the original source substance due to surface effects has been explained by some mathematical models based on Langmuir equation.[10]

New evidence is emerging on the nature and properties of high dilution of homoeopathic medicines. These findings indicate that the science of Homoeopathy is a form of Nanomedicine, with the medicines capable of initiating changes in the physiological and biochemical dynamics of the animal as a complex adaptive system.[11] Several lines of evidence suggest that homoeopathic high dilutions do have pharmacological action.[10]

In the present work, we have prepared Allium cepa extract, studied the antioxidant activity, measured the total flavonoid and phenolic content, carried out its phytochemical analysis and compared it with the mother tincture (MT) of Allium cepa and homoeopathic dilutions 30C Allium cepa and 200C Allium cepa.


  Methodology Top


Allium cepa MT, 30C and 200C (Dr. Reckeweg) have been used for all studies. All other chemicals have been procured from Sigma-Aldrich. All reagents have been prepared following standard protocols. Allium cepa MT has been used as the positive control.

Preparation of onion extract

The onion bulb was washed with freshly prepared sterile distilled water. The outer covering of the bulb was manually peeled off, and the fleshy part of the onion was rewashed with freshly prepared sterile distilled water and weighed. The onion bulb was cut into small parts and squashed. To the squashed preparation, 90% ethanol (200 mL) was added for 8 h with 10 min interval shaking. The extraction was filtered using muslin cloth and Whatman No. 1 filter paper. The filtrate was evaporated at 45°C to dryness and was kept in a sterile bottle and refrigerated until use.

Determination of the total phenolic content

The concentration of phenolics in the samples was determined using spectrophotometric method of Singleton and Rossi.[12] The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained. A 1 mg/mL methanol solution of the extract was prepared for analysis. The reaction mixture was prepared by mixing 0.5 mL of methanol solution of extract/samples, 2.5 mL of 10% Folin–Ciocalteu's reagent in water and 2.5 mL 7.5% NaHCO3. The blank is prepared by mixing 0.5 mL methanol, 2.5 mL 10% Folin–Ciocalteu's reagent dissolved in water and 2.5 mL of 7.5% of NaHCO3. The samples were incubated in a thermostat at 45°C for 45 min and the absorbance determined spectrophotometrically at λmax 765 nm. The same procedure is repeated for the standard solution of gallic acid and the calibration line is construed. Based on the measured absorbance, the concentration of phenolics was read (mg/mL) from the calibration line, and the content of phenolics in the samples was expressed in terms of gallic acid equivalent (mg of GA/mL of sample).

Determination of the total flavonoid content

The total flavonoid content was measured with the aluminium chloride colorimetric assay.[13] One millilitre of methanol and 1 mL of standard quercetin solution (5, 10, 20, 40, 60, 80 and 100 μg/mL) were added to the test tubes. This was followed by 4 mL of distilled water and 0.3 mL of 5% sodium nitrite solution in each. A volume of 0.3 mL of 10% aluminium chloride was added after 5 min followed by 2 mL of 1 M sodium hydroxide. The volume was made up to 10 mL with distilled water and the contents were mixed well. An orange yellowish colour developed. The absorbance was measured at 510 nm spectrophotometrically. The samples were prepared in triplicate. A calibration curve was plotted using the values for standard quercetin. The data of total flavonoid content were expressed as milligram of quercetin equivalents/mL of sample.

Determination of the antioxidant potential

The ability of the plant extract/sample to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals is assessed by the standard method.[14] A simple method that has been developed to determine the antioxidant activity of foods utilises the stable DPPH radical. The odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm and is purple in colour. The colour turns from purple to yellow as the molar absorptivity of the DPPH radical at 517 nm reduces when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H. The resulting decolourisation is stoichiometric with respect to number of electrons captured. The samples are prepared in triplicate for each analysis and the mean value of absorbance is obtained. The stock solution of the extract was prepared in methanol to achieve the concentration of 1 mg/mL. Dilutions were made to obtain concentrations of 1000, 900, 800, 700, 600, 500 and 400 μg/mL. Diluted solutions (1 mL each) were mixed with 1 mL of methanolic solution of DPPH in concentration of 1 mg/mL. After 30 min incubation in darkness at room temperature (23°C), the absorbance was recorded at 517 nm. Control sample contained all the reagents except the extract. Percentage inhibition was calculated and IC50 values were estimated from the percentage inhibition versus concentration plot.

Qualitative phytochemical analysis

Plants contain many bioactive chemical substances that produce definite physiological and biochemical actions in the human body. These bioactive constituents are alkaloids, tannin, flavonoids, phenolic compounds, etc.[15],[16] Since Allium cepa has received considerable attention in recent years due to diverse pharmacological properties including antioxidant and antitumor activity, a preliminary phytochemical study to determine the phytoconstituents present was undertaken for the extract prepared and the homoeopathic formulations. The ethanol extract was considered suitable since the homoeopathic formulations are in ethanol medium. All tests were performed on the blank. The MT is used as the positive control.

Test for steroids

Salkowski reaction

To the different sample solutions, chloroform was added followed by concentrated sulphuric acid along the sides of the tube. A red-brown colouration indicated the presence of steroids.

Test for alkaloids

The samples were dissolved separately in 1% HCl. One millilitre of the solution was taken and a few drops of the following reagents were added and various tests performed.

Dragendroff's test

One millilitre of Dragendroff's reagent was added to the different sample solutions. Formation of reddish brown precipitate indicated the presence of alkaloids.

Mayer's test

One millilitre of Mayer's reagent was added to the different sample solutions. Formation of cream colour precipitate indicated the presence of alkaloids.

Hager's test

One millilitre of Hager's reagent was added to the different sample solutions. Formation of yellow colour precipitate indicated the presence of alkaloids.

Wagner's test

One millilitre of Wagner's reagent was added to the different sample solutions. Formation of reddish brown precipitate indicated the presence of alkaloids.

Test for tannins

Ferric chloride test

Different sample solutions were treated with ferric chloride solution; appearance of blue and green colours indicated the presence of hydrolysable and condensed tannins.

Lead acetate test

Small quantity of the sample solutions was dissolved in distilled water and 10% lead acetate solution was added to them, a white precipitate indicated the presence of phenolics and tannins.

Test for flavonoids

Alkaline reagent test

To the different sample solutions, a few drops of sodium hydroxide solution were added. Formation of intense yellow colour, which turned colourless after addition of few drops of dilute hydrochloric acid, indicated the presence of flavonoids.

Shinoda test

To the different sample solutions, a few magnesium turnings were added followed by a few drop of concentrated hydrochloric acid. Appearance of a crimson red colour indicated the presence of flavonoids.

Test for carbohydrates

Molisch's test

The solution to be tested are mixed with a small amount of Molisch's reagent (alpha-naphthol dissolved in ethanol) in a test tube and mixed well. A small amount of concentrated sulphuric acid was slowly added down the sides of the sloping test tube. Appearance of purple ring at the junction indicated the presence of carbohydrates.

Fehling's test (for reducing sugars)

The sample solutions are mixed with a small amount of Fehling's solution and heated. Appearance of a red precipitate indicated the presence of a reducing sugar.

Test for saponin glycosides

Froth formation test

A small quantity of the samples was diluted with 20 mL of distilled water and shaken vigorously; formation of 1 cm layer of foam which is stable for 10 min indicated the presence of saponins.

Test for amino acids

Ninhydrin test

A solution of ninhydrin in ethanol is added to the sample solutions. Appearance of a purple colour indicated the presence of amino acids.

Test for cardiac glycosides

Keller–Killiani test

Glacial acetic acid (0.4 mL) and a few drops of 5% ferric chloride solution are added to the sample solutions. Concentrated sulphuric acid (0.5 mL) is added along the side of the test tube carefully. The formation of blue colour in the acetic acid layer confirmed the presence of cardiac glycosides.

Test for anthraquinone glycosides

Hydroxyanthraquinone test

To 1 mL of the samples, a few drops of 10% potassium hydroxide solution were added. The formation of a red colour confirmed the presence of anthraquinone glycosides.

Test for proteins

Biuret test

To 2 mL of the sample solutions, 5 drops of 1% copper sulphate solution are added followed by 2 mL of 10% NaOH. The contents are mixed thoroughly. Formation of a purple or violet colour confirmed the presence of proteins.


  Results Top


Total phenolic content

The total phenolic contents of the samples using Folin–Ciocalteu's reagent are expressed in terms of gallic acid equivalent in [Figure 1]. The reagent which is a mixture of phosphotungstic acid and phosphomolybdic acid which after oxidation of the phenols is reduced to a mixture of blue oxides of tungsten and molybdenum. The blue colouration produced has a maximum absorption in the region of 765 nm and is proportional to the total quantity of phenolic compounds originally present. The total phenolic content for Allium cepa extract and various homoeopathic formulations are presented in [Table 1] and [Figure 2].
Figure 1: Total phenolic content expressed as milligram of gallic acid equivalents. Standard curve for gallic acid

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Table 1: Total phenolic content for the Allium cepa extract and various homoeopathic formulations

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Figure 2: Variation in the total phenolic content expressed as milligram of gallic acid equivalents for different samples

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Total flavonoid content

The concentration of flavonoids in Allium cepa extract was determined using spectrophotometric method with aluminium chloride. The standard curve for quercetin is provided in [Figure 3]. The total flavonoid content was expressed in terms of microgram quercetin equivalent in [Figure 4]. The results for the total flavonoid content for Allium cepa extract and various homoeopathic formulations are presented in [Table 2].
Figure 3: Total flavonoid content expressed as milligram of quercetin equivalents. Standard curve for quercetin

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Figure 4: Variation in total flavonoid content expressed as microgram of quercetin equivalents

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Table 2: Total flavonoid content for the Allium cepa extract and various homoeopathic formulations

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Antioxidant potential

The scavenging activity percentage (AA%) was determined using equation 1 according to Mensor et al.[14] Antioxidant (DPPH scavenging) activity of Allium cepa extract is presented as percentage of DPPH radicals inhibition in [Figure 5] Ascorbic acid is used as a reference. The IC50 value of the extract is found to be 915 μg/mL. The DPPH radical scavenging activity for Allium cepa extract and various homoeopathic formulations is presented in [Table 3]. Allium cepa extract and MT show appreciable antioxidant activity while the homoeopathic formulations 30C and 200C do not show any appreciable antioxidant activity.
Figure 5: Antioxidant (2,2-diphenyl-1-picrylhydrazyl scavenging) activity of Allium cepa extract presented as percentage of 2,2-diphenyl-1-picrylhydrazyl radicals inhibition

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Table 3: The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity for the Allium cepa extract and various homoeopathic formulations

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Phytochemical analysis

Although quantitative analysis is more sensitive to very small amount of the active constituents, qualitative analysis is the first step in any analysis. The qualitative analysis of onion extract and Allium cepa preparations available in the market showed the presence or absence of phytochemicals as shown in [Table 4]. The phytochemical evaluation of the various phytoconstituents is shown in [Figure 6]. We see the presence of various important bioactive constituents in the sample solutions of ultra-dilute homoeopathic formulations. This is a very important finding given that mathematically we do not expect any active material present in these ultra-dilute solutions. More research needs to be carried out in this direction.
Table 4: Qualitative phytochemical analysis of extract of onion and Allium cepa preparations available in the market

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Figure 6: Phytochemical evaluation of bioactive phytoconstituents by colour reactions. (1) Steroids - Salkowski test, (2) Alkaloids - Dragendroff test, (3) Mayer test, (4) Hager test, (5) Wagner test, (6) Tannins - Ferric chloride test, (7) Lead acetate test, (8) Flavonoids - Alkaline reagent test before HCl addition, (9) Alkaline reagent test after HCl addition, (10) Shinoda's test; (11) Sugars - Molisch's test, (12) Fehling's test, (13) Saponins - Foam test, (14) Amino acids - Ninhydrin test, (15) Cardiac glycosides - Keller–Killiani test; (16) Anthraquinone glycosides - Hydroxyanthraquinone test, (17) Proteins - Biuret test

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  Discussion Top


Our qualitative and quantitative studies show that homoeopathic preparations of Allium cepa MT, 30C and 200C show the presence of many important phytochemicals even in the ultra-dilute preparations. This study validates the claim made by a scientist that homoeopathic formulations retain the starting material in spite of being ultra-dilute. This has been attributed to the succussion process followed to prepare different potencies. The succussion given to the liquid mass is expected to produce particles of varied shapes and sizes due to shearing forces generated during the pounding of the liquid container against an elastic stop. Mathematical modelling and study of physiochemical properties suggest that surface effects are responsible for the transfer of active ingredients during successive dilutions such that even ultra-dilute solutions show retention of active ingredients.


  Conclusion Top


The controversy regarding Homoeopathy medicines having high potencies i.e., 30C and 200C which involve huge dilution factors (10 − 60 and 10 − 400, respectively), such that theoretically there should be no measurable remnants of the starting materials is being disputed by many scientists. The results for the quantitative evaluation of flavonoid and phenolic content and the qualitative phytochemical analysis of Allium cepa extract, MT, 30C and 200C show that various bioactive phytochemicals such as alkaloids, tannins, saponins, carbohydrates, cardiac glycosides and proteins are retained even in ultra-dilute solutions. The prepared Allium cepa extract and MT show good antioxidant properties, whereas Allium cepa 30C and 200C show limited antioxidant potential. Recent studies indicate that nanoparticles of active material exist in solutions of homoeopathic formulations. Further interdisciplinary research is necessary to unravel the mystery of these wonderful formulations.

Financial support and sponsorship

University of Delhi under Innovation Project Scheme for SSC 302.

Conflicts of interest

None declared.



 
  References Top

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Smith C, Lombard KA, Peffley EB, Liu W. Genetic analysis of quercetin in onion (Allium cepa L.) 'Lady Raider'. Tex J Agric Nat Resour 2003;16:24-28.  Back to cited text no. 4
    
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Nausheen QN, Ali SA, Subur K. Cardioprotective and antioxidant activity of onion (Allium cepa) Leaves extract in doxorubicin induced cardiotoxicity in rats. Ann Exp Biol 2014;2:37-42.  Back to cited text no. 5
    
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Jung JY, Lim Y, Moon MS, Kim JY, Kwon O. Onion peel extracts ameliorate hyperglycemia and insulin resistance in high fat diet/streptozotocin-induced diabetic rats. Nutr Metab (Lond) 2011;8:18.  Back to cited text no. 6
    
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Bystrická J, Musilová J, Tomáš J, Tóth T, Kavalcová P, Šiatkovský O. Intake of heavy metals in selected varieties of onion (Allium cepa L.) Grown in the different locations 2015;26:17-21.  Back to cited text no. 7
    
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Upadhyay RP, Nayak C. Homeopathy emerging as nanomedicine. Int J High Dilution Res 2011;10:299-310.  Back to cited text no. 8
    
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Bellavite P, Marzotto M, Olioso D, Moratti E, Conforti A. High-dilution effects revisited 1. Physicochemical aspects. Homeopathy 2014;103:4-21.  Back to cited text no. 10
    
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Chikramane PS, Suresh AK, Bellare JR, Kane SG. Extreme homeopathic dilutions retain starting materials: A nanoparticulate perspective. Homeopathy 2010;99:231-42.  Back to cited text no. 11
    
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Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 1965;16:144-58.  Back to cited text no. 12
    
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Sen S, De B, Devanna N, Chakraborty R. Total phenolic, total flavonoid content, and antioxidant capacity of the leaves of Meyna spinosa Roxb. an Indian medicinal plant. Chin J Nat Med 2013;11:149-57.  Back to cited text no. 13
    
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Mensor LL, Menezes FS, Leitão GG, Reis AS, dos Santos TC, Coube CS, et al. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001;15:127-30.  Back to cited text no. 14
    
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Murugan T, Wins JA, Murugan M. Antimicrobial activity and phytochemical constituents of leaf extracts of Cassia auriculata. Indian J Pharm Sci 2013;75:122-5.  Back to cited text no. 15
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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