Phytochemical qualitative analysis-Glycoside identification test for homoeopathic mother tincture and dilution

November 10, 2024 admin Homeopathic Pharmacy 0

Dr Aswin Anandh. M

 ABSTRACT: Homoeopathic medicines are prepared from the various sources. One of the major source is plants, more than 6000 plant varieties are used as medicine. The Homoeopathic system of medicine has their own principle “Similia similibus curentur” & own technique for preparation of medicine is Potentization by two methods – “SUCCUSSION” & “TRITURATION”. As we know that whole system of Homeopathy in trouble proving their scientificity to modern world. Ever disturbing controversy is that Homoeopathic medicines have no curative power in it. To overcome those phytochemical constituents of plants such as Alkaloids, Glycosides, Terpenoids, Phenols, Saponins etc. said to be therapeutic in extracts of plants. There is specific chemical identification test to identify the presence of phytochemical constituents in Homoeopathic dilutions.

KEYWORDS: Glycoside, Homeopathy, Flavonoids, Mother tincture, Dilution, Phytochemical, Plant extract

INTRODUCTION:
Glycoside: Organic natural compounds present in a lot of plants and some animals, these compounds upon hydrolysis give one or more sugars (glycone) B- form and non sugar (aglycone) or called genin. Glycosides play numerous roles in living organisms. In plants, they often serve as defense mechanisms against herbivores and pathogens. Some glycosides are also involved in plant growth regulation and metabolism. The first glycoside identified was amygdalin, isolated from almonds by French chemists Pierre Robiquet and Antoine Boutron-Charlard in 1830.

PROPERTIES:

Glycosides exhibit a variety of physical and chemical characteristics that are impacted by the glycone and aglycone constituents. The following are some common properties:

  1. Stability : Under normal circumstances, glycosides are rather stable, but they can be degraded to produce sugar and aglycone by bases, acids, or enzymes called glycosidases.
  1. Solubility : Because glycosides have a hydrophilic sugar component, they are typically soluble in water. The type of aglycone determines how soluble they are in organic solvents.
  1. Taste : Glycosides can be tasteless, sweet – glycyrrhizin, bitter – cardiac glycosides, or neither.
  1. Optical activity : Because the sugar component of many glycosides contains chiral centers, they display optical activity.
  1. Biological activity : Glycosides have important biological and pharmacological effects. Examples of this include saponins, which are used as detergents, and cardiac glycosides, which are used to treat heart disease.

Classification of Glycosides:

Based on

  1. The nature of Glycone
  2. Number of sugars
  3. Nature of the glycoside
  4. Botanical source
  5. Therapeutic use
  6. The type of Glycosidic Bond
  7. The Glycosidal Linkage and
  8. Chemical nature of Aglycone

Glycosides can be of several classes such as iridoid, alcoholic, anthraquinone, flavonoid, coumarin, chromone, cardiac, steviol, cyanogenic, phenolic, saponins, iridoid, thio, among others. But mainly classified as

  1. On nature of Glycone
    Glucoside: If glucose group is present in glycoside.
    • Fructoside : If fructose group is present in glycoside.
    • Glucuronide: If glucuronic acid is present.
  2. Number of sugars
  • One sugar – monosides – e.g. Salicin.
  • Two sugar – Biosides – e.g. Diosmin.
  • Three sugars – Triosides – e.g. Digoxin.
  1. Nature of the Glycoside
  • Primary glycosides: Originally present in the plant –e.g. Purpurea
  • Secondary glycosides: Resulted from removal of one sugar from the primary glycosides –e.g. Digitoxin
  1. Botanical source
  • Digitalis glycoside
  • Senna glycoside
  1. Therapeutic use
  • Analgesic glycoside
  • Purgative glycoside
  • Cardiac glycoside
  1. Type of glycosidic bond
  • Depending on whether the glycosidic bond lies “above” or “below” the plane of the cyclic sugar molecule, glycosides are classified as:

»α-glycosides

»β-glycosides

  1. Glycosidal linkage
  • Aglycone- O- Sugar O-glycosides
  • Aglycone- C- Sugar C-glycosides
  • Aglycone- S- Sugar S-glycosides
  • Aglycone- N- Sugar N-glycosides
  1. On the basis of Aglycone

The presence of nature of aglycone group classes are as follows:
• Steroidal and triterpenoid glycosides
• Anthraquinone glycosides
• Cyanogenetic and cyanophoric glycosides
• Saponin glycosides
• Flavone glycosides
• Coumarin glycosides
• Cardiac glycoside

S.N CLASS EXAMPLES
1 ANTHRAQUINONE GLYCOSIDES SENNA ANGUISTIFOLIA,
RHEUM OFFICINALE (Rhubarb)
2 CYANOGENETIC & CYANOPHORIC GLYCOSIDES AMYGDALUS AMARA (Bitter almond),
PRUNUS VIRGINIANA (Wild black cherry)
3 SAPONIN GLYCOSIDES DIOSCOREA VILLOSA (China root)
4 FLAVONE GLYCOSIDES GINKGO BILOBA
5 COUMARIN GLYCOSIDES APIUM GRAVEOLENS (Celery)
6 CARDIAC GLYCOSIDES DIGITALIS PURPUREA, THEVETIA PERUVIANA, SCILLA MARITIMA
7 STEROIDAL GLYCOSIDES SOLANUM LYCOPERSICUM, VITIS VINIFERA

CHEMICAL TESTS FOR GLYCOSIDES

  1. ANTHROQUINONE GLYCOSIDE
BORNTRAGER’S

TEST

 Add 5–10 ml of diluted HCl to 1 ml of sample; boil for 10 minutes on a water bath, then filter. After extracting the filtrate using CCl4 and benzene, mix with an equal volume of ammonia solution and shake. Formation of pink or red colour in ammonical layer due to presence of anthraquinone moiety
  1. SAPONIN GLYCOSIDE
FOAM TEST To 1ml of drug add 10–20 ml of water, shake for few minutes. Formation frothing which persists for 60–120 s in presence of saponins.

 

Trichloro acetic acid test Sample

+

addition of saturated solution of trichloro acetic acid

 Forms Colored precipitate
Zimmermann test Meta dinitrobenzene solution was added to the alcoholic solution of drug containing alkali, on heating It forms violet color in presence of Keto steroid
  1. TEST FOR CARDIAC GLYCOSIDES
Keller – Killiani Test • An equal volume of water and 0.5 milliliter of a strong lead acetate solution were added to the drug’s alcoholic extract, shaken, and filtered.
• A similar volume of chloroform was extracted with the filtrate.
• After the chloroform extract was evaporated until it was completely dry, the residue was dissolved in three milliliters of glacial acetic acid and a few drops of FeCl3 solution were added.
• After that, the mixture was poured into a test tube with two milliliters of concentrated H2SO4.		 Reddish brown layer is formed, which turns bluish green after standing due to presence of digitoxose.
3. STEROID AND TRITERPENOID GLYCOSIDE
Libermann burchard test
The drug’s alcoholic extract was dried out and then extracted with CHCl3. A few drops of anhydrous acetic acid were then added, and finally, concentrated H2 SO4 from the test tube’s side wall was added to the CHCl3.
Formation of violet to blue coloured ring at the junction of two liquid, indicate the presence of steroid moiety.
Salkowaski test
The drug’s alcoholic extract was dried out and then extracted using CHCl3. Conc. H2 SO4 from the test tube’s sidewall was added to the CHCl3 extract.Formation of yellow coloured ring at the junction of two liquid, which turns red after 2 min, indicate the presence of steroid moiety.
Antimony trichloride test
Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3, add saturated solution of SbCl3 in CHCl3 containing 20% acetic anhydride. Formation of pink colour on heating indicates presence of steroids and triterpenoids
Legal Test • An equal volume of water and 0.5 milliliter of a strong lead acetate solution were added to the drug’s alcoholic extract, shaken, and filtered.
• An equivalent volume of chloroform was extracted from the filtrate, and the chloroform extract was evaporated until it was completely dry.
• To create an alkaline solution, the residue was dissolved in two milliliters of pyridine, and two milliliters of sodium nitroprusside were added after that.		 Formation of pink color in presence of glycosides or aglycon moiety.
Baljet test Thick section of leaf of digitalis or the part of drug containing cardiac glycoside, when dipped in sodium picrate solution It forms yellow to orange color in presence of aglycones or glycosides.
3,5 – Dinitro benzoic acid test To the alcoholic solution of drug few drops of NaOH followed by 2% solution of 3,5-dinitro benzoic acid was added. Formation of pink color indicates presence of cardiac glycosides.
  1. TEST FOR COUMARIN GLYCOSIDES
Fecl3 Test To the concentrated alcoholic extract of drug few drops of alcoholic FeCl3 solution was added. Formation of deep green color, which turned yellow on addition of conc. HNO3, indicates presence of coumarins.
Fluorescence Test The alcoholic extract of drug was mixed with 1N NaOH solution (one ml each). Development of blue-green fluorescence indicates presence of coumarins.
  1. TEST FOR CYNOPHORIC GLYCOSIDES
Sodium pictrate Test Powdered drug was moistened with water in a conical flask and few drops of conc. Sulphuric acid was added.

Filter paper impregnated with sodium picrate solution followed by sodium carbonate solution was trapped on the neck of flask using cork.

 Formation of brick red color due to volatile HCN in presence of cynophoric glycosides takes place.
  1. TEST FOR FLAVONOID GLYCOSIDE
Ammonia Test Filter paper dipped in alcoholic solution of drug was exposed to ammonia vapor. Formation of yellow spot on filter paper
Shinoda Test To the alcoholic extract of drug magnesium turning and dil. HCl was added, formation of red colour indicates the presence of flavonoids. To the alcoholic extract of drug zinc turning and dil. HCl was added Formation of deep red to magenta colour indicates the presence of dihydro flavonoids
Vanillin Hcl Test Vanillin HCl was added to the alcoholic solution of drug. Formation of pink colour due to presence of flavonoids.

SIMILAR STUDIES:

  1. Qualitative phytochemical evaluation of in-house prepared coleus aromaticus mother tincture as per homoeopathic pharmacopoeia of India (HPI) guidelines – Dr. Aswin Anandh. M

Result:Homoeopathic mother tincture is prepared by maceration method as given in HPI and phytochemical qualitative analysis is done. It demonstrates the absence of saponins and presence of alkaloids, flavonoids, tannins, terpenoids, glycosides, carbohydrates, reducing sugar and proteins. It is necessary to carry out additional research using GCMS techniques, other chromatography techniques, and phytochemical quantitative analysis.

  1. Qualitative and quantitative analysis of phytochemicals in ficus religiosa – Dr. K Rathna Kumari

Result: This research sheds light on the phytochemicals found in medications, such as cardiac glycosides, tannins, flavonoids, and alkaloids. Phytochemicals have also been found in potencies that surpass the Avogadro limit. Strong evidence from this study indicates that the mother tincture of Ficus religiosa contains phytochemicals in varying potencies; therefore, homoeopathic medicines are not placebos.

  1. 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

Result: The prepared extract and the Mother Tincture have confirmed the presence of multiple phytochemicals, including glycosides, saponins, steroids, flavonoids, alkaloids, carbohydrates, and amino acids. Avogadro’s limit calculations indicate that source material shouldn’t be present in preparations that are diluted more than 12C. Interestingly, despite being ultra-high dilutions, Allium cepa 30C and 200C formulations produced positive results for numerous phytochemicals.

CONCLUSION:
As Homoeopathy evolves, Homeopathic system should be outstanding in this scientific world. We need more Research work in proving efficacy of Homoeopathy. Main criticism in Homoeopathy is that no curative power in medicine. By performing certain analysis, we can prove that therapeutic phytochemical constituent present in homoeopathic dilutions too.

 BIBLIOGRAPHY:

  1. Ikan R. Naturally Occurring Glycosides. John Wiley & Sons; 1999.
  2. “Glycoside | Biochemistry.” Encyclopedia Britannica, britannica.com/science/glycoside.
  3. Aswin Anandh M, Sandhya SV, Amutha WP, Sreeja S. Qualitative phytochemical evaluation of in-house prepared coleus aromaticus mother tincture as per homoeopathic pharmacopoeia of India (HPI) guidelines. Int J Hom Sci 2024;8(3):340-344. DOI: 33545/26164485.2024.v8.i3f.1236
  4. Kytidou, Kassiani, et al. “Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics.” Frontiers in Plant Science, vol. 11, 7 Apr. 2020, www.ncbi.nlm.nih.gov/pmc/articles/PMC7154165/, https://doi.org/10.3389/fpls.2020.00357. Accessed 12 Mar. 2021.
  5. Shaikh JR, Patil M. Qualitative Tests for Preliminary Phytochemical screening: An Overview. International Journal of Chemical Studies. 2020 Mar 1;8(2):603–8.
  6. Brito-Arias M. Synthesis and Characterization of Glycosides. Springer Nature; 2022.
  7. Kaya GI, Melzig MF. Quantitative Determination of Cucurbitacin E and Cucurbitacin I in Homoeopathic Mother Tincture of Gratiola Officinalis L. by HPLC. PubMed. 2008 Dec 1;63(12):851–3.
  8. Oe, Schultz, and Gmelin R. “[Homeopathic Mother Tinctures from Mustard Oil Glycoside-Containing Plants].” PubMed, vol. 94, no. 1, 1 Jan. 1955, pp. 12–4. Accessed 10 Apr. 2024.
  9. Adamczak, Artur, et al. “Antibacterial Activity of Some Flavonoids and Organic Acids Widely Distributed in Plants.” Journal of Clinical Medicine, vol. 9, no. 1, 31 Dec. 2019, www.ncbi.nlm.nih.gov/pmc/articles/PMC7019947/, https://doi.org/10.3390/jcm9010109. Accessed 21 Sept. 2020.
  10. Kumari DrKR. Qualitative and Quantitative Analysis of Phytochemicals in Ficus Religiosa. International Journal of Homoeopathic Sciences. 2022 Oct 1;6(4):113–7.
  11. Arora E, Sharma V, Khurana A, Manchanda A, Sahani D, Abraham S, et al. 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 Journal of Research in Homoeopathy. 2017;11(2):88.

Dr. Aswin Anandh. M PG Scholar
Department of Homoeopathic Pharmacy
Sarada Krishna Homoeopathic Medical College (Affiliated to the Tamilnadu Dr. M.G.R. Medical University, Chennai), Kulasekharam, Kanniyakumari district, Tamilnadu, India.
Email : aswinmuthaian@gmail.com

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