Histochemical Localization of Primary and Secondary Metabolites in Tiliacora racemosa Colebr.


Yogeshwari C1*, Kumudha P2

1Research Scholar, PG and Research Department of Botany, Vellalar College for Women (Autonomous), Thindal, Erode – 638012, Tamil Nadu, India.

2Associate Professor, PG and Research Department of Botany, Vellalar College for Women (Autonomous), Thindal, Erode – 638012, Tamil Nadu, India.

*Corresponding Author E-mail: yobotnet@gmail.com



Tiliacora racemosa commonly known as Perungkattukodi in Tamil, has been used in many Ayurvedic preparations and is also a plant of ethnomedicinal use. In the present study, histochemical localization of various primary and secondary metabolites of leaf and stem of Tiliacora racemosa was carried out with the aim of contributing to its quality control in herbal industries. The various metabolites such as carbohydrates, starch, proteins, alkaloids, flavonoids, phenols, tannins, saponins and lipids were localized using various specific chemical reagents microscopically. The results of this study could be useful for identification of the plant species and also for the determination of the authenticity of the drug in herbal industry.


KEYWORDS: Tiliacora racemosa, Perungkattukodi, histochemical, metabolites, quality control.




The use of medicinal plants has been encouraged by the World Health Organization since it realized that about 80% of the population turns to the traditional medicinal system to get rid of diseases1. In recent years, there has been a rapid increase in the standardization of selected medicinal plants of potential therapeutic significance2 which could be useful for the determination of the authenticity of medicinal plant species and the identification of the plant organs where the highest concentrations of active substances are present, especially when the plants are fragmented for use in herbal drugs3.


Chemical studies performed using histochemical techniques allow a quick and inexpensive preliminary evaluation of bioactive phytoconstituents in medicinally important plant species4,5. Beyond that, it allows to elucidate the aspects referring to storage and secretion of secondary metabolites, which could lead to the correct localization and extraction of medicinal chemicals6.


Tiliacora racemosa commonly known as Perungkattukodi in Tamil, belongs to the Family Menispermaceae. The plant has been used in many Ayurvedic preparations and the paste of leaves has been used to treat cuts and wounds and roots for treating snakebite7. The plant is shown to possess many potential alkaloids8, esters9 and lactones10. The aim of this study was to investigate the histochemical characteristics of the leaf and stem of Tiliacora racemosa as a means of providing information for quality control in the herbal industry.



Collection and authentication of plant material:

The plant specimens for the proposed study were collected from Guruvareddiyur section, Chennampatti range, Western Ghats, Tamil Nadu, India. The collected plant was identified with the help of Flora11 and authenticated by the Botanical Survey of India (Southern circle), Coimbatore. The voucher number of the specimen is BSI/SRC/5/23/2016/Tech./1521.


Histochemical localization of different metabolites:

The required samples of different organs were cut and removed from the plant and fixed in FAA (Formalin-5ml+ Acetic acid-5ml + 70% Ethyl alcohol-90ml). After fixation, they were washed thoroughly with water. In the present investigation, temporary mounts of sections were employed and for this, free hand sections were taken and treated with different chemical reagents to localize different primary and secondary metabolites.


The following standard solutions were employed in the histochemical tests: Lugol Iodine12 to reveal starch, Molish reagent to reveal carbohydrates13, potassium hydroxide and copper sulphate solution to stain proteins14, Wagner's and Dragendorff's  reagent to stain alkaloids15, lead acetate which reveals flavonoids15, ferric chloride and sodium carbonate which stains tannins16, alcoholic ferric chloride to stain phenols17, concentrated sulphuric acid which reveals saponins18 and iodine water for localization of lipids19.



The main classes of primary and secondary metabolites localized from the stem and leaf of Tiliacora racemosa after histochemical analysis are presented in Table 1. Carbohydrates were localized in the parenchymatous ground tissue of leaf and pith and vascular region of the stem which appeared as purple patches in response to Molish reagent in leaf and stem (Fig. 1A, 2A). The reactions for starch were positive for Lugol iodine solutions which presented a dark blue patches in the collenchyma, phloem tissues and pith region of the stem and sclerenchymatous bundle sheath and parenchymatous ground tissue of the leaf (Fig. 1B, 2B).  The presence of starch granules can be important for taxon identification3. Biuret test performed by immersing the sections in strong potassium hydroxide solution followed by addition of few drops of aqueous solution of 1% copper sulphate showed positive results for proteins and were localized as blue patches in vascular region (Fig. 1C, 2C).



Table 1: Histochemical localization of various metabolites in Tiliacora racemosa

Metabolite group


Colour observed


Molish reagent



Lugol iodine

Dark blue


Potassium hydroxide and aqueous solution of 1% copper sulphate solution



Wagner's reagent

Dragendorff's reagent

Reddish brown

Reddish orange


Lead acetate



Alcoholic ferric chloride



Ferric chloride and sodium carbonate

Dark blue


conc. Sulphuric acid

Blue to bluish black


Iodine water


























Figure 1: Histochemical tests performed on transverse sections of Tiliacora racemosa leaf.  A- Carbohydrates; B- Starch; C- Proteins; D- Alkaloids in reaction with Wagner's reagent; E- Alkaloids in reaction with Dragendorff's reagent; F- Flavonoids; G- Phenols; H- Tannins; I- Saponins; J- Lipids. Vb- vascular bundle; X- xylem; Pa- parenchymatous ground tissue; Sc- sclerenchymatous bundle sheath; LEp- lower epidermis.



















Figure 2: Histochemical tests performed on transverse sections of Tiliacora racemosa stem.  A- Carbohydrates; B- Starch; C- Proteins; D- Alkaloids in reaction with Wagner's reagent; E- Alkaloids in reaction with Dragendorff's reagent; F- Flavonoids; G- Phenols; H- Tannins; I- Saponins; J- Lipids. Ep- epidermis; Vb- vascular bundle; X- xylem; XFi- xylem fibres; MR- medullary rays; Sc- sclerenchymatous bundle sheath; Co- cortex; Pi- pith.


It is useful to locate the chemical compounds in the cells of the herbal drugs. Histochemical techniques are fast and cheap methods that can be used to identify bioactive classes of phytocompounds in tissues and cell compartments precisely20,21. The reactions with Wagner's and Dragendorff's reagent were positive indicating the presence of alkaloids in the sclerenchymatous bundle sheath surrounding the vascular bundles in the leaf and stem (Fig. 1D&E, 2D&E). The presence of alkaloids is a chemotaxonomic feature of Menispermaceae, allowing the separation of genera according to the structural class of alkaloids being biosynthesized18. Lead acetate revealed the presence of flavonoids in bundle sheath of leaf which showed yellow colour (Fig. 1F).  The vascular region of stem reacted positive to lead acetate indicating the presence of flavonoids (Fig. 2F).


Alcoholic ferric chloride produced a stable green colour with phenolic compounds which were localized in xylem vessels, bundle sheath and ground tissue of leaf and collenchyma and vascular tissues of stem (Fig. 1G, 2G). Tannins were localized in bundle sheath and xylem fibres of leaf and collenchyma of stem which appeared dark blue in reaction with ferric chloride and sodium carbonate (Fig. 1H, 2H). The reactions with concentrated sulphuric acid revealed positive results for saponins which produced blue to bluish black colour in vascular and ground tissues (Fig. 1I, 2I ) whereas the reactions with iodine water were positive indicating the lipophilic substances in the parenchymatous ground tissues of leaf and vascular and pith region of stem (Fig. 1J, 2J). Localization of primary and secondary metabolites with various histochemical indicators in leaf and stem of Tiliacora racemosa indicate that these reagents can selectively identify specific substances within intact cells and tissues. Hence these techniques can be used in herbal industries for providing quality control measures.



Histochemical standardization of Tiliacora racemosa could be useful to identify and determine the authenticity of this drug in herbal industry.



1.     Pacheco-silva NV and Donato AM. Morpho-anatomy of the leaf of Myrciaria glomerata. Revista Brasileira de Farmacognosia. 26(3); 2016: 275–80.

2.     Reddy YSR, Venkatesh S, Ravichandran T, Subburaju T and Suresh B. Pharmacognostical studies on Wrightia tinctoria bark. Pharmaceutical Biology. 37(4); 1999: 291–95.

3.     Andrade EA, Folquitto DG, Camargo Luzc LE, Paludod KS, Faragob PV and Budel JM. Anatomy and histochemistry of leaves and stems of Sapium glandulosum. Revista Brasileira de Farmacognosia. 27; 2017: 282-289.

4.     Matiasa LJ, Mercadante-Simõesa MO, Royob VA, Ribeiroc LM, Santosa AC and Fonseca JMS. Structure and histochemistry of medicinal species of Solanum. Revista Brasileira de Farmacognosia. 2015; http://dx.doi.org/10.1016/j.bjp.2015.11.002.

5.     Araújo ND, Coelho VPM, Ventrella MC and Fátima Agra M. Leaf anatomy and histochemistry of three species of Ficus sect . Americanae supported by light and electron microscopy. Microscopy and microanalysis. 20; 2014: 296–304.

6.     Kuster VC and Vale FHA. Leaf histochemistry analysis of four medicinal species from Cerrado. Revista Brasileira de Farmacognosia. 26; 2016: 673-678.

7.     Yogeshwari C and Kumudha P. Ethnobotanical survey of medicinal plants for skin diseases and poisonous bites in Chennampatti range, Erode district, Tamilnadu. International Journal of ChemTech Research. 10(7); 2017: 925-930.

8.     Guha KP, Das PC and Mukherjee B. Structure of Tiliamosine : A new diphenyl bisbenzylisoquinoline alkaloid from Tiliacora racemosa. Tetrahedron Letters. 17(47); 1976: 4241–44.

9.     Joseph Selvaraj S, Alphonse I and John Britto S. Isolation and characterization of novel esters from aerial parts of Tiliacora acuminata. Indian Journal of Chemistry. 48B(July); 2009: 1038–40.

10.   Joseph Selvaraj S, Alphonse I and John Britto S. A new lactone from aerial parts of Tiliacora acuminataIndian Journal of Chemistry. 47B(June); 2008: 942-944.

11.   Gamble JS. Flora of the Presidency of Madras, Neeraj Publishing House, Delhi. 2014.

12.   Jensen WA. Botanical histochemistry: Principles and Practice. W. H. Freeman and Company, San Francisco. 1962.

13.   Kokate CK, Purohit AP and Gokhale SB. Pharmacognosy. Nirali Prakashan, Pune. 2014.

14.   Serra JA. Histochemical tests for proteins and amino acids; the characterization of basic proteins. Stain technology. 21(1); 1946: 5-18.

15.   Evans WC. Trease and Evans Pharmacognosy. Elsevier, New Delhi, India. 2005.

16.   Krishnamurthy KV. Methods in plant histochemistry. S. Viswanathan (Printers & Publishers) Private Limited, Madras, India. 1988.

17.   Mace ME. Histochemical localization of phenols in healthy and diseased banana roots. Physiologia Plantarum, 16(4); 1963: 915-925.

18.   Cavalcanti AC, Gomes ANP, Porto NM, Agra MF, Moura TFAL and Oliveira EJ. Phamacognostic evaluation of Cissampelos sympodialis Eichl leaves. South African Journal of Botany, 93; 2014: 70–78.

19.   Khandelwal KR. Practical Pharmacognosy. Pragati Books Pvt. Ltd.,  India. 2008.

20.   Araujo ARB, Royo VA, Mercadante-Simões MO, Fonseca FSA, Ferraz VP, Oliveira DA, Menezes EV, Melo Júnior AF and Brandão MM. Physicochemical profile of the oil from the seed of Tontelea micrantha ( Celastraceae ). South African Journal of Botany. 112; 2017:112–17.

21.   Furr M and Mahlberg PG. Histochemical analyses of laticifers and glandular trichomes in Cannabis sativa. Journal of natural products. 44(2); 1981: 153–59.





Received on 23.01.2018       Modified on 10.02.2018

Accepted on 28.02.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(2): 152-156.

DOI: 10.5958/0975-4385.2018.00022.5