Preliminary pytochemical screening and HPTLC finger printing of Clerodendrum inerme (L.) Gaertn.

 

Hurakadli Swathi Basavaraj1, Hebbar Chaithra S.2, Ravikrishna S.3, Suchitra Narayan Prabhu4

1PG Scholar, Department of Dravyaguna, SDM College of Ayurveda, Udupi

2Associate Professor, Department of Dravyaguna, SDM College of Ayurveda, Udupi

3Associate Professor, Department of Agadatantra, SDM College of Ayurveda, Udupi

4 Research Officer, S.D.M Centre for Research in Ayurveda and Allied Sciences, Udupi, Karnataka.

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

 

ABSTRACT:

Objective: The present work was carried out to perform the powder microscopy, HPTLC finger printing and preliminary phytochemical evaluation of whole plant of Clerodendrum inerme (L.) Gaertn.

Method: The study was carried out in terms of powder microscopy, HPTLC fingerprinting was done using CAMAG HPTLC system equipped with Linomat 5 applicator, TLC scanner 4 and WIN CATS-4 software were used and preliminary phytochemical parameters were analysed.

Results: Powder microscopic features shows presence of epidermal cells containing chlorophyll and stomata, parenchyama cells of abundant fragments of outer layer of roots in association with exodermis, presence of vessels, parenchyama cells with starch, bundle of fibres interspersed with starch grains in a surrounding are seen. Preliminary phytochemical analysis showed the presence of alkaloid, steroid, carbohydrate, tannin, coumarins, phenols and resin. And HPTLC documentation revealed presence of many phytochemicals with different Rf values and HPTLC densitometric scan of the plates showed numerous bands and peaks.

Conclusion: Various characters that were observed in this study will help in identification of C. inerme (L.) Gaertn. in crude form. It can be concluded that HPTLC fingerprint analysis of whole plant extract of C. inerme (L.) Gaertn. can be used as a diagnostic tool for the correct identification of the plant and it is useful as a phytochemical marker and also a good estimator of genetic variability in plant populations.

 

KEYWORDS: Clerodendrum inerme (L) Gaertn., Kshudra Agnimantha, Whole plant, Powder microscopy, Preliminary phytochemical analysis, HPTLC fingerprinting.

 

 


 

 

 

 

 

INTRODUCTION:

Clerodendrum inreme (L.) Gaetrn. belonging to Lamiaceae family1 is known as Kshudra agnimantha in lexicons (Nighantu)2 is a straggling shrub with mucilaginous matter and foetid smell when crushed. This is commonly seen along sea coast, often cultivated as a hedge plant. Is native to Deccan plateau and Carnatic region and in Sri lanka.3 As per the ethno-botanical studies carried out, clinical application of C. inerme (L.) Gaertn., leaves are used as febrifuge, alternative, controls epilepsy, heals fractures, malaria, oedema, atrophic rhinitis, diabetes mellitus, gastric diseases rheumatic swellings and burning sensations. Oral intake of juice helps in relieving muscular pains and stiffness of legs (in tetanus) thus an anti-inflammatory drug. The methanol extract of roots contains verbascoside which exhibits analgesic and antimicrobial activities. The fruits are used in food poisoning.4

 

In spite of the numerous medicinal uses attributed to C. inerme (L.) Gaertn., there is no pharmacognostic report on its whole plant to determine phytochemical standards required for quality control of the crude drug. Taking this into the account it was thought worthwhile to generate data relating to powder microscopy, HPTLC fingerprinting and preliminary phytochemical evaluation, that may be useful as a reference in future development of a monograph on C. inerme (L.) Gaertn., and also to keep a check on intentional/unintentional adulteration.

 

Therefore, the main aim of the present investigation was to study the phytochemical screening along with powder microscopy and high-performance thin layer chromatography (HPTLC) studies on whole plant of C. inerme (L.) Gaertn. Knowledge of chemical constituent of the plant is not only essential for the discovery of therapeutic agents but also for economical source of the alkaloids carbohydrates etc.5


 


 

Fig. 1a. Epidermal cells in surface viewviewtransverse

 

Fig. 1b. Parenchymal cells of cork

 

Fig. 1c.Exodermis

 

 

Fig. 1d. Vessels

 

 

Fig. 1e. Parenchyma cells with content 

 

 

Fig. 1f. Bundle of fibres

 


Plate 1: Powder microscopy of Clerodendruminerme (L.) Gaertn. whole plant

 

 


MATERIALS AND METHOD:

Ř Collection of plant material:

The whole plant of C. inerme (L.) Gaertn. was collected as study sample  from backwaters of Pitrody, Udupi district, Karnataka. The authenticity of the drug sample was confirmed botanically by the experts and their characters were compared with various floras and standard herbarium sample available at S.D.M Centre for Research in Ayurveda and Allied Sciences, Udupi, Karnataka. Later the sample was shade dried and a coarse powder (Sieve No.20) of the same was prepared and further processing was facilitated at S.D.M Centre for Research in Ayurveda and Allied Sciences, Udupi. The powdered whole plant of C. inerme (L.) Gaertn was subjected for extraction to obtain the aqueous and alcohol extracts. Cold maceration technique was adapted to obtain aqueous and alcoholic extract of C. Inerme (L.) Gaertn. (Sample Code: 882/17042601)

 

Ř Preparation of plant material:

Shade dried whole plant powder (600g.) was placed in 5.0 L. of water and 500 g. of drug was placed in 5.0 L. of alcohol for 24 hours with occasional shaking in two different flat bottom flask. After 24 hours the mixture was filtered and then it was concentrated to dryness on a water bath. The obtained residues were weighed and kept in dry containers and stored in desiccator were used for further studies.

 

a)   Powder microscopy:

The powdered sample was passed through 355 µm (Sieve no.80). A pinch sieved sample was warmed with drops of chloral hydrate on a microscopic characters were observed. And the characters were observed. And the observation was also photographed under the Zeiss AXIO trinocular microscope attached with Zeiss Axio Cam camera under bright field light. Magnifications of the figures are indicated by the scale bars.

 

b)   High-performance thin layer chromatography fingerprinting:

HPTLC Profile (High Performance Thin Layer Chromatography) HPTLC studies were carried out in the following steps;

 

·      Sample Preparation:

1g. sample was suspended in 20.0ml alcohol (99.9%) at room temperature kept for 24 hours, followed by filtration. Later filtrate was reduced to 10.0ml on a water bath.

 

·      Sample Application:

3,6,9 µl sample were applied on pre-coated silica gel F254 on aluminium plates to a band width of 7 mm using Linomat 5 TLC applicator attached to CAMAG HPTLC system, which was programmed through WIN CATS software.

 

·      Development of Chromatogram:

After the application of sample, the chromatogram was developed in twin trough glass chamber 10 x 10 cm saturated with Toluene: Ethyl acetate: Formic acid (5.0: 5.5: 1.0) for 15 minutes.

 

 

 

·      Detection of Spots

The air-dried plates were viewed in ultraviolet radiation to mid-day light (Figure 2). The chromatograms were scanned by densitometer at UV 254nm, 366nm and 620nm. The Rf values and finger print data were recorded by WIN CATS software. Rf, colour of the spots and densitometric scan were recorded.

 

c)    Preliminary phytochemical evaluation6,7,8,9:

Preliminary phytochemical testes are used to detect the presence of various phytochemical groups, which is the indicative of type of phytochemicals present in the plant. These tests indicate the presence of different class of constituents present in the extract.

 

The following tests have been carried out for alcoholic and aqueous extracts. 

1.    Tests for alkaloids:

·      Dragendroff’s test:

To a few mg of extract dissolved in alcohol, a few drops of acetic acid and Dragendroff’s reagent were added and shaken well. An orange red precipitate formed indicates the presence of alkaloids.

 

·      Wagners’s test:

To a few mg of extract dissolved in acetic acid, a few drops of Wagner’s reagent was added. A reddish brown precipitate formed indicates the presence of alkaloids.

 

·      Mayer’s test:

To a few mg of extract dissolved in acetic acid, a few drops of Mayer’s reagent was added. A dull white precipitate formed indicates the presence of alkaloids.

 

·      Hager’s test:

To a few mg of extract dissolved in acetic acid, 3 ml of  Hager’s reagent was added, the formation of yellow precipitate indicates the presence of alkaloids.

 

2.    Tests for carbohydrates:

·      Molisch’s test:

To the extract, 1 ml of α-naphthol solution and   conc.  sulphuric acid were  added along  the sides of  test  tube. Violet colour formed at the junction of the two liquids indicates the presence of carbohydrates.

 

·      Fehling’s test:

A few mg of extract was mixed with equal quantities of Fehling’s solution A and B. The mixture was warmed on a water bath. The formation of a brick red precipitate indicates the presence of carbohydrates.

 

·      Benedict’s test:

To 5 ml of Benedict’s reagent, a few mg of extract was added, and boiled for two minutes and cooled. Formation of a red precipitate indicates the presence of carbohydrates.

 

3.    Test for steroids:

·      Libermann-Burchard test:

To the extract was dissolved in chloroform, 1 ml of acetic acid and 1 ml of acetic anhydride  were  added, then heated on a water bath and cooled. Few drops of conc. Sulphuric acid were added along the sides of the test tube. Appearance of bluish green colour indicates the presence of steroids.

 

·      Salkowski test:

The extract was dissolved in chloroform and equal volume of conc. Sulphuric acid was added. Formation of bluish red to cherry red colour in chloroform layer and green fluorescence in the acid layer indicates the presence of steroids.

 

4.    Test for saponins:

To a few mg of extract, distilled water was added and shaken. Stable froth formation indicates the presence of saponin.

 

5.    Test for tannins:

To the extract, a few drops of dilute solution of ferric chloride was added, formation of dark blue colour shows the presence of tannins.

 

6.    Test for flavonoids

Shinoda’s test:

To the extract in alcohol, a few magnesium turnings and few drops of conc. hydrochloric acid were added and heated on a water bath. Formation of red to pink colour indicates the presence of flavonoids.

 

7.    Test for phenol

To the extract in alcohol, added two drops of alcoholic ferric chloride. Formation of blue to blue black indicates the presence of phenol.

 

8.    Test for coumarins

To the extract in alcohol, a few drops of 2 N sodium hydroxide solution was added. Dark yellow colour formation indicates the presence of coumarins.

 

9.    Test for triterpenoids

The extract was warmed with tiny bits and few drops of thionyl chloride. Formation of pink colour indicates the presence of triterpenoids.

 

 

 

10. Test for carboxylic acid

Extract dissolved in water is treated with sodium bicarbonate. Brisk effervescence indicates the presence of carboxylic acid.

 

11. Test for resin

Few mg of the sample was mixed with water and acetone. Turbidity indicates the presence of resin.

 

12. Test for quinone

A few mg of alcohol extract was treated with 0.5% of sodium hydroxide. Deep coloration like pink, purple or red indicates the presence of quinone.

 

13. Test for amino acids

Few mg of the sample was mixed with ninhydrine reagent. Purple colour indicates the presence of amino acids.

 

RESULTS AND DISCUSSION:

a)   Powder microscopical features of whole plant of C. inerme (L.) Gaertn:

Epidermal cells containing chlorophyll and stomata are seen in the surface area. Parenchyama cells of cork are thick walled rectangular with brown matter dispersed, walls are suberized. Abundant fragments of outer layer of roots in association with exodermis with wavey walls. At times vessels are seen. Parenchyama cells with starch content are seen dispersed. Bundle of fibres interspersed with starch grains in a surrounding are seen. (Plate 1)

 

b)   HPTLC:

Rf values and colour of the spots in chromatogram developed in Toluene: Ethyl acetate: Formic acid (5.0: 5.5: 1.0) for alcoholic extract of  whole plant of C. ineme (L.) Gaertn. were recorded in Table (Table 1 and Plate 2). HPTLC documentation revealed presence of many phytochemicals with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under Short UV (254 nm), long UV (366 nm) and post derivatisation (62 0nm).10 (Table 1 and Plate 2).

 

Densitometric scan at  254nm, 366nm and  620nm (after derivatisation), for alcoholic extract of  whole plant of  C. ineme (L) Gaertn. revealed 5 peaks, 6 peaks and 8 peaks corresponding to different constituents in ethanol extracts with maximum Rf. (Plate 3, Plate 4, Plate 5)

 

 

 

 

TABLE. 1: Rf values of alcohol extract of Clerodendrum inerme (L.) Gaertn. whole plant

Short UV

Long UV

Post  derivatisation

-

-

0.09 (L. purple)

-

-

0.26 (D. purple)

-

0.56 (FL. purple)

-

-

-

0.58 (D. purple)

0.63 (L. green)

0.63 (FD. pink)

0.63 (L. purple)

-

-

0.66 (L. purple)

0.68 (D. green)

0.68 (FD. red)

0.68 (L. purple)

-

0.75 (FD. brown)

-

0.79 (D. green)

-

-

-

0.83 (FD. red)

-

0.87 (D. green)

0.87 (FD. red)

0.87 (D. purple)

-

0.91 (FL. pink)

0.91 (D. purple)

-

0.96 (FD. red)

-

 

c)    Preliminary phytochemical evaluation:

Preliminary phytochemical analysis showed the presence of alkaloid, steroid, carbohydrate, tannin, coumarins, phenols and resin indicating the active compounds. (Table 2)

 

 

 

 

 

 

 


 

 

 

 

 

Track  1-Clerodendrum inerme – 3µl

Track  2-Clerodendrum inerme – 6µl

Track  3-Clerodendrum inerme – 9µl

Solvent system Toluene: Ethyl Acetate: Formic acid (5:5.5:1.0)

Plate 2. HPTLC photo documentation of ethanolic extract of C. inerme (L.) Gaertn.

 

 

TABLE. 2 : Showing qualitative phyto-chemical analyses of ethanolic extracts of C. inerme (L.) Gaertn. whole plant

Sl.No

Tests

Colour if positive

Observations

Clerodendruminerme(L.) Gaertn.

1

Alkaloids

 

Dragendrof’s test

Orange precipitate

Red solution

Negative

 

Wagners test

Red precipitate

Red solution

Positive

 

Mayers test

Dull white precipitate

Dull white precipitate

Positive

 

Hagers test

Yellow precipitate

 

Negative

2

Steroids

 

Liebermann- buchard test

Bluish green

Green solution

Positive

 

Salkowski test

Bluish red to cherry red

Green solution at the upper layer and clear at the base layer.

 

Negative

3

Carbohydrate

 

Molish test

Violet ring

Violet ring

Positive

 

Fehlings test

Brick red precipitate

Ink blue solution

Negative

 

Benedicts test

Red precipitate

Bluish green solution

Negative

4

Tannin

 

With FeCl3

Dark blue or green or brown

Dark Brown

Positive

5

Flavanoids

 

Shinoda’s test

Red to pink

Light brown

Negative

 

6

Saponins

 

With NaHCO3

Stable froth

No froth

Negative

7

Triterpenoids

 

Tin and thionyl chloride test

Red

Grey precipitate

Negative

8

Coumarins

 

With 2 N NaOH

Yellow

Yellow

Positive

9

Phenols

 

With alcoholic ferric chloride

Blue to blue black, brown

Black solution

Positive

10

Carboxylic acid

 

With water and NaHCO3

Brisk effervescence

no effervescence

Negative

11

Resin

 

With aqueous acetone

Turbidity

Turbidity

Positive

12

Quinone

 

5% NaOH

Pink/purple/red

Light Yellow

Negative

13

Amino acids

 

Ninhydrine reagent

Purple color

Light Yellow

Negative

 

 

 


 

Fig 3a. C. inerme (L.) Gaertn. (3µl)

Plate 3.Densitometric scan at 254nm

 

 

 

Fig. 4a. C. inerme (L.) Gaertn. (6µl)

Plate 4. Densitometric scan at 366nm

 

Fig 5a. C. inerme (L.) Gaertn. (9µl)

Plate 5.Densitometric scan after derivatisation at 620nm

 

CONCLUSION:

From the present investigation, powder microscopic features of whole plant of C. ineme (L.) Gaertn. shows epidermal cells, parenchyama cells in association with exodermis, presence of vessels, parenchyama cells, Bundle of fibres (Plate 1). The proposed HPTLC fingerprinting screening showed 4 major sports under short UV, 8 spots under long UV and 8 spots Post derivatisation (Plate 2). And Densitometric scan at 254nm, 366nm and 620nm (after derivatisation), for alcoholic extract of whole plant of  C. ineme (L) Gaertn. revealed 5 peaks, 6 peaks and 8 peaks corresponding to different constituents in ethanol extracts with maximum Rf. (Plate 3, Plate 4, Plate 5) Confirming the presence of number of active constituents in whole plant of C. ineme (L.) Gaertn. This method was found to be simple, precise, and accurate which can be used for the quality control of the raw materials as well as formulations. And Preliminary phytochemical analysis showed the presence of alkaloid, steroid, carbohydrate, tannin, coumarins, phenols and resin indicating the active compounds were not disturbed during the preparation and storage Hence, the present study attempts to outline basic requirements necessary to develop scientific/technical standards to justify the medicinal plant worth exploring for further research work and also to keep a check on intentional/unintentional adulteration.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.     http://florakarnataka.ces.iisc.ac.in/hjcb2/herbsheet.php?id=4284&cat=1

2.     Kamat S.D. Dhanvantari Nighantu, Chaukhamba Sanskrit Pratishthan New Delhi:.2002

3.     GopalkrishnaBhat k. Flora of Udupi, Indian Naturalist. Udupi: 2003

4.     Khare C.P. Indian Medicinal Plants. Springer. New Delhi: 2007

5.     Sirsi M. Indian J Pharmacol.1964; 26(5):129-131

6.     Dey P, Mukherjee M, Preliminary phytochemical studies of leaf extracts of Molineria recurvate, Journal of pharma. Res.2012;4,7: 3727-3730

7.     Anyanwu GI, Dawet A. Pharmacological and phytochemical screening of Hyptissuaveolens Poit (Lamiaceae) for bioactivity in rodents. International Journal of Research in Plant Science 2012; 2(3):50-55.

8.     Anonymous, Who guidelines on safety monitoring of Herbal medicines in pharmacovigialance system, Geneva, World Health Organization, (2004) 4 : 114.

9.     Ravi Sunder Prajapati, G.P. Richhariya, HPTLC Fingerprint Profile and Preliminary Phyto-chemical analysis of Nimba (Azadirachtaindica) Leaf and Stem Bark, Research Journal of Pharmacognosy and Phytochemistry. 8(2): April- June, 2016

10.   Tripathi Manoj and Sikarwar, R.L.S. Pharmacognostic Standardization of stem bark of Erythrina variegate L. Journal of the Indian Botanical Society ( 2014), 93 (3 and 4),248-253.

 

 

 

 

 

 

 

 

 

Received on 15.02.2018       Modified on 18.03.2018

Accepted on 11.04.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(2): 145-151.

DOI: 10.5958/0975-4385.2018.00021.3