INTRODUCTION:

Humanity has relied on nature for its various needs from immemorial times. One of the important applications is using plants as medicine to treat diseases and various illnesses. According to WHO, about 80% of the world’s population still depends on natural (herbal) medicines for its primary healthcare needs.¹

Lantana camara Linn. is a flowering ornamental plant belonging to the family Verbenaceae; it was probably introduced in India before the nineteenth century. Lantana camara is distributed throughout India in moderate to high rainfall and well-drained regions; considered a weed, it has been found to have fundamental medicinal properties. In India, the ash of the entire plant is used externally for chronic ulcers.²

The decoction of the leaves and the stem is used externally as an excellent wash for eczema or chronic inflammation of the skin.³

Powdered fresh leaves are used for sprains. The flowering tops in decoction are used for coughs, colds, fever, jaundice and chest diseases.³

The decoction of the root is recommended for use against infections of the respiratory tract, mumps, and gargles and is a good mouthwash for ulcers of the mouth.³

Leaves extract of Lantana camara is reported to possess anticancer activity⁴^,⁵^,⁶^,⁷, active against murine tumors⁸, extracts of leaves and roots reported to possess antibacterial activity^9,10, hypoglycemic¹¹ and wound healing^12,13 activity.

Little work has been reported on proper identification and characterization of the roots.

In this work, an attempt has been made to identify and characterize detailed pharmacognostic properties of the roots of Lantana camara, macroscopic characters, detailed and reproducible microscopic features, preliminary phytochemical and physiochemical properties, which can be utilized to confirm the identity of the plant material for authentication and standardization.

Taxonomy:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Lamiales

Family: Verbenaceae

Genus: Lantana

Species: Lantana camara Linn.

MATERIALS AND METHODS:

Collection of specimen:

The plant specimen for the proposed study was collected from the Naubad Forest range in Bidar District, Karnataka.

Care was taken to select healthy plants and normal organs.

Authentication:

The plant under study was authenticated by Prof B.S. Sajjan, Head Dept. of Botany B.V. Bhoomreddy College Bidar, with herbarium reference number BVBCol/ 3-2010.

Chemicals and Reagents:

Acetic acid, Butyl alcohol, Potassium iodide, Magnesium metal, Ferric chloride, Chloroform and Sulphuric acid were procured from S.D. Fine Chemicals, Mumbai, Maharashtra, India.

Formalin, Methanol and Hydrochloric acid were procured from Nice Chemicals, Kochi, Kerala, India.

Ethyl alcohol obtained from Nirani Sugars, Mudhol, Karnataka India.

All reagents used are of analytical grade and prepared using standard procedures prescribed in Pharmacopeia and reference books.

Macroscopic (Roots)^14,15:

The length of the roots is around 20-40cm and about 0.2-0.5cm thick; they are usually creamish brown externally, the bark is thin, the outer surface is rough with longitudinal wrinkles having a hard fracture, with characteristic odour and pungent taste.

Microscopy (Roots)^16,17:

The required root samples were cut, removed from the plant, and fixed in FAA (Formalin-5ml+ Acetic acid-5ml + 70% Ethyl alcohol-90ml). After 24 hrs. of fixing, the specimens were dehydrated with graded series of tertiary – Butyl alcohol as per the schedule given by Sass, 1940¹⁸. Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60 ºC) until TBA solution attained supersaturation. The specimens were cast into paraffin blocks.

Sectioning:

The paraffin-embedded specimens were sectioned with the help of a Rotary Microtome.

The thickness of the sections was 10-12µm. Dewaxing of the sections was by the customary procedure as mentioned by Johansen¹⁹. The sections were stained with Toluidine blue as per the method published by O’Brien et al.²⁰. Since Toluidine blue is a polychromatic stain, the staining results were excellent, and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. Wherever necessary, sections were stained with safranin, fast green, and IKI (starch).

The microscopic study was performed by taking the root specimen in two different sizes, a thin root measuring about 500µm and a thick root measuring about 2.4mm thick.

Powder Microscopy:

Glycerine-mounted temporary preparations were made from macerated/cleared materials. Powdered material was cleared with NaOH and mounted in a glycerine medium after staining. Different cell components were studied and measured.

Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with a Nikon labphoto 2, microscopic unit. For normal observations, a bright field was used. Polarized light was employed to study crystals, starch grains, and lignified cells.

Phytochemical Studies:^21–24

The extracts so obtained were subjected to preliminary phytochemical screening as follows.

1. Test for alkaloids:

Wagner’s Test: To the 2-3 ml of filtrate, a few drops of Wagner’s reagent were added. The formation of a reddish-brown precipitate indicates the presence of alkaloids.

2. Test for Flavonoids: To 1ml of solvent extract, 50% methanol 2ml and add metal magnesium to that add 5-6 drops of concentrated HCL. The formation of a red colour solution indicated the test as positive for the presence of flavonoids.

3. Tests for Phenols :

FeCl₃ test: Two to three drops of FeCl₃was added to 1ml of the sample. Phenolic compounds produce a deep violet colour or black precipitate with ferric ions.

4. Test for Saponins:

Foam Test:-The solvent extract was diluted with 20 ml of distilled water and shaken in a graduated cylinder for 15 minutes. A one cm layer of foam formation indicates the presence of Saponins.

5. Test for Tannins:

The sample was stirred with distilled water (10ml) and then filtered. A few drops of 5% ferric chloride were then added. Black or blue-green colouration or precipitate was taken as a positive result for tannins’ presence.

6. Test for terpenoids :

Salkowski Tests: To 2 ml of solvent extract, 2 ml chloroform and 2 ml concentrated H₂SO₄ were added and shaken well. The formation of reddish-brown colour indicated the test as positive for the presence of terpenoids.

7. Test for Anthroquinone glycoside:

To the extract solution (1ml), 5% H₂SO₄(1ml) was added. The mixture was boiled in a water bath and then filtered; Filtrate was then shaken with an equal volume of chloroform and kept to stand for 5min, then the lower layer of chloroform was shaken with half of its volume with dilute ammonia; the formation of rosepink to red colour of the ammonical layer indicates the presence of anthraquinone glycosides.

8. Test for steroids:

Salkowski Tests: To 2ml of solvent extract, 2ml chloroform, and 2ml concentrated H₂SO₄were added. The chloroform layer appears red, and the acid layer’s greenish-yellow fluorescence indicated the test positive for steroids.

Physicochemical Studies:

Extractive Values:

Five grams of the powdered drug was extracted by cold maceration method by using different solvents distilled water, Alcohol 90%, and Chloroform.

Determination of Total ash:^{25, 26}

About 2g powdered drug sample was weighed and ignited in a flat, thin porcelain dish until fumes ceased to evolve, then lower the dish and heated more strongly until all the carbon is burnt off and cooled the dish in a desiccator. Weigh the dish containing ash and calculate the percentage of total ash with reference to the air-dried sample of the crude drug.

Acid insoluble ash:

Acid insoluble ash is determined by dissolving ash in dilute hydrochloric acid (10% m/m), the liquid is filtered through an ash less filter paper and thoroughly washed with hot water. The filter paper is ignited in the original dish, cooledand weighed.

RESULTS:

Figure: 1 Root of Lantana camara

T.S. of Thin Root

Figure:2 T.S. of Thin root (Rd: Rhysidome ; Co: Cortex; Px : Protoxylem; Sx: Secondary xylem; Ve: Vessel; SPh: Secondary phloem; Pe: Periderm; GR: Growth rings; )

Thin-root:

The thin root, measuring about 500µm thick, consists of a wavy intact rhizodermal layer, a narrow cortical zone of shrunken cells and disintegrated phloem. The secondary xylem is a circular, dense, solid cylinder of fibres and a few vessels. The fibres are thick-walled with a narrow lumen. The vessels in the central portion of the xylem cylinder are narrow and scattered. Towards the periphery, the vessels are wide, circular and thin-walled; they occur in a single ring.

The secondary xylem is compact and circular, measuring1.9 mm in diameter. It exhibits three or four distinct growth rings. The growth rings seemingly ring porous. Growth ring boundaries are demarcated by thick-walled fibres; at the beginning of each growth occurs a ring of wide thin-walled vessels; external to the zone of wide vessels, these are abruptly smaller, sparsely distributed vessels. The vessels are circular or ovate, thick-walled and solitary. The wider vessels are up to 120µm in diameter; the narrow vessels are up to 40µm wide.

The primary xylem is fairly preserved in the central part. It consists of 5 or 6 exarch xylem strands.

T.S. of thick root:

Figure 3. T.S. of Thick root(Co: Cortex; GR: Growth rings; Pe: Periderm; GRB: Growth ring boundary; MX: Metaxylem; NVe: Narrow vessel; Rd: Rhysidome; Px: Protoxylem; Sph: Secondary phloem; Sx: Secondary xylem; Ve: Vessel)

Thick root: The thick root is 2.4mm thick. It consists of a reasonably wide periderm with a rough and fissured surface. The narrow cortical zone consists of one or three layers of cells. The secondary phloem is wider and continuous throughout the Vascular growth rings cylinder. The phloem elements occur in compact radial files and include sieve elements and parenchyma cells.

Powder Microscopy:

Figure: 4 Powder Microscopy: Wide vessel elements and xylem fibres, Narrow tailed vessel elements, fibres and Parenchyma cells

(LWP: Lateral wall pits; NF: Narrow fibres; NVe: Narrow vessel elements; Pa: Parenchyma; PP: Perforation plate; Ta: Tail; WF: Wide fibre; WVe: Wide Vessel element; )

Figure: 5, Xylem fibres and narrow tailed vessels

(LWP: Lateral wall pits; NF; Narrow fibres; NVe: Narrow vessel elements; Pa: Parenchyma; PP: Perforation plate; Ta: Tail; WF: Wide fibre: WVe: Wide vessel element; )

Figure6:Narrow tailed Vessels, (LWP: Lateral wall pits: Pp: Perforation plate; Ta: Tail)

Table 1. Results of Phytochemical tests:

Phytochemical tests	Hexane	Ethyl Acetate	Methanol	Distilled water
Alkaloids	+ve	-ve	-ve	-ve
Flavanoids	-ve	-ve	+ve	+ve
Phenols	-ve	-ve	+ve	+ve
Saponin	-ve	-ve	-ve	-ve
Tannins	-ve	-ve	-ve	-ve
Terpenoids	+ve	+ve	-ve	-ve
Anthroquinone	-ve	-ve	-ve	-ve
Steroids	-ve	-ve	+ve	-ve

(+ve: Positive; -ve: Negative)

Table 2. Results of Extractive values:

Solvent	Extract Recovered	Percentage Solubility
Distilled Water	0.53	10.6
Alcohol 90%	0.89	17.8
Chloroform	0.88	17.6

Table 3. Results of Ash values

Test	Value obtained
Total Ash	4.2%
Acid insoluble Ash	2.5%

DISCUSSION:

Leaf extracts of Lantana camara were reported for antiprolifirative activity against HEP-2 ( laryngeal cancer ) and Ncl H 292 ( lung cancer ) cell lines.⁴

Leaves of Lantana camara were reported to exhibit cytotoxic effect on vero cell line.⁵

Methanolic extracts of different parts of Lantana camara exhibited significant cytotoxic activity against five different human cancer cell lines.⁶

Methanolic extracts of Lantana camara leaves show cytotoxic effect against MCF-7 cell line.⁷

Oleanonic acid isolated from Lantana camara was screened for anti-cancer activity against murine tumour and three other human cancer cell lines. The extracts have shown good cytotoxic activity against A375 (Malignant skin Melanoma ) cancer cells.⁸

Ethanolic extracts of Lantana camara leaves and roots have been reported to possess anti-bacterial activity.²⁷

Methanolic extracts of different parts of Lantana camara have shown anti-bacterial activity against Gram positive Bacillus cereus and Gram negative Salmonella typhi.¹⁰

Antihyperglycemic activity of methanol extracts of Lantana camara leaves was reported in alloxan induced diabetic rats.¹¹

Hypoglycaemic activity of methanolic extracts of Lantana camara fruits was reported in streptozocin induced diabetic rats.²⁸

Aqueous extracts of Lantana camara was reported for anti-inflammatory activity in albino rats.²⁹

Wound healing property of ethanolic leaf extracts of Lantana camara was reported in adult male Wister rats.³⁰

Topical application of aqueous extracts of Lantana camara leaves remarkably decreased the number and duration of epileptic seizures as compared to sodium valproate in kainite treated mice.³¹

Standardization of herbal medicines is the process of prescribing a set of standards or inherent characteristics, constant parameters, definitive qualitative and quantitative values that carry an assurance of quality, efficacy, safety and reproducibility. It is the process of developing and agreeing upon technical standards.

Specific standards are worked out by experimentation and observations, which would lead to the process of prescribing a set of characteristics exhibited by the particular herbal medicine. Hence standardization is a tool in the quality control process.³²

Identification and authentication are two major steps in the quality evaluation of herbal medicines. These methods are very important in herbal drug authentication and quality control, and they continue to provide the main methods used in pharmacopoeias worldwide. A sufficient number of tests should be performed on representative samples as necessary to establish the identity of the ingredients. Although the focus here is on tests that establish identity, the unique properties of the material are also considered in selecting tests. The botanical identity—scientific name (genus, species, subspecies/variety, author, and family)—of each medicinal plant under cultivation should be verified and recorded. If available, the local and English common names should also be recorded.³³

The scientific pharmacognostic study of a medicinal plant begins with the proper identification of the genus and species of the selected plant by comparison with the available literature and references by an expert botanist and taxonomist. The morphological features are properly observed and compared with references to identify the proper genus and species, the microscopic study is vital to scientifically establish the identity of a plant in a reproducible and cost effective way. In the present study after confirming the plant root morphologically, for microscopic study of the root slides of the transverse sections and the powdered root were prepared by specific methods and photomicrographs were taken and labelled identifying the distinctive features; all these can be utilized to set parameters for standardization of the root drug.

CONCLUSION:

Lantana camara, once considered an obnoxious weed, has been used to treat different ailments in the traditional system of medicine. Based on this, it has been scientifically investigated for most of its medicinal properties, and different phytoconstituents have been isolated and linked adequately to its pharmacological activity.

In the present scenario, there is a need to obtain the crude drug from an authentic source and confirm it according to scientific parameters. An attempt has been made to establish parameters to scientifically identify and authenticate the root parts of Lantana camara pharmacognostically and phytochemically. This would be helpful to obtain the crude drug from its origin in pure form, free from other species and adulterants. This data can be utilized to design a scientific monograph for the crude drug,

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 14.11.2022 Modified on 16.01.2023

Res. J. Pharmacognosy and Phytochem. 2023; 15(2):99-104.

DOI: 10.52711/0975-4385.2023.00015