INTRODUCTION:

Plectranthus amboinicus (Lour.) Spreng. belongs to family Lamiaceae, and is commonly known as Parnayavani in Sanskrit, in English as country borage. Its different synonyms are found in Ayurvedic classics as Chornika, Karpooravalli, Sugandhi, Parnika, Himasagar, Ashmari bheda. It is a perennial plant distributed throughout tropical Africa, Asia, Australia, and America, including Brazil¹. It is a large succulent aromatic fleshly perennial herb, possessing short soft erect hairs with distinctive smelling leaves. This plant is distributed throughout India, and is cultivated in gardens. This herb has therapeutic and nutritional properties attributed to its natural phytochemical compounds which are highly valued in the pharmaceutical industry. It is widely used in folk medicine to treat conditions like cold, asthma, constipation, headache, cough, fever and skin diseases. The leaves of the plant are often eaten raw or used as flavoring agents, or incorporated as ingredients in the preparation of traditional food.

This plant is commonly used in Chinese folk medicine for the treatment of cough, fever, sore-throats, mumps, and mosquito bite. In Taiwan, Plectranthus amboinicus (Lour.) Spreng. (PA) is a common folk medicine for summer cold, scald, wounds, and bites from bugs or mosquitoes^2,3.

In Ayurveda, the leaves are bitter, acrid, thermogenic, aromatic, anodyne, appetizing, digestive, carminative, stomachic, anthelmintic, constipating, deodorant, expectorant, lithontriptic, diuretic and liver tonic. They are effective in cephalalgia, otalgia, anorexia, dyspepsia, flatulence, colic, diarrhoea and cholera especially in children; halitosis, convulsions, epilepsy, cough, chronic asthma, hiccough, bronchitis, renal and vesical calculi, strangury, hepatopathy, malarial fever, antispasmodic and cathartic⁴. Expressed juice is applied round the orbit to relieve the pain in conjunctivitis⁵.

The juice of leaves with honey is helpful in cold and cough, the concentrated decoction consumed while warm is effective in respiratory infections. The juice mixed with sugar is a powerful carminative. These health benefits are reported as traditional or folkloric usage⁶. The juice of the leaves is applied on the affected part of the skin in cases of Insect bites, allergic skin thickening, itching and skin irritation²⁵. To reduce ‘Kapha’ (mucus, phlegm) in the body, a decoction prepared by boiling P. amboinicus leaves in water and added with cardamom, cloves and a teaspoon of honey is quite helpful if taken twice a day for 3 days⁷.

In Malaysia, bruised leaves are applied to burns and their poultice on centipede and scorpionbites. The leaf juice heals chapped lips and in java it is used to treat cracks at the corners of the mouth⁸.

Many pharmacological properties have been reported including antimicrobial, antifungal, anti-inflammatory, antidiabetic, anxiolytic, antineoplastic, analgesic, antimalarial, antibiofilm efficacy, diuretic, wound healing activity, and antiplatelet aggregation activity due to the presence of various important constituents or secondary metabolites such as flavonoids, glycosides, phenols, tannins, and steroids, which have been identified through various spectroscopic methods⁹. However a comprehensive pharmacognostical²⁵ and phytochemistry²⁶ related work yet to be done of the species.

The present study was therefore undertaken to evaluate the various qualitative parameters of Plectranthus amboinicus (Lour.) Spreng., the finding of which will be helpful in setting standards for this medicinal plant

The taxonomical position of Plectranthus amboinicus Lour:

Division : Magnoliophyta

Kingdom : Plantae

Clade : Angiosperms

Class : Magnoliopsida

Order : Lamiales

Family : Lamiaceae

Genus : Plectranthus

Species: Plectranthus amboinicus

Synonyms: Coleus amboinicus Lour.¹⁰

Vernacular name:

English: Country borage, Indian borage, Indian mint

Hindi: Patta ajavayin, Patharchur, Amroda, Pathercheer

Sanskrit: Karpuravalli, Sugandhavalakam, Parnayavani

Bengali: Amalkuchi

Kannada: Doddapatre, doddapatre soppu

Malayalam: Panikoorka

Gujarati: Ovapan

Marathi: Pathurchur¹¹

MATERIALS AND METHODS:

The plant material was collected at mature stage from coastal region of East Midnapur District, West Bengal, INDIA. It was authenticated through detailed Taxonomic study and confirm from the regional flora. The voucher specimen of the plant was deposited to the institute herbarium. This samples were washed under running tape water for 5 minutes followed by sterile distilled water for 3 times. Half of the fresh drug sample used for morphological study and half of them were air dried and pulverized to obtain 60 mesh size and stored in air tied container to avoid any contamination due to moisture. The macroscopy and organoleptic study of the crude drug were performed in terms of its shape, size, colour, odour, taste etc. For microscopy and anatomical study (transverse section), fine hand section of fresh parts of leave sample (petiole, mid rib, laminae) were done, washed, mounted in 50% glycerin solution and observed the microscopical characters with digital microscope^12-15. For powder microscopy powdered sample was treated with different solutions, strained and mounted following standard method and observed under a compound microscope at projection 10x and 40x for rapid and accurate determination of their identity following the standard methods^16-18,26,30. Phytochemical analysis was done on the basis of protocol prescribed by WHO on quality control methods for herbal materials (2011)¹⁹ and Ayurvedic Pharmacopoeia (2001)²⁰. For chemical profiling of the plant 5g of the crud sample was taken in a round bottom flask and 20ml of CH₃OH was added to it. The mixture was boiled for one hour under reflux and filtered (Whatman No. 40 filter paper). The filtrate was taken for preliminary phytochemical analysis.The methanolic extract, obtained as described in the previous section was utilised for preliminary phytochemical testing - 2ml of the extract being taken for each test²⁹. The results are given in Table 1. The extract concentrated and then carried out High Performance Thin Layer Chromatography (HPTLC) following the Standard methods^21,22,27,28.

RESULTS

Macroscopy:

Leaves are soft, simple, fleshy, thick, broad, ovate and covered with hairs, numerous at lower portion. Taste aromatic, odour pleasant, length 2.5-4cm long, with prominent petiole, apex acute. Serrate margins, petiole prominent. Ventral side of the leaf is dark green and dorsal side of the leaf is light green (Fig.1).

Fig.1: Morphology of Plectranthus amboinicus Lour. leaf

Transverse section of leaf:

The transverse section of leaf displays the dorsiventral nature with distinct adaxial and abaxial surfaces and the following tissues are present in midrib and lamina sections (Fig.2).

Midrib:

The transverse view of midrib shows a plano-convex shape with flat posterior side and hemispherical anterior side. Midrib consists of rectangular to polygonal epidermal cells with skinny and a little wavy anticlinal walls. It also encloses the small, single and less distinguished vascular bundles with 4-6 rows of xylem whereas only a thin arc of phloem is present. The stomata are mainly of diacytic type as shown by surface preparation and lamina is in continuation with midrib region.

Epidermis: It consists of only a single layer of rectangular cells with cuticle. Numerous trichomes of both covering and glandular are found. The two epidermises are almost identical to each other but later are more discontinuous due to presence of numerous trichomes and stomata.

Trichomes:

Both upper and lower epidermis are densely covered with trichomes of various types, mainly covering, glandular trichomes. Covering trichome sare unicellular and multicellular in nature (up to 6 cells) and are present with acute apex and comparatively wide basal cells.The base of glandular trichomes is made up of 2-3 cells with unicellular head.

Mesophyll:

This area is further divided into two regions depending upon type of cells present. Region near to upper epidermis contains single layer of compact and elongated cells and forms the adaxial palisade mesophyll, whereas towards lower epidermis more spherical parenchymatous cells containing chloroplast with almost negligible intercellular space are present and makes the abaxial spongy mesophyll of the leaf.

(A) (B)

(C) (D)

(E) (F)

Fig.2:Transverse section of Plectranthus amboinicus leaf

A. Transverse section of midrib of the leaf; B. Midrib of the leaf showing pith; C. Transverse section of lamina of the leaf; D. Different types of covering trichomes attached to epidermis; E. Multicellular covering trichomes attached to epidermis; F. Glandular and multicellular trichomes attached to epidermis.

Powder microscopy:

(A) (B)

(E) (F)

(G) (H)

(i)

Fig. 3: Powder microscopy of Plectranthus amboinicus Lour. Leaf A., B., C., D. Multi cellular uniseriate trichomes; E., F. Glandular trichomes; G., H. Diacytic stomata; I. Phloem fiber

(J) (K)

(L) (M)

(N) (O)

Fig. 4: Powder microscopy of Plectranthus amboinicus Lour. Leaf J., K. Epidermal cells; L. Annular vessels;M. Scalariform vessel;

N., O. Prismatic crystals

Powder Microscopy:

Plectranthus amboinicus Lour. Leaf powder shown multi cellular uniseriate trichomes with broad base, few trichomes are slightly bent and few are straight, numerous glandular trichomes, diacytic stomata, long thick walled phloem fibers with large lumen, epidermal cells and mucilage containing cells, group of annular vessels, scalariform vessels and numerous prismatic crystals of various sizes (Fig.3,4).

HPTLC Profile of Plectranthus amboinicus Lour. (Leaf):

Sample preparation:

1g of the sample was subjected to reflux with methanol for 1 hour and extract was filtered using filter paper. The filtrate was concentrated (c = 1g/10mL) and taken for the following TLC profile²⁷.

Chromatography experimental:

Stationary Phase:

Precoated (support on Aluminum Sheets) Silica Gel Plate. Specification: TLC Silica Gel 60 F₂₅₄, Mfg. by Merck, 26.09.2016, Batch No. 1.05554.0007.

Mobile Phase:

Hexane: Chloroform: Ethylacetate: Methanol: Formic acid (3: 2: 4: 0.5: 0.5) (v/v) [G R grade solvent used, mfg. by Merck, India].

Sample application:

Applied volume 5µL as 6mm band and applied at 15 mm from the base of the plate. Plate size was 5x10cm³⁴.

Development:

Developed up to 90mm in CAMAG Twin trough chamber, Plate preconditioning (temp 25°C and relative average humidity was 42%).

Photography of Developed HPTLC Plate:

254 nm and 366 nm.

Fig.5: HPTLC of Plectranthus amboinicus Lour. leaf

Result of Preliminary Phytochemical Analysis:

Table 1. Preliminary Phytochemical Tests of Methanolic Extracts of leafs of Plectranthus amboinicus

Sl. No.	Test/Reagent used	Observation
1a.	Alkaloids - Mayer’s test (in dil. HCl)	Negative
1b.	Alkaloids - Dragendorff’s test (in dil. HCl)	Negative
2a.	Carbohydrates – Molisch’s test	Negative
2b.	Reducing sugars - Fehling’s test	Negative
3a.	Flavonoids – (Lead acetate test)	Positive
3b.	Flavonoids - (Dil. NaOH, dil. HCl)	Positive
4.	Terpenoids/ Steroids - (Salkowski test)	Positive
5.	FeCl₃test for Phenolic compounds/ Tannins	Positive
6.	Glycoside (Acetic acid, FeCl₃, H₂SO₄)	Positive

Table -2 R_f values:

Sample	R_f values
Sample	254 nm	366 nm
Plectranthus amboinicus (Leaf)	0.28, 0.46, 0.58	0.28, 0.38, 0.46, 0.58, 0.74

DISCUSSION AND CONCLUSION:

Each medicinal plant and the specific plant part used as crude drug material contain active or major chemical constituents with a distinctive profile that can be used for chemical quality control and quality assurance²³. In the present study, the macroscopic, microscopic study of the leaf and its components reveals the characteristics feature in identifying the specimen, and also reveal the presence of contaminant or deterioration in a sample. The microscopical and anatomical study gives a preliminary idea about the nature and disposition of cells, tissues and cell inclusions. The Phytochemical screening revealed the presence of phenolic compounds, tannins, glycoside, in high concentrations in methanolic extracts. Thus the presence of wide varieties of secondary metabolites in the leaves of Plectranthus amboinicus (Lour.) Spreng. is proved to be effective against several diseases. HPTLC is a valuable quality assessment tool for the identification and quantification of chemical constituents present in plant drugs^31-34. The retention factor (Rf) values obtained from it can be used to identify compounds due to their uniqueness for each compound (Table 2). In the present study, the Rf values of individual compounds appearing as spots vertically have been noted (the less polar compounds moving higher up the plates resulting in higher Rf values), which may thus be used as a quality control profile for this drug and in near future this investigation will further helps the research scholars to isolate the important compounds present in this plant species.

ACKNOWLEDGEMENT:

We do hereby; express our deep sense of gratitude and appreciation to the Director of CARI, Kolkata for permitting us to undertake this research work, thus enabling us to get the feel of doing the work and kept going till completion.

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Received on 15.02.2021 Modified on 07.05.2021

Research J. Pharm.and Tech 2022; 15(2):717-722.

DOI: 10.52711/0974-360X.2022.00119