1. INTRODUCTION:

The traditional medicine is the current need of the existing health care system and the various traditional systems of medicine are proving their effectiveness in improving the standards of existing treatment methods. Siddha system of medicine is one among the oldest systems of medicine. Kodiveli Churnam is a compound Siddha formulation made up of Plumbago zeylanica (root-35g), Pongamia pinnata (root-35g), Chukrasia tabularis (stem bark-35g), Piper longum (fruit-17.5g), Brassica juncea (seed-17.5g), Nigella sativa (seed-17.5g), Zingiber officinalis (rhizome-17.5g) and Terminalia chebula (pericarp-17.5g). The formulation has been used traditionally for arthritis and the chronic inflammatory disorders¹. Plumbago zeylanica L., which is already considered to be a medicinal plant of higher importance has exhibited a wide range of activities such as anti-fungal, anti-bacterial, anti-oxidant, anti-diabetic, hepatoprotective, anti-inflammatory, wound healing and cytotoxicity².

Whereas the second constituent of the formulation, Pongamia pinnata and its various preparations have exhibited a similar broad spectrum of biological activities like antioxidant, antimicrobial, anti-inflammatory and anti-diabetic activities³. In the countries of largest population such as India and China, the barks of Chukrasia tabularis, the third constituent of the formulation have been used traditionally as anti-diarrheal and anti-influenza drugs. It is also utilised for fever like conditions due to antipyretic properties. As it is a well known astringent which heals the scars and stretch marks it is also used in the conditions such as insect bites and allergies and their decoction is used to relive minor skin irritations⁴. In the series of the above constituents, Piper longum is the commonly used herb to treat chronic bronchitis, constipation, asthma, gonorrhoea, diarrhoea, paralysis of the tongue, cholera, viral hepatitis, chronic malaria, stomach ache, respiratory infections, cough, tumours, bronchitis and diseases of the spleen. It has the most efficient phytoconstituents such as piperine, sylvatin, piperlongumine, sesamin, pipermonaline, diaeudesmin and piperundecalidine⁵. Next to Piper longum, Brassica juncea seeds which are commonly called as mustard seeds have a plethora of biological activities to combat severe illnesses like obesity, diabetes, depression, cataract and cancer⁶. Nigella sativa Linn. seeds, often known as black seed or black cumin, are used in traditional (herbal) medicine around the world to cure and prevent a variety of ailments and conditions, including asthma, diarrhoea, and dyslipidaemia. Thymoquinone, the primary component of the essential oil but also found in the fixed oil, has been demonstrated to be responsible for much of the biological activity of the seeds. The seeds' crude extracts (and some of its active ingredients, such as volatile oil and thymoquinone) have been shown to protect against nephrotoxicity and hepatotoxicity caused by disease or pollutants. Anti-inflammatory, analgesic, antipyretic, antibacterial and anticancer properties are all present in the seeds/oil. The oil lowers blood pressure while increasing breathing⁷. Zingiber officinalis is an unavoidable herb with numerous medicinal properties used commonly throughout the world. Immunomodulatory, anti-tumorigenic, anti-inflammatory, anti-apoptotic, anti-hyperglycemic, anti-lipidemic and anti-emetic activities are among the principal pharmacological properties of ginger and substances derived from it⁸. Terminalia chebula Retz. (T. chebula) is a member of the Combretaceae family and is one of the most widely utilised medicinal plants in Ayurveda, Siddha, Unani, and Homeopathy. It is known in Tibet as the "King of Medicines" and is placed first in the Ayurvedic materia medica due to its amazing wound-healing abilities and vast range of therapeutic characteristics. Antibacterial, antifungal, antiviral, antidiabetic, antimutagenic, antioxidant, antiulcer, and wound healing activities are all found in T. chebula. It also protects heart from injury and is used to treat renal problems. In traditional medicine, it is a moderate, safe, and effective laxative. T. chebula and its phytoconstituents have a nontoxic therapeutic effect. T. chebula is a component in the well-known herbal remedy Triphala, which is used to cure enlargement of the liver, stomach problems and eye pain⁹.

As Kodiveli churnam has the active parts of all the above-mentioned plants, it is expected to provide a wide range of pharmacological effects. Hence as a part of the study, standardization of Kodiveli churnam was done. The microscopic and phytochemical studies are essential to authentify this Ayurvedic preparation available in the market under various trade names. Considering this need, powder macroscopy, microscopy and HPTLC fingerprinting were carried out for its standardization.

2. MATERIALS AND METHODS:

2.1 Preparation of Kodiveli Churnam:

The active parts of the above plants represented in Table1 were collected, authenticated, shade dried and powdered. The prepared churnam was passed through the sieve no. 85 to get a uniform powder. The finally prepared churnam was stored in the air tight container and stored at cool dry place.

Table 1: List of constituents used to prepare Kodiveli churnam

S.no.	Source	Active Part	Quantity
1	Plumbago zeylanica	Root	35g
2	Pongamia pinnata	Root	35g
3	Chukrasia tabularis	Stem bark	35g
4	Piper longum	Fruit	17.5g
5	Brassica juncea	Seed	17.5g
6	Nigella sativa	Seed	17.5g
7	Zingiber officinalis	Rhizome	17.5g
8	Terminalia chebula	Pericarp	17.5g

2.2 Macroscopy and Microscopy:

The macroscopic characteristics of the formulation were observed visually and the microscopic characteristics were observed using compound microscope^10-22.

2.3 Preparation of Extracts for TLC/HPTLC:

The Kodiveli chooram at a quantity of 4 g was soaked overnight in ethanol and heated using water bath for 30 minutes and filtered. The filtrate was further concentrated by heating on water bath and made up to 10ml in a standard flask separately^23-25.

2.4 Method for Development of TLC/HPTLC:

The aluminium chromatographic plates with 0.2mm thickness pre coated silica gel 60F254 were used to develop the TLC/HPTLC finger prints of the Kodiveli Churnam. The CAMAG Linomat IV Automatic Sample Spotter (Camag Muttenz, Switzerland) equipped with syringe (Hamilton, 100μL) was used to spot the samples and the fingerprint was developed in the twin trough glass chamber. The mobile phase used was Toluene : Ethyl acetate: Formic acid : Methanol in the ratio of 6.5:2.5:0.5:0.2v/v/v/v. CAMAG TLC scanner II equipped with CATS 3 software was used to scan the developed TLC plates. The developed spots were observed at various UV (254, 366 nm) and Visible (520 nm) wavelengths^26-28.

3. RESULTS:

3.1 Macroscopy and Microscopy:

The finally prepared Kodiveli Churnam has an appearance of moderate to fine powder and yellowish brown in colour as shown in Figure 1. It has a characteristic odour and slightly pungent taste. As represented in Figure 2, under microscope showed different shapes and sizes of thick walled, lignified, isolated or groups of sclereids with pits and wide lumen; a few fragments of palisade- like testa with distinct parenchyma in surface view; numerous mostly single, isolated, oblong, rounded to oval, sac- shaped starch grains with rarely 2 component and a few groups of starch grains; a very few palisade-like testa in sectional view; few pitted parenchyma cells in different shapes and sizes; few brownish mass contents; few fragments of lignified, spiral and reticulated vessels associated with parenchyma cells; few fragments of thin walled polygonal parenchyma cell of testa in sectional view; a few fragments of lignified testa in surface view; a few fragments of lignified tracheids with wide lumen; a very few prismatic crystals of calcium oxalate; a very few uniseriate trichome with blunt tips; few fragments of thick walled, polygonal parenchyma cells of cork in surface view; few isolated or in groups, different shapes and sizes of thick walled, lignified stone cells with lumen and distinct pits; a few oil globules; a few fragments of endosperms cells in surface view and a few fragments of asepted fibres with narrow lumen.

Figure 1: Kodiveli churnam

Figure 2: Powder microscopy of Kodiveli churanam

1. Sclereids, 2.Testa in surface view, 3 and 4 Starch grains, 5. Palisade- like testa, 6. Pitted parenchyma, 7. Brown contents, 8. Vessels with parenchyma, 9. Parenchyma cells of testa, 10. Testa in surface view, 11. Aleurone grains, 12. Tracheids, 13. Prismatic crystals, 14. Trichome, 15.Cork, 16. Stone cells, 17. Oil globules, 18. Endosperm cells, 19. Fibres.

3.2 Chemical Characterisation:

3.2.1 Thin Layer Chromatography:

The best resolution of the spots of phytoconstituents present in the ethanolic extract of Kodiveli chooram (EEKC) was attained using the solvent system containing the mixture of Toluene: Ethyl acetate: Formic acid: Methanol in the ratio of 6.5:2.5:0.5:0.2 v/v/v/v. The spots observed in triplicate in the range of 254nm, 366nm and 520nm (derivatized using vanillin sulphuric acid) were shown in Figure 3. The respective Rf values of the spots and their colours were represented in Table 2.

Figure 3: TLC photo-documentation of Kodiveli churnam

Table 2: The Rf values and the colour characteristics of the TLC bands

	λ=254 nm		λ=366 nm		λ=520 nm (Derivitized)
Band No.	Rf	Colour	Rf	Colour	Rf	Colour
1	0.04	Dark	0.09	Dark blue	0.07	Pink
2	0.14	Dark	0.28	Salmon	0.13	Blue
3	0.25	Dark	0.34	Red	0.19	Light blue
4	0.37	Light	0.47	Cyan blue	0.24	Light pink
5	0.48	Dark	0.55	Brown	0.27	Pink
6	0.56	Dark	0.63	Green	0.42	Violet
7	0.62	Light	0.69	Light red	0.48	Yellow
8	0.79	dark	0.75	Red	0.66	Purple
9	-	-	0.78	blue	0.73	Purple
10	-	-	-	-	0.78	Light purple
11	-	-	-	-	0.85	Purple

3.2.2 High Performance Thin Layer Chromatography

The Densitometric scanning followed by High Performance Thin layer Chromatography of the EEKC at 254 nm revealed 11 peaks and the Rf values 0.14, 0.56, 0.48 were found to be the peaks of maximum height. At 366 nm 10 peaks were obtained and the Rf values of 0.64, 0.34, 0.48, 0.79 were found to be the major peaks. At 520 nm 14 peaks were obtained and the Rf values of 0.67, 0.74, 0.87 were found to be the major peaks. The other details of the obtained HPTLC finger print were represented in Figure 4.

4. DISCUSSION:

In traditional medicine most of the formulations available in the market are in the form of churna. Mostly they appear similar and mild variations are observed in their colour, texture and odour. So it is mandatory to identify a specific formulation in terms of organoleptic properties to confirm the authenticity. In this study the colour, texture and odour are the unique blend of its raw drugs. Pharmacognostic studies help us to identify the presence of specific microstructures of raw drugs in the finished formulation. In powder microscopy presence of testa and stone cells represents fruit and seeds of Piper longum, Brassica juncea and Nigella sativa. Starch grains and fibres represent the root of Plumbago zeylanica and Zingiber officinalis. Oil globules, starch, prismatic crystals and stone cell represent the root of Pongamia pinnata. Cork, trichome and brown content representing the stem bark of Chukrasia tabularis. Sclereids, starch grains, brown contents and stone cells representing the pericarp of Terminalia chebula. Other than the pharmacognostic techniques the HPTLC finger printing is mandatory as an advanced technique for the identification of any herbal formulation. The specific solvent system used in this study captures most of the phytoconstituents and hence it was selected for finger printing analysis. Visualisation at three wavelengths will ensure the capture of most of its phytoconstituents presents in this formulation.

5. CONCLUSION:

It is concluded that the present study analysed the organoleptic, macro-microscopic characters and TLC/HPTLC finger print of Kodiveli churnam and the results obtained may help in preparation, standardization and validation of the polyherbal formulation which will help to ensure the quality of this formulation.

6. CONFLICT OF INTEREST:

None.

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Received on 23.04.2022 Modified on 29.11.2022

Research J. Pharm. and Tech 2023; 16(11):5058-5062.

DOI: 10.52711/0974-360X.2023.00819