INTRODUCTION:

The measurement of Siddha drug standards is the most important factor. Drugs are utilized either unadulterated or after undergoing certain changes. They may be herbal, metal, mineral or animal origin. Siddha medicines become cherished throughout the world in those days.

The most recent methods should be used for the evolution of standards because the system deals with the use of multiple plant medications, increasing the likelihood of adulteration and substitution¹. Treatment options for Siddha comprise 32 external and 32 internal medications. Theeneer (distillate) is one of the internal medicines in which coarsely grounded drugs are immersed in water for about 8 hrs then it is distilled into the distillation apparatus ². Depending on the kind of raw drug and formulations used, the distillate's effectiveness varies. "Ekamooligai theeneer" is the name given to the mixture of solitary herbs after which it is distilled, generally used on long duration basis as blood purifiers, rejuvenators, tonics and digestives³. Sombu Theeneer is the formulation which is quoted in the Siddha classical text Siddha Vaithya Thiratu indicated for sinusitis, abdominal colic, bronchial asthma, fever and dysuria⁴. Drugs must be standardized by having their identities verified and their standards and purity determined. The potency and safety of pharmaceuticals can be impacted by inadequate quality control, which could result in adverse health effects for users⁵. The markets for traditional and herbal medicines in India offer substantial export opportunities. Strict quality standards, quality control, safety and efficacy are necessary for export to multiple nations⁶. Pharmacopoeial Laboratory for Indian Medicine (PLIM), Ghaziabad was tasked by the Department of Ayush with creating a testing protocol⁷. The current volume of the protocol for testing Ayurvedic, Siddha, and Unani products includes methods for determining these safety and quality parameters as well as limits for heavy metals, microbial load, pesticide residue and aflatoxins. The parameters are tested for different categories of single and compound formulations. For the purpose of apprising the necessity of standardization of medicines, the present study aims to exhibit standardized parameters for the Siddha formulation Sombu Theeneer in accordance with the PLIM guidelines.

MATERIALS AND METHODS:

Procurement of the test drug:

The test drug was purchased from GMP certified company IMPCOPS (Indian Medical Practitioners Co-operative Pharmacy and Stores) and used for analysis.

Table 1. Ingredients of the test drug – Sombu Theeneer

S. no

Ingredients of the test drug

Quantity

Sombu

(Foeniculum vulgare Mill.)

20 ser (560 gm)

Water

1 Padhaku (10.7 litres)

Physicochemical analysis:

Organoleptic characters of Sombu theeneer:

Organoleptic features such as nature, odour, property, state, and appearance were evaluated.

Solubility Profile:

The solvents used for the solubility test were DMSO, hexane, water, ethanol, and chloroform.

Determination of Specific gravity:

Specific gravity bottle was calibrated by adding freshly boiled and cooled water to it at a temperature of 25ºC, then weighing the contents. Followed by this the test item was filled and the weight of the Specific gravity bottle along with the test sample was weighed. The weight of a given volume of a liquid at 25ºC (unless otherwise noted) is compared to the weight of an equivalent volume of water at the same temperature; all weights are taken in air. This process determines a liquid's specific gravity ⁸.

Test for acid and basic radicals:

A 250 ml clean beaker was filled with precisely measured 25 ml of Sombu Theeneer and 50 ml of distilled water for the biochemical analysis. Then, for roughly ten minutes, it was thoroughly boiled. Next, it was allowed to cool and filter, and then 100 millilitres of distilled water were added to it to make it up to 100 millilitres. In this analysis, the test for specific acid radical carried with respect to sulphate, chloride, carbonate and phosphate. Further test for basic radicals proceeded for identification of calcium, magnesium, ammonium, mercury, arsenic, iron, zinc and miscelleneous such as starch, reducing sugar, alkaloids, tannic acid and unsaturated compound.

TLC analysis:

Using silica gel 60F254 (7X6 cm; Merck), the test sample was subjected to thin layer chromatography (TLC) in accordance with a traditional one-dimensional ascending method. The sample was cut with regular household scissors. Soft pencil was used to mark plates. A 10-micro litre sample was applied using a micro pipette, which was positioned five tracks apart at a distance of one centimetre each. Following the run plates' drying and observation using visible light, they were placed in the twin trough chamber equipped with the designated solvent system. Light long-wave UV light (365 nm) and short-wave UV light (254 nm) ⁹.

HPTLC analysis:

The HPTLC method is an advanced, automated, and sophisticated separation technique that was developed from TLC. The use of auto samplers and pre-coated HPTLC graded plates allowed for precise, sensitive, and significant separation on both a qualitative and quantitative level. HPTLC, or high performance thin layer chromatography, is an invaluable instrument for evaluating botanical materials in an economical and efficient manner. High levels of selectivity, sensitivity, and speed are provided by the HPTLC method in addition to one-step sample preparation. Therefore, it is convenient to use this method for regular quality control analysis. It offers a phytochemical chromatographic fingerprint that can be used to verify the identity and potency of phytotherapeutics¹⁰.

HPTLC- Chromatogram Development:

It was done in chambers with CAMAG Twin Troughs. The method of elution of the sample was determined by the component's adsorption capacity. Plates were removed from the chamber and dried following elution.

HPTLC- Scanning:

Plates were scanned at 366 nm under UV light. Software called CAMAG was used to integrate the data that were scanned. To identify the phytoconstituents in each sample, a chromatographic finger print was created and the corresponding Rf values were recorded¹¹.

Heavy metal analysis:

One of the most popular and trustworthy methods for identifying metals and metalloids in environmental samples is atomic absorption spectrometry (AAS). AAS Model AA 240 Series was used to determine the sample's overall heavy metal content. In order to ascertain the test item's concentrations of heavy metals like lead, cadmium, arsenic, and mercury. The test sample was digested using 1mol/L HCl in order to measure the levels of mercury and arsenic. Similarly, 1mol/L of HNO3 was used to digest the sample in order to determine the levels of lead and cadmium. Standards like As and Hg require a 100 ppm sample in 1mol/L HCl, and metals like Cd and Pb require a 100 ppm sample in 1mol/L HNO3^12,13.

Test for Specific pathogen:

Using the pour plate method, the test sample was directly inoculated into the designated pathogen medium (EMB, DCC, Mannitol, Cetrimide). For observation, the plates were incubated for 24 to 72 hours at 37°C. Existence of a particular pathogen recognized by its unique colour in relation to the pattern of colony formation in every type of media. The pathogens Salmonella, E-coli, Pseudomonas aeruginosa and Staphylococcus aureus were tested.

Pesticide residue Analysis:

Acetone was used to extract the test samples, and then they were homogenized for a short while. Acetone was then added to the test mixture after more filtering was permitted. The test sample was heated in a rotary evaporator set to a maximum temperature of 40ºC until the solvent had nearly evaporated. Add a few millilitres of toluene to the residue and heat it once more until the acetone is completely gone. After being dissolved in toluene, the residue will be filtered through a membrane filter ¹⁴.

Aflatoxin Analysis:

A standard aflatoxin solution in volumes of 2.5 µL, 5 µL, 7.5 µL, and 10 µL was applied to the surface of a pre-coated TLC plate. In the same way, the test sample was positioned and when the solvent front has moved at least 15 cm from the origin, the spots were dried and then chromatogram was developed in an unsaturated chamber with a solvent system made up of a combination of chloroform, acetone, and isopropyl alcohol (85: 10: 5). After taking the plate out of the developing chamber and labelling it with the solvent, it was air dried. Examine the plate under 365 nm of UV light to find the spots ^15,16.

RESULTS:

Organoleptic characters of Sombu theeneer:

The organoleptic characters of Sombu theeneer are mentioned in the table 2. It reveals that Sombu theeneer is a pale white coloured non- viscous liquid with strong characteristic odour and liquid consistency.

Table 2. Organoleptic characters

State	Liquid
Appearance	White coloured
Nature	Clear liquid
Odor	Strong Characteristic
Property	Non-Viscous

Solubility Profile of Sombu theeneer

The sample was soluble in ethanol, DMSO and water. It was insoluble in chloroform, hexane and ethyl acetate as listed in table 3.

Table 3. Solubility Profile

S. No	Solvent Used	Solubility
1	Chloroform	Insoluble
2	Ethanol	Soluble
3	Water	Soluble
4	Hexane	Insoluble
5	Ethyl acetate	Insoluble
6	DMSO	Soluble

Determination of Specific gravity:

Sombu Theeneer had a specific gravity of 0.9224.

Results on acid and basic radicals:

The trial drug's chemical analysis shows that it contains alkaloids, carbonate, ammonium, and iron. As listed in table 4.

Results of HPTLC chromatogram

Four noticeable peaks are found in the sample, which indicates that it contains four different types of phytocomponents, according to an HPTLC finger printing analysis. The peaks have Rf values between 0.10 and 0.12. As listed in table 5 and figure 1.

Figure 1. HPTLC finger printing

Table 4. Test for acid and basic radicals

S. no	Experiment	Observation	Inference
I.	Test For Acid Radicals
1.	Test for Sulphate: In a test tube, 2 millilitres of the previously prepared extract were added to 2 millilitres of a 4% ammonium oxalate solution.	No cloudy appearance	Lack of sulphate
2.	Test for Chloride: Dil. HNO₃ was added to 2 ml of the previously prepared extract until the effervescence subsided. Silver nitrate solution (two millilitres) was then added.	No cloudy appearance	Lack of chloride.
3.	Test for Phosphate: Two millilitres of the extract were treated with two millilitres each of con. HNO₃and ammonium molybdate solution.	No cloudy yellow appearance.	Lack of phosphate.
4.	Test for Carbonate: Magnesium sulphate solution was used to treat two millilitres of the extract.	Cloudy appearance present.	Carbonate is present
II.	Test for Basic Radicals
1.	Test for Calcium: A solution of 4% ammonium oxalate was added to two millilitres of the extract.	No Cloudy appearance.	Lack of calcium
2.	Test for Magnesium: Excess drops of sodium hydroxide solution were added to 2 millilitres of extract.	No white precipitate.	Lack of magnesium
3.	Test for Ammonium: Few millilitres of Nessler's reagent and an excess of sodium hydroxide solution were added to two millilitres of extract.	Brown colour appeared.	Ammonium is present.
4.	Test for Mercury: A solution of sodium hydroxide was added to two millilitres of the extract.	No yellow precipitate was appeared.	Lack of mercury
5.	Test for Arsenic: A solution of sodium hydroxide was added to two millilitres of the extract.	No brownish red precipitate was obtained.	Lack of Arsenic
6.	Test for Iron: a. Add 2 ml of ammonium thiocyanate solution to the 2 ml of extract. b. 2 ml of ammonium thiocynate solution and 2 ml of con. HNO3 were added to the 2 ml of extract.	Mild red colour appeared. Blood red colour appeared.	Iron is present. Iron is present.
7.	Test for Zinc: Excess sodium hydroxide drops were added to 2 millilitres of the extract solution.	White precipitate was not appeared.	Lack of zinc.
III.	Miscellaneous
1.	Test for Starch: A weak solution of iodine was used to treat 2 millilitres of the extract.	No blue colour developed	Lack of starch
2.	Test for Reducing sugar: In a test tube, add 5 ml of Benedict's qualitative solution, boil it for 2 minutes, then add 8–10 drops of extract and boil it for an additional 2 minutes. It was noticed that the colour changed.	No brick red colour developed.	Lack of Reducing sugar
3.	Test for the Alkaloids: Picric acid (2 ml) was added to 2 ml of extract.	Yellow colour developed.	Alkaloid is present.
4.	Test for Tannic acid: 2 millilitres of ferric chloride solution were added to 2 millilitres of extract.	No Black colour precipitate was obtained.	Lack of tannic acid.

Table 5. Peak table analysis of Sombu Theeneer

Peak	Start Rf	Start Height	Max Rf	Max Height	Max %	End Rf	End Height	Area	Area %
1	0.10	6.3	0.04	406.8	27.51	0.02	322.2	11488.8	37.55
2	0.01	315.4	0.00	432.1	29.22	0.01	321.2	3971.7	12.98
3	0.03	342.7	0.04	351.6	23.78	0.08	285.0	8868.4	28.98
4	0.12	275.8	0.13	288.1	19.48	0.18	5.8	6268.4	20.49

Results of Heavy metal analysis:

The current investigation's findings unequivocally demonstrate that the sample contains no traces of heavy metals like arsenic, cadmium, or mercury, but it does contain lead at a level of 1.180 PPM as listed in the table 6.

Table 6. Heavy metal analysis

Name of the Heavy Metal	Absorption Max l max	Result Analysis	Maximum Limit^12,13
Lead	217.0 nm	1.180	10 ppm
Arsenic	193.7 nm	BDL	3 ppm
Cadmium	228.8 nm	BDL	0.3 ppm
Mercury	253.7 nm	BDL	1ppm

BDL: Below Detection Level

Results on Specific pathogen:

Result analysis on Specific pathogen test of Sombu Theeneer indicates the absence of pathogens such as Salmonella, E-coli, Pseudomonas aeruginosa and Staphylococcus aureus. As listed in table 7 . In all of the plates that were inoculated with the test sample, there were no colonies or growths visible.

Table 7. Specific pathogen analysis of Sombu Theeneer

Organism	Specification	Result	Method
Salmonella	Absent	Absent	As per AYUSH specification
E-coli	Absent	Absent
Pseudomonas aeruginosa	Absent	Absent
Staphylococcus aureus	Absent	Absent

Results on Pesticide residue Analysis

The findings demonstrated that the sample submitted for analysis contained no traces of pesticide residues, including pyrethroids, organo chlorine, phosphorus and carbamates as listed in table 8.

Table 8. Pesticide residue analysis of Sombu Theeneer

Pesticide Residue	Sample	Ayush Limit (mg/kg)¹⁴
I.Organo Chlorine Pesticides
Alpha BHC	BQL	0.1mg/kg
Beta BHC	BQL	0.1mg/kg
Gamma BHC	BQL	0.1mg/kg
Delta BHC	BQL	0.1mg/kg
DDT	BQL	1mg/kg
Endosulphan	BQL	3mg/kg
II.Organo Phosphorus Pesticides
Malathion	BQL	1mg/kg
Chlorpyriphos	BQL	0.2 mg/kg
Dichlorovos	BQL	1mg/kg
III. Organo carbamates
Carbofuran	BQL	0.1mg/kg
III.Pyrethroid
Cypermethrin	BQL	1mg/kg

BQL- Below Quantification Limit

Results on Aflatoxin Analysis

When compared to the standard, the test sample loaded on TLC plates did not exhibit any spots, indicating that it was free of aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2. As listed in table 9.

Table 9: Aflatoxin Analysis

Aflatoxin	Sample	Ayush Specification Limit^15,16
B1	Not Detected – Absent	0.5 ppm (0.5mg/kg)
B2	Not Detected – Absent	0.1 ppm (0.1mg/kg)
G1	Not Detected – Absent	0.5 ppm (0.5mg/kg)
G2	Not Detected – Absent	0.1 ppm (0.1mg/kg)

DISCUSSION:

Theeneer is the most potent formulation due to its extraction technique, shows quick action, easy to metabolize¹⁷. Sombu Theeneer is the Siddha formulation in which Sombu (Foeniculum vulgare Mill.) is the key ingredient that has acrid with a sweet taste and hot potency, these properties neutralize the kabam humour. The individual drug shows significant presence of pharmacologically active phytoconstituents such as Trans anethole, Estragole, Methylchavicol, Coumarins, Anisaldehyde, Scopoletin, Bergapten, Umbelliferone, Xanthotoxin, Eugenol ^18,19. Trans-anethole is the chief component of several essential oils which has anti inflammatory, neuro protective, anti oxidative and anti genotoxicity activity ^20-22. Methylchavicol is a special metabolite belongs to phenylpropanoids found in essential oils of medicinal plants possess anti microbial activity ²³. Anisaldehyde has acaricidal activity ²⁴ and Coumarins possess anti breast cancer activity, antiviral, antibacterial, antifungal, antiparasitic, analgesic, anti inflammatory, anticoagulant, anti oxidative, anti neurodegenerative, anti depressive and antidiabeticproperties^25,26. Scopoletin shows antimicrobial, analgesic, anti-oxidant, anti-inflammatory, anti-amyloidogenic, anti-aging, anti-cholinesterase, diuretic, anti-tumor, hepatoprotective, cardioprotectiveactions ²⁷. Umbelliferone shows anti-inflammatory, anti cancerous, anti diabetic, molluscicidal ²⁸. Bergapten is a potent anti histamine, antimicrobial, neuroprotective, anti inflammatory, anti cancer and anti diabetic agent ^29,30. Eugenol has a great role in anti viral, anti inflammatory, anti bacterial and anti oxidative activities³¹. The formulation ‘Sombu Theeneer’ did not show signs of toxicity and ensured safety for clinical usage³². The results obtained from the HPTLC analysis of the sample Sombu Theeneer contains four different types of phytocomponents. Each peak's Rf value falls between 0.10 and 0.12. Result analysis on acid radical analysis of the formulation Sombu Theeneer reveals the presence of carbonates. Further test on basic radicals reveals the presence of ammonium and iron. Other tests reveal the presence of alkaloids. The sample has no traces of heavy metals like arsenic, cadmium, or mercury, according to the heavy metals analysis results, but lead is present at a level of 1.180 PPM. Outcome of specific pathogen test of Sombu Theeneer indicates the absence of pathogenic microbes such as Salmonella, E-coli, Pseudomonas Aeruginosa and Staphylococcus Aureus. Pesticide residue analysis signifies that there were no traces of pesticides residues such as pyrethroids, organo chlorine, phosphorus and carbamates. Further the formulation Sombu Theeneer was free from Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2. It was concluded that the formulation Sombu Theeneer complies with the standard as prescribed by the guideline.

CONCLUSION:

There has been a worldwide effort to supervise the quality and administer the expanding industry of traditional medications. Health care agencies and executives from several countries have demonstrated a dynamic commitment to provide standardized botanical drugs. The present investigation results reveals that the formulation Sombu Theeneer follow the standard outlines in the Ayush guideline.

COMPETING INTEREST:

There is no conflict of interest among the authors.

FUNDING:

No funding was received for the study.

ACKNOWLEDGEMENT:

I would like to thank all the faculties of Department of Maruthuvam.

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Received on 17.12.2023 Modified on 12.04.2024

Research J. Pharm. and Tech 2024; 17(11):5261-5266.

DOI: 10.52711/0974-360X.2024.00805