Over the past few years, there has been a significant resurgence in the popularity of herbal medicine on a global scale, as more and more people are turning to natural remedies for their health concerns. One of the ancient Indian medical systems known as the Siddha system, predominantly practiced in the southern regions of India, has garnered considerable interest. This growing fascination is evident as numerous Siddha formulations are being manufactured and made available in the market. However, despite the immense potential that these traditional herbal remedies hold, a major obstacle to their widespread acceptance is the absence of standardized quality control measures.

Although numerous herbal formulations are available in the market, many of them do not have well-defined quality control standards. This underscores the urgent necessity to create fast, sensitive, and precise quality control methods specifically designed for Siddha formulations, utilizing modern technological innovations. It is crucial to ensure that herbal medicines meet stringent quality standards, as the effectiveness and safety of these remedies directly depend on their composition and quality.^[1]

Given these circumstances, the primary focus of this study is to establish the standardization and quantification of phytochemical constituents in "Pun Purai Ennai," a polyherbal Siddha formulation renowned for its potential wound-healing properties. Through meticulous analytical methods and advanced technology, our objective is to gain a comprehensive understanding of the composition and quality of this ancient formulation, thereby enhancing its credibility and trustworthiness as a therapeutic option in contemporary medicine. The valuable insights gained from this research endeavor promise to promote wider acceptance and utilization of Siddha formulations, ensuring consistent and safe medicinal effects.

MATERIALS AND METHODS

Procurement of formulation

In this study, Pun Purai Ennai was sourced from the Government Siddha Medical College in Chennai.

Preparation of pun purai ennai

Pun purai ennai is a Siddha medicine, prepared using ingredients given in Table 1. Heat the oil and powdered ingredients except kungiliyam, in a sand gritty consistency. Unmount the stove add the kungiliyam finally and dissolve.

Table 1: Ingredients Of Pun Purai Ennai

Organoleptic characters ^[2]

Organoleptic characters of the pun purai ennai were noted. Organoleptic parameters are varna (colour), gandha (odour), ruchi (taste), and texture were analyzed.

Physiochemical analysis ^[3,4]

Physical parameters

Specific Density

The specific gravity of the oil was measured using the pycnometer method, and the specific density was calculated from these measurements.

pH

The pH of the oils was assessed using a pH meter.

Refractive Index

The refractive index was measured at room temperature with an Abbe refractometer.

Viscosity

The viscosity of the oil sample was evaluated using an Ostwald viscometer.

Boiling Point

The boiling point of the oil sample was determined using a thermometer with a precision of ±1 degree Celsius. It was observed that the boiling points were affected by the degree of unsaturation in the fatty acids present in the oils.

Spreadability

The spreadability of the oil was determined by utilizing glass plates coated with a 1% aqueous gelatin solution and was allowed to solidify on a tray floating on an ice bath for 1 h. The film was then dried at room temperature for two days using a dust-free cabin. Then plates were placed on graph paper on a flat surface and 10 µl of the oil was placed using a microliter pipette. After 5 min, the diameter of the circular spreading area was read.

Chemical Parameters

Determination of Free Fatty Acid

The free fatty acid (FFA) content in the oil sample was evaluated by titration using phenolphthalein as an indicator until a pink hue was observed. A precise amount of 0.002 kg of the oil sample was accurately weighed and placed into a volumetric flask.

Determination of Saponification Value

The saponification value was calculated by combining 2 g of the oil sample with 25 ml of 1.0% alcoholic KOH in a volumetric flask. The mixture was boiled for 45 minutes, ensuring complete saponification, aided by a condenser. After cooling, 1 ml of phenolphthalein was added, and the solution was titrated with 0.5N HCl until the pink color disappeared. A blank determination was performed simultaneously. The saponification value was calculated using the following formula:

Saponification value = 56.1 × N × (V2 - V1) / W

Where N is the normality of the HCl solution used.

V1 is the volume of HCl used in the analysis (ml).

V2 is the volume of HCl used in the blank (ml).

W is the weight of the sample (g).

Determination of Iodine Value

To determine the iodine value, 1 g of oil was mixed with 15 ml of carbon tetrachloride and 25 ml of Hanus reagent in a 500 ml volumetric flask. After allowing the mixture to stand in the dark for 30 minutes, 20 ml of 10% KI and 150 ml of distilled water were added. The solution was then titrated with 0.1 N thiosulphate until a faint yellow color faded. Then, 1.5 ml of starch indicator was added, and titration was continued until the blue color disappeared. A blank determination was conducted alongside the test. The iodine value was calculated using the formula:

Iodine value = 12.69 × (V2 - V1) × N / W,

Where, W is the weight of the sample (g),

V1 is the volume of HCl used in the test (ml),

V2 is the volume of HCl used in the blank (ml), and N is the normality of HCl.

Determination of Peroxide Value

The peroxide value, which measures the peroxide content in the oil, was determined by assessing the iodine released from potassium iodide. A known quantity of the oil sample was dissolved in acetic acid and chloroform, followed by the addition of saturated KI. The iodine liberated from KI due to the oxidative action of peroxides in the oil was measured by titration with standard sodium thiosulphate, using starch solution as an indicator. A blank titration was also performed. The peroxide value was calculated as follows:

PV (meq/kg oil) = (S - B) × W × N

Here, B is the volume of sodium thiosulphate utilized for the blank solution,

W is the weight of the oil sample,

S is the volume of sodium thiosulphate consumed by the oil sample, and

N is the normality of the standard sodium thiosulphate solution.

Determination of Acid Value

To determine the acid value, 3 g of oil samples were placed in 250 ml conical flasks. Freshly neutralized ethyl alcohol (30 ml) was added to ensure the complete dissolution of the samples. The solution was then boiled, cooled, and treated with 1 ml of phenolphthalein indicator. It was subsequently titrated with 1 N sodium hydroxide solution until a persistent light pink color appeared. The acid value was calculated using the equation:

Acid value = 2.82 × V × 100 / W × 1000 × 4

where V is the titre value of 1 N NaOH, and

2.82 is the equivalent weight of oleic acid.

Determination of Total Alkali

The total alkali content was determined by titrating the excess acid in the aqueous phase with a standard volumetric NaOH solution. The sample was mixed with ethanol and 1 N H2SO4 solution, heated until dissolved, and then titrated with 1 N NaOH using phenolphthalein as an indicator.

Foamability Test

For the foamability test, approximately 0.5 g of the sample was added to a 100 ml standard flask containing 100 ml of double-distilled water. The mixture was vigorously shaken for two minutes to generate foam and then allowed to stand for 10 minutes. The height of the foam was measured carefully.

Phytochemical screening ^[5]

The preliminary phytochemical evaluation was conducted for the pun purai ennai formulation following standard procedures. The evaluation involved a series of tests aimed at detecting the presence of various compounds in the sample, including carbohydrates, alkaloids, triterpenoids, glycosides, steroids, phenolic compounds, tannins, saponins, flavonoids, proteins, amino acids, and both fats and fixed oils.

Quantification of phytochemical constituents ^[6,7]

Quantification of Tannins

Tannins were quantified using the Folin-Ciocalteu method. A 10 ml volumetric flask was used to mix 0.1 ml of the sample with 7.5 ml of distilled water, 0.5 ml of Folin-Ciocalteu phenol reagent, and 1 ml of 35% sodium carbonate. The mixture was left to stand for 30 minutes. Reference standards, ranging from 0.07 to 1 mg/ml of tannic acid, were prepared in a similar manner. After incubation, the absorbance was read at 750 nm using a UV/Visible spectrophotometer. The tannin content was expressed as mg tannic acid equivalents per gram of the sample.

Quantification of Alkaloids

To quantify alkaloids in the sample, 0.63 g of the PPE sample was mixed with 200 ml of 10?etic acid in ethanol. The mixture was allowed to stand for 4 hours and then heated to reduce the volume to one-quarter of the original. The solution was filtered, and 15 drops of concentrated NaOH were added, followed by a 3-hour resting period. After incubation, the precipitate was collected, and the supernatant was discarded. The residue was washed with 20 ml of 0.1M ammonium hydroxide, filtered, and weighed to determine the alkaloid content.

Quantification of total phenol content ^[8,9]

The total phenolic content was determined using Folin Ciocalteu's method. Aliquots of 1 ml and gallic acid standards (ranging from 0.007 mg/ml to 1 mg/ml) were mixed with 5 ml of distilled water and 0.5 ml of Folin Ciocalteu's reagent. After 5 minutes, 1.5 ml of 20% sodium carbonate was added, and the volume was adjusted to 10 ml. The tubes were incubated for 2 hours, resulting in an intense blue color. Absorbance at 750 nm was measured using a UV-visible spectrophotometer. Triplicate experiments were conducted with a reagent blank, and the total phenolic content was expressed as mg gallic acid equivalent weight per 100 g of the sample.

Quantification of total flavonoid content ^[9,10,11]

The total phenolic content was determined using Folin Ciocalteu's method. Aliquots of 1 ml and gallic acid standards (ranging from 0.007 mg/ml to 1 mg/ml) were mixed with 5 ml of distilled water and 0.5 ml of Folin Ciocalteu's reagent. After 5 minutes, 1.5 ml of 20% sodium carbonate was added, and the volume was adjusted to 10 ml. The tubes were incubated for 2 hours, resulting in an intense blue color. Absorbance at 750 nm was measured using a UV-visible spectrophotometer. Triplicate experiments were conducted with a reagent blank, and the total phenolic content was expressed as mg gallic acid equivalent weight per 100 g of the sample.

Quantification of cyanogenic glycosides content ^[12]

The cyanogenic glycoside content in the sample (PPE) was determined through a distillation process after autolysis. The sample was mixed with 2.5% sodium hydroxide and an antifoaming agent (tannic acid). The resulting distillate was combined with ammonium hydroxide and potassium iodide. Titration with 0.02M silver nitrate was performed until continuous turbidity appeared, indicating the endpoint. The cyanogenic glycoside content in the PPE sample was then calculated using the following formula:

Cyanogenic Glycoside (mg/100g) = (Titre value (Cm³) ×1.08×Exact volume / Aliquot volume (Cm³) × sample weight (g)) × 100Mineral and metal analysis ^[13]

Qualitative test

• Silicate Test

A small portion of the sample was vigorously shaken with either concentrated hydrochloric acid (HCl) or sulfuric acid (H2SO4). The persistence of insoluble or slightly soluble residues after shaking indicates the presence of silicates in the sample.

• Phosphate Test

To test for phosphates, 2 ml of the sample was mixed with 2 ml of ammonium molybdate solution and 2 ml of concentrated nitric acid (Conc. HNO3). The appearance of a cloudy yellow solution confirms the presence of phosphate.

• Sulphate Test

2 ml of the sample was treated with 2 ml of diluted HCl until effervescence ceased, followed by the addition of 2 ml of barium chloride solution. A white precipitate that does not dissolve in concentrated HCl confirms the presence of sulphate.

• Chloride Test

2 ml of the sample was combined with 2 ml of dilute nitric acid (HNO3) until effervescence ceased. Then, 2 ml of silver nitrate was added. The formation of a curdy white precipitate indicates the presence of chloride.

• Sulphide Test

A foul-smelling gas, reminiscent of rotten eggs, was produced when 1 gm of the oil sample was combined with 2 ml of concentrated HCl, indicating the presence of sulphide compounds.

• Iodide Test

Upon mixing 2 ml of the sample with dilute nitric acid and 0.5 ml of silver nitrate solution, the formation of a pale-yellow, curdy precipitate indicates the presence of iodide.

• Iron Test

1 ml of the sample was treated with 1 ml of potassium ferrocyanide solution. The presence of an intense blue precipitate, which is insoluble in dilute hydrochloric acid, confirms the presence of iron.

• Calcium Test

The addition of 2 ml of 4% ammonium oxalate solution to 2 ml of the sample resulted in the formation of a white precipitate, indicating the presence of calcium.

• Sodium Test

2 ml of the sample was made into a paste using HCl and introduced into the blue flame of a Bunsen burner. The appearance of a yellow flame indicates the presence of sodium.

• Potassium Test

The sample was treated with 2 ml of sodium nitrite solution, followed by 2 ml of cobalt nitrate in 30% glacial acetic acid. The formation of a yellow precipitate indicates the presence of potassium.

• Zinc Test

5 ml of the sample was treated with 2 ml of potassium ferrocyanide solution, resulting in a white precipitate that is insoluble in dilute hydrochloric acid, confirming the presence of zinc.

• Magnesium Test

2 ml of the sample was treated with 1 ml of dilute ammonia solution. The appearance of a white precipitate suggests the presence of magnesium.

• Lead Test

2 ml of the sample was mixed with 2 ml of potassium iodide solution. The formation of a yellow precipitate indicates the presence of lead.

• Cobalt Test

Upon treatment with concentrated HCl, 2 ml of the sample developed a bluish-purple color, indicating the presence of cobalt.

• Nickel Test

2 ml of the sample was treated with aqueous ammonia, leading to the formation of a light green precipitate, which indicates the presence of nickel.

• Bismuth Test

2 ml of the sample was mixed with ammonium hydroxide, resulting in a white precipitate, indicating the presence of bismuth.

• Silver Test

When treated with aqueous ammonia, 2 ml of the sample formed a brown precipitate, which confirms the presence of silver.

• Sulphur Test

2 ml of the sample was combined with sodium nitroprusside, producing a violet color that indicates the presence of sulphur.

• Selenium Test

2 ml of the sample was treated with perchloric acid, followed by the addition of hydrazine to form selenium acid. The appearance of a red color indicates the presence of selenium.

• Chromium Test

Treatment of 2 ml of the sample with dilute HCl resulted in a blue color, indicating the presence of chromium.

• Manganese Test

2 ml of the sample was exposed to hydrogen sulphide gas or sodium hydroxide, leading to a pink or white precipitate, confirming the presence of manganese.

• Mercury Test

The addition of potassium iodide solution to 2 ml of the sample resulted in a red precipitate, indicating the presence of mercury.

• Cadmium Test

2 ml of the sample was treated with sodium hydroxide, leading to the formation of a white precipitate, which indicates the presence of cadmium.

Quantification test ^[14]

Samples of about 100mg PPE were digested in Teflon vessels with 1 ml ultrapure nitric acid using a microwave digestion unit for 45 mins. After digestion, samples were made up to 50ml in standard measuring flasks. Calibration standards were prepared in a range starting from 0.25 ?g/ml using ultrapure nitric acid, including a blank. The analysis was conducted using an Agilent ICPOES 5100 VDV instrument, operating at an RF power of 1.2 kW, with a plasma gas flow rate of 12 L/min and a nebulizer gas flow rate of 0.70 L/min, as the samples were introduced through a nebulizer and spray chamber.

RESULTS AND DISCUSSION

Organoleptic characters ^[2,15]

The organoleptic characters of pun purai ennai were illustrated in the following table 2.

Table 2: Organoleptic Characters Of Pun Purai Ennai

Pun Purai Ennai, with its oily texture, offers a protective barrier for wounds. Its application forms a thin layer that shields the wound from contaminants and maintains a moist environment. The oil's moisture-retaining properties prevent excessive drying and support tissue proliferation.

Physicochemical analysis ^[16,17,18]

The physicochemical analysis of pun purai ennai was presented in Table 3 and Table 4.

Table 3: Physical Analysis Of Pun Purai Ennai

Pun Purai Ennai's physical analysis revealed key properties: specific gravity of 1.074 kg/m?3; indicated solute presence, pH of 9.5 showed an alkaline nature with potential antimicrobial effects. A refractive index of 1.46 suggested oil saturation, a viscosity of 46.05 mm?2;/s demonstrated moderate absorption, and a boiling point above 234°C indicated thermal stability. A spreadability of 2 cm denoted moderate skin spreadability, offering insights into solute content, nature, saturation, absorption, and spreadability.

Table 4: Chemical Analysis Of Pun Purai Ennai

The analysis offers valuable insights into Pun Purai Ennai's composition and attributes. A low free fatty acid value signifies stability and reduced rancidity risk. The saponification value indicates the average molecular weight of the fatty acids present, whereas a low iodine value implies a lower degree of unsaturation and reduced susceptibility to oxidation. Low peroxide and acid values indicate stable storage and minimal spoilage. The total alkali value suggests low alkaline content. A foam height of 1 cm indicates moderate foaming ability. These findings collectively inform about the quality, stability, and potential wound healing applications of Pun Purai Ennai. The physicochemical parameters of the tested sample were compared against the standard ranges outlined in the Codex Alimentarius (Standard for Named Vegetable Oils) and the research conducted by M. O. Aremu et al. (2015). It was found that the values of the tested parameters fell within the acceptable range specified by both references.

Phytochemical screening ^[19]

The phytochemical constituent present in Pun Purai Ennai is given in Table 5.

Table 5: Phytochemical Screening Of Pun Purai Ennai

+ indicates the presence of active constituents

- indicates the absence of active constituents

The analysis of Pun Purai Ennai identified diverse phytochemicals including alkaloids, glycosides, carbohydrates, steroids & triterpenoids, phenolics, tannins, flavonoids, fats & fixed oils, and proteins. These compounds possess therapeutic potential contributing to wound-healing effects. While free amino acids were absent, tannins, phenolics, flavonoids, and fatty acids offer wound-healing benefits. Antioxidant properties of phenolics and flavonoids reduce oxidative stress, aiding tissue repair. Anti-inflammatory attributes mitigate inflammation, expediting healing. Though saponins weren't detected, other phytochemicals indicate valuable wound-healing potential in Pun Purai Ennai.

Quantification of phytochemical constituents

Quantification of tannins ^[20,21]

The quantification of tannins in Pun Purai Ennai and the results of the quantification are presented in Table 6 and Fig. 1.

Table 6: Estimation Of Total Tannin Content Of PPE

Quantification of alkaloids ^[22]

The quantification of alkaloids in Pun Purai Ennai is presented in Fig. 2.

% Alkaloid = Weight of alkaloid / Weight of sample × 100.

% Alkaloid = 0.49/ 0.63* 100 = 77.77%

Fig. 2: Estimation of the alkaloid content of PPE

Quantification of total phenol content ^[23,24]

The quantification of total phenol content in Pun Purai Ennai was estimated and the results of the quantification are presented in Table 7 and Fig. 3.

Table No.7: Estimation Of Total Phenolic Content Of PPE

Figure no.3: Estimation of total phenolic content of PPE

Quantification of total flavonoid content ^[25]

The quantification of total flavonoid content in Pun Purai Ennai was estimated and the results of the quantification are presented in Table 8, and Fig. 4.

Table 8: Estimation Of Total Flavonoid Content Of PPE

Quantification of cyanogenic glycosides content ^[26]

The quantification of cyanogenic glycoside content in Pun Purai Ennai were presented in Fig. 5.

Cyanogenic glycoside (mg/100g) = Titre value (cm3) ×1.08×Exact volume / Aliquot volume (cm3) × sample weight (g))

Cyanogenic glycoside (mg/100g) = 1.3×1.08×20 / 21.4×1 = 1.31mg/100g

       
           
       
    Fig. 5: Estimation of total glycoside content of PPE

Mineral and metal analysis

Qualitative test

Evaluation of the presence of various metals and minerals in pun purai ennai was illustrated in Table 9.

Table 9. Test for metals and minerals

The metal and mineral analysis of PPE shows the presence of silicate, phosphate, iodide, iron, zinc, magnesium, lead, bismuth, silver, sulphur, manganese, and cadmium. These elements are believed to play significant roles in wound healing, including tissue regeneration, collagen synthesis, antimicrobial effects, and immune system support. The absence of Sulphate, chloride, sulphide, calcium, sodium, potassium, cobalt, nickel, selenium, chromium, and mercury

Quantitative test ^[27]

The quantification of heavy metals in Pun Purai Ennai was evaluated and the results of this evaluation were determined and illustrated in Table 10.

Table No.10: Quantification Of Minerals And Heavy Metals In Pun Purai Ennai

BDL – Below Detection Level; ppm – parts / million