Phytochemical and Spectral Characterisation of Standardized Moringa oleifera and Curcuma longa Extract Fractions

Materials

All chemicals, including chloroform, hydrochloric acid, ethanol, petroleum ether, sulfuric acid, 5% ferric chloride, toluene, ethyl acetate, acetic acid, and formic acid, were procured from Merck and Cosmo Chem Pvt. Ltd. All solvents are of analytical grade.

Methods

Collections and Drying

Moringa oleifera (leaves) and Curcuma longa (rhizome) was harvested from the Latur district of Maharashtra, India, and subsequently shade-dried under controlled settings to prevent exposure to direct sunlight. The desiccated plant matter was subsequently pulverized into a coarse powder. Fine particles were eliminated by running the powdered substance through a 120-mesh sieve.

Standardization of plants materials

Determination of foreign organic matter

A thin layer of 5 g of coarsely powdered, air-dried drug material was distributed on a clean surface. The specimen was inspected visually and using a 6× magnifying lens. Foreign organic materials were meticulously isolated to the fullest extent practicable. The quantity of foreign organic matter was ascertained by weighing the isolated material and expressing it as a proportion of the initial sample weight [18].

Determination of moisture content

An accurately weighed, clean, dry weighing bottle equipped with a glass stopper was utilized. Approximately 2 g of the sample was introduced into the bottle, uniformly distributed to a maximum depth of 10 mm, after which the bottle was sealed and weighed. The filled bottle was thereafter positioned in an oven and dehydrated until a stable weight was achieved. Upon drying, the bottle was extracted from the oven and permitted to cool to ambient temperature within a desiccator. The loss on drying was determined and represented as a percentage weight loss (% w/w) [19].

Ash value

The residual ash from the burning of the medicinal plant material was assessed utilizing three distinct methodologies: total ash, acid-insoluble ash, and water-soluble ash, in accordance with established protocols [20].

Determination of Total ash

Approximately 2 g of the air-dried crude medication was precisely measured and positioned in a pre-weighed silica dish. The material was burned at a temperature not above 450 °C until devoid of carbon. Upon permitting the dish to attain room temperature, the mass of the ash was documented. The total ash content was determined based on the air-dried substance and expressed as % w/w [21].

Determination of Water- soluble ash

The ash produced by the aforementioned process was subjected to boiling with 25 mL of water for 5 minutes, thereafter filtered, and the insoluble residue was gathered on ashless filter paper. The residue was rinsed with hot water and incinerated for 15 minutes at a temperature not surpassing 450 °C. Upon cooling, the mass of the insoluble residue was documented. The water-soluble ash was determined by subtracting the weight of the insoluble residue from the total ash weight and was represented as % w/w relative to the air-dried medication.

Determination of Acid -insoluble ash

The ash was subjected to boiling with 25 mL of 2 M hydrochloric acid for 5 minutes, thereafter filtered, and the insoluble residue was retained on ashless filter paper. The residue was rinsed with hot water, incinerated at a temperature not beyond 450 °C, cooled in a desiccator, and subsequently weighed. The acid-insoluble ash concentration was determined and represented as a percentage by weight (% w/w) relative to the air-dried medication.

Extractive values

Multiple extractive values, encompassing water-soluble and alcohol-soluble extractive values, were assessed utilizing established pharmacopoeial methodologies.

Determination of water-soluble extractive value

One hundred milliliters of chloroform water was incorporated into 1.5 g of air-dried, coarsely powdered medication and macerated in a sealed container for 24 hours. The mixture was agitated intermittently for the initial 6 hours and subsequently permitted to rest for the ensuing 18 hours. Following filtration, 25 mL of the filtrate was placed in a shallow, flat evaporating dish, evaporated to dryness, and subsequently dried at 105 °C until a consistent weight was achieved. The water-soluble extractive value was determined and represented as % w/w relative to the air-dried substance [24].

Determination of Alcohol-soluble extractive value

In a sealed flask, 1.5 g of air-dried, finely powdered medication was macerated with 100 mL of ethanol of appropriate concentration for 24 hours. The mixture was agitated intermittently for the initial 6 hours and subsequently permitted to rest for the ensuing 18 hours. Following meticulous filtration to avert ethanol loss, 25 mL of the filtrate was placed in a shallow, flat evaporating dish, evaporated to dryness, and subsequently dried at 105 °C until a consistent weight was achieved. The ethanol-soluble extractive value was determined and represented as % w/w relative to the air-dried substance [25].

Extraction

Sequential extractions were conducted utilizing solvents of ascending polarity: petroleum ether (60–80 °C), ethanol, and water. The procedure was conducted in several batches utilizing a Soxhlet extractor to guarantee thorough extraction for each solvent fraction. At intervals, aliquots from the siphon tube were applied to thin-layer chromatography (TLC) plates and subjected to iodine vapor exposure. The lack of colorful dots signified the completion of extraction with the corresponding solvent. The solvent from each extract was then eliminated using distillation, utilizing lower pressure as required, and the concentrated residue was permitted to air dry. The desiccated extract was subsequently stored in hermetically sealed containers to maintain its integrity. The identical approach was adhered to for each subsequent solvent. The residual plant material (marc) was meticulously oven-dried to eliminate any remaining solvent before to advancing to the subsequent extraction phase. The desiccated plant material underwent reflux with distilled water for around three hours to provide the aqueous extract.

Phytochemical Test

The phytochemical tests are given in table 1  

Table 1: Phytochemical Test

Characterization of Bioactive Fractions by TLC

Subsequent to the pharmacological assessment of the ethanol fractions, the active fractions underwent thin-layer chromatography (TLC) for the identification of phytocomponents. The TLC examination of Moringa oleifera extract, employing the solvent system chloroform: methanol: water (2:1:2), identified three unique spots. Conversely, thin-layer chromatography of Curcuma longa extract, utilizing chloroform: methanol (9.8:2), revealed several spots, signifying the presence of various components.

Table 2: TLC studies of plant extracts

Column Chromatography of Active Extracts

Initial phytochemical analysis of Moringa oleifera and Curcuma longa extracts revealed the existence of several phytochemicals in both ethanol and chloroform fractions. Column chromatography was conducted on these active extracts to isolate specific phytometabolites for subsequent study.

High Performance Thin Layer Chromatography (HPTLC) Fingerprint Analysis

1.      Preparation of Extract
Each plant extract was dissolved in its respective solvent at a concentration of 5 mg/mL.

2.      Application  of Extract
The obtained extracts (5 mg/mL) were applied to HPTLC plates (10 × 10 cm) with a Linomat syringe. The samples were applied in bands measuring 5–6 mm in width, with a separation of 6 mm between them.

3.      Development of the Chromatogram
The HPTLC plates were generated in a CAMAG twin-trough chamber with an optimum solvent mixture, as determined for TLC analysis. Upon reaching a development of 80 mm, the plates were extracted from the chamber and permitted to air-dry.

4.      Scanning of the Chromatogram

The prepared plates were analyzed with a CAMAG HPTLC Scanner in absorbance mode at wavelengths of 254, 366, and 560 nm. The scanned data were examined utilizing the WinCATS planar chromatography manager software. Fingerprint chromatograms were produced to document the Rf values, band colors, and other distinctive characteristics of the chemicals found in the extracts. Bands at 254, 366, and 560 nm were seen without any supplementary derivatizing agents [26]

  Structural Elucidation of Isolated Fractions

The fractions associated with TLC spots were subsequently analyzed using spectroscopic methods, including UV–Vis, mass spectrometry (MS), Fourier-transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (^1H NMR), to ascertain their structural characteristics.

Collections and Drying

The specimens of Moringa oleifera (leaves) and Curcuma longa (rhizome) were successfully gathered from the Latur region of Maharashtra, India. Shade drying in controlled settings produced dried plant material with negligible browning and no evidence of microbial contamination. The desiccated plants were further pulverized into coarse powder, and finer particles were eliminated by sifting through a 120-mesh screen. The resultant powdered substance exhibited a consistent texture, making it appropriate for subsequent extraction and phytochemical investigations.

Authentication

The identities of the collected plants were verified by comparing their morphological characteristics. Authentication was confirmed by the Botanical Survey of India, Pune, Maharashtra state, India (BSI/WRC/Iden.Cer./2021).

 Standardization of plants materials

 Determination of foreign organic matter

The examination of foreign organic matter indicated that Moringa oleifera leavest comprised 0.5% w/w of foreign material, but Curcuma longa rhizome exhibited a reduced contamination level of 0.1% w/w. Both plant materials comply with the acceptable purity standards, since the allowable limit for foreign organic materials does not exceed 2% w/w.

Determination of moisture content

The moisture content of the extracts was assessed, yielding findings within acceptable parameters. The loss on drying (LOD) analysis indicated that Moringa oleifera leaves initially possessed a moisture content of 0.00% w/w, which marginally rise to 0.19% after 1 hour and stabilized at 0.20% from 2 to 4 hours. Likewise, Curcuma longa (rhizome) commenced at 0.00% w/w, escalated to 0.20% at 1 hour, and attained 0.22% at 2 hours, sustaining this concentration through 3 and 4 hours. The results demonstrate negligible and consistent moisture loss over time for both extracts, affirming that all plant extracts remain below the permitted limits of detection.

Determination of Ash value

The ash content of the plant materials was analysed to determine their inorganic residue profiles. The leaves of Moringa oleifera demonstrated a total ash content of 2.0% w/w, an acid-insoluble ash of 1.8% w/w, and a water-soluble ash of 0.5% w/w. In contrast, Curcuma longa (Rhizome) exhibited a marginally elevated total ash level of 3.0% w/w, comprising acid-insoluble ash at 1.6% w/w and water-soluble ash at 0.2% w/w. The results reveal significant disparities in the inorganic makeup of the two plant materials.

Determination of Extractive values

The extractive qualities of the plant materials were assessed to determine the solubility of their contents in various solvents. The leaves of Moringa oleifera had ethanol-soluble and water-soluble extractive values of 10.5% w/w and 7.9% w/w, respectively. In contrast, Curcuma longa (rhizome) exhibited marginally elevated values, with an ethanol-soluble extractive value of 12.5% w/w and a water-soluble extractive value of 8.1% w/w, signifying superior solubility of its components in both solvents compared to Moringa oleifera.

Extraction

Extraction was conducted in several batches utilizing different solvents. The characteristics and yield percentages of each extract are summarized below. The extraction investigation of Moringa oleifera had shown that all solvent fractions yielded dark green, semisolid, and viscous extracts. Ethanol produced the maximum yield at 12.14%, followed by ethyl acetate at 7.45%, chloroform at 4.58%, aqueous extract at 5.05%, and petroleum ether, which exhibited the lowest yield at 3.56%.

Yield of Various Extracts from the leaves powder (Moringa Oleifera)

The extraction of Moringa oleifera with various solvents produced dark green, semisolid, and viscous extracts. Of the solvents evaluated, ethanol yielded the greatest percentage (12.14%), signifying its superior efficacy in extracting bioactive compounds. Petroleum ether yielded the least (3.56%), whereas chloroform, ethyl acetate, and aqueous extracts had reasonable yields. The results indicate that solvent polarity markedly affects the extraction efficiency of Moringa phytoconstituents.

Yield of Various Extracts from rhizome powder (Curcuma longa)

All solvent fractions of Curcuma longa yielded yellowish, semisolid extracts. Ethanol had the maximum extraction yield at 9.85%, followed by chloroform at 7.45%, ethyl acetate at 6.52%, petroleum ether at 6.22%, and the aqueous extract, which exhibited the lowest yield at 6.07%.

Preliminary Phytochemical Screening

The initial phytochemical study of the extracts revealed the existence of many bioactive compounds, such as triterpenoids, steroids, glycosides, saponins, alkaloids, flavonoids, tannins, proteins, free amino acids, carbohydrates, and vitamins. The data indicate that both plant extracts are abundant in various phytometabolites with potential pharmacological properties.

Phytochemical test of Moringa oleifera Extract

The initial phytochemical analysis of the extract revealed the presence of flavonoids, as validated by a positive H₂SO₄ test. Tests for terpenoids, alkaloids, steroids, and tannins yielded negative results, indicating their absence or levels below detectable thresholds in the extract. The data suggest that flavonoids are likely the primary bioactive components responsible for the extract's pharmacological activity. Additional chromatographic and spectroscopic investigations are necessary to thoroughly describe these flavonoid molecules.

Figure 2: [a] TLC chromatograph of Moringa oleifera extract [b] TLC chromatograph of Curcuma longa extract powder

Rf values of plant extracts

The Rf values for Moringa oleifera extract are 0.31, 0.45, and 0.56, as determined by TLC analysis, whereas the Rf values for curcuma longa extract are 0.22, 0.32, and 0.43. The Thin Layer Chromatography (TLC) examination of diverse plant extracts was conducted at different detection wavelengths, demonstrating unique Rf values for each extract. Moringa oleifera (MEF1), detected at 366 nm, yielded 7 spots with Rf values of 0.07, 0.32, 0.47, 0.56, 0.66, 0.71, and 0.80. Finally, Curcuma longa (CEF3), identified at 366 nm, exhibited 8 spots with Rf values of 0.07, 0.26, 0.32, 0.43, 0.50, 0.62, 0.81, and 0.84. Each plant extract displayed distinct Rf patterns, underscoring the variability in their chemical contents.

High Performance Thin Layer Chromatography (HPTLC)

HPTLC fingerprints study

The HPTLC fingerprint analysis at 366 nm exhibited clearly defined and consistent chromatographic patterns for both samples. Moringa oleifera (MEF1) displayed seven unique bands with Rf values between 0.07 and 0.80, signifying the existence of several phytoconstituents, primarily flavonoid compounds. Curcuma longa (CEF3) exhibited nine distinct bands with Rf values ranging from 0.07 to 0.84, indicating a chemically diverse profile aligned with curcuminoids and associated compounds. The unique HPTLC fingerprints validate the complexity, identity, and consistency of both extracts, affirming their appropriateness for quality control and additional pharmacological research.

Figure 3: [a] HPTLC chromatogram of MEF1 extract measured at 366nm [b] HPTLC chromatogram of CEF3 extract measured at 366nm

Figure 2: [a] TLC chromatograph of Moringa oleifera extract [b] TLC chromatograph of Curcuma longa extract powder

Rf values of plant extracts

The Rf values for Moringa oleifera extract are 0.31, 0.45, and 0.56, as determined by TLC analysis, whereas the Rf values for curcuma longa extract are 0.22, 0.32, and 0.43. The Thin Layer Chromatography (TLC) examination of diverse plant extracts was conducted at different detection wavelengths, demonstrating unique Rf values for each extract. Moringa oleifera (MEF1), detected at 366 nm, yielded 7 spots with Rf values of 0.07, 0.32, 0.47, 0.56, 0.66, 0.71, and 0.80. Finally, Curcuma longa (CEF3), identified at 366 nm, exhibited 8 spots with Rf values of 0.07, 0.26, 0.32, 0.43, 0.50, 0.62, 0.81, and 0.84. Each plant extract displayed distinct Rf patterns, underscoring the variability in their chemical contents.

High Performance Thin Layer Chromatography (HPTLC)

HPTLC fingerprints study

The HPTLC fingerprint analysis at 366 nm exhibited clearly defined and consistent chromatographic patterns for both samples. Moringa oleifera (MEF1) displayed seven unique bands with Rf values between 0.07 and 0.80, signifying the existence of several phytoconstituents, primarily flavonoid compounds. Curcuma longa (CEF3) exhibited nine distinct bands with Rf values ranging from 0.07 to 0.84, indicating a chemically diverse profile aligned with curcuminoids and associated compounds. The unique HPTLC fingerprints validate the complexity, identity, and consistency of both extracts, affirming their appropriateness for quality control and additional pharmacological research.

Figure 3: [a] HPTLC chromatogram of MEF1 extract measured at 366nm [b] HPTLC chromatogram of CEF3 extract measured at 366nm