INTRODUCTION:

The development of Ayurveda, Unani, Siddha, homoeopathy, and other natural herb-based health sciences (Ayush) is mostly attributed to India. Ayurvedic medicine recognized as ‘alternative system of medicine’ have a huge antiquity for the preventative treatment for diseases. In many cultures, using medicinal herbs for therapeutic purposes is a secular practice.¹A plant-based medical system is very important because of its effectiveness and safety. The rising prerequisitefor safer drugs, considerable attention should be given to standards, quality, and effectiveness of the herbal preparations.²

Ayurvedic formulations utilize the quality regulation parameters to ensure the developed formulation stand by to the standards stated in Ayurveda. Furthermost the quality control test reported in ancient literature based on visual inspection and without valid scientific proof. Therefore, standardization of developed formulation with reliable quality protocol using modern analytical techniques is todays need.³Standardization of drug includes confirmation of its identity, quality and purity throughout shelf-life, storage, distribution and biological activity. However, the quantity of marker compounds would serve as an extra criterion in assessing the sample quality, the fingerprint profiles serve as guidelines for the phytochemical analysis of the medicine to ensure its quality. Ayurvedic formulations based on plants include pills, powders, asavas, aristhas, and semisolids like avlehas and ghritas.^2,3

The formulation of Asavas includes the drug soaking for specific period in sugar or jaggery solution.That undergoes a fermentation process generating alcohol for specific period of time that enables active constituent extraction contained in drug.The alcohol generated, acts as a preservative.^4,5Aloe vera is the main ingredient of Kumariasava(KS), a well-known Ayurvedic herbal composition that is sold over-the-counter under a number of brands. It’s a self-fermented galenical used to treat cough, weak digestion, urinary tract issues, and slow liver. It contains approximately 40–50 crude medicines.^6,7The present research was aimed to standardize the developed KS formulation for its physicochemical, spectral analysis, TLC, HPTLC and HPLC evaluation and its comparative analysis with standard marker compound.

EXPERIMENTAL:

Formulation of Kumaryasava:

The Kumaryasava (KS) formulation was prepared by using the composition mentioned in Table no. 1 and further analysed for analytical markers in formulation. Commercially available brand Dhootpapeshwar Kumaryasavawas procured from local marketplace.^8,9

Table 1. Composition of Kumaryasava formulation

Sr. No.	Ingredient	*Kumaryasava* formulation	Quantity (Kg)
1	Kumari rasa	ü	4lit
2	Haritaki	ü	1 kg
3	Dhataki	ü	256 gm
4	Jatiphala	ü	16 gm
7	Jatamansi	ü	16 gm
8	Guda	ü	1.2 kg
9	Madhu	ü	1 kg
10	Water	ü	4l

Standardization using Physico-chemical Parameters:

Numerous characteristics, including formulation specific gravity pH, alcohol content, total solids content and the quantity of reducing and non-reducing sugars, were examined in the developed KS formulation sample.¹⁰

Preparation of Extract:

The developed KS formulation about was boiled and evaporated on a water bath to reduce half of its initial volume, kept aside to get cooled and further diluted with distilled water to 100ml. Furthermore; extract successively extracted with different mentioned solvents such as petroleum ether (60–80^◦C), chloroform, ethyl acetateand ethyl ether respectively. Finally, all the obtained extracts in various solvents were dried at room temperature under reduced pressure.¹⁰

Qualitative chemical examination:

Chemical tests and TLC studies were used to qualitatively assess each extract for the presence of several phytoconstituents such as tannins, phytosterols, carbohydrates, alkaloids, phenolic compounds, anthraquinone glycosides and saponins.¹²

TLC studies:

The extracts in various mentioned solvents were analysed for TLC with stationary phase Silica gel GF-254 and mobile phase chloroform: ethyl acetate (75:25). The developed spots on TLC plates were inspected in UV and visible light. Amongst all extract of chloroform has reported well-resolved fluorescent components fractions I–V and hence it was considered for further analysis.

HPTLC studies:

HPTLC fingerprint analysis of chloroform extract of KSand standard aloin marker were noted at 254 and 366 nm. The isolated fractions of chloroform extract of KS III–V were exposed to HPTLC fingerprints with similar experimental circumstances as TLC.¹¹

HPLC studies:

According to the results of HPTLC studies it was noted that fraction-V specified anthraquinones which is a foremost part of aloevera. Fraction V (chloroform extract) was additionally investigated with HPLC utilizing column Phenomenex Kinetex XB- C18 (25 cm×4.6mm, i.d.), 10µm and (60:40) methanol: water with sample flow rate: 1 ml/minat 250nm.^11,12

Spectral Studies:

UV spectra of fraction III-V were prepared and recorded in solvents methanol and ethanol. FTIR spectrums for standard aloin and Fraction III-V recorded on FTIR spectrophotometer (Alpha Brooker, Tokyo, Japan). NMR spectra were recorded of neat sample. 1H (400MHZ) and 13C (100.60 MHZ) NMR were performed on NMR spectrometer (BRUKER, Zürich, Switzerland) operating at 600 MHz with cryoprobe prodigy (150 MHz for 13C; chemical shift (_) = ppm; coupling constants (J) = Hz).¹³

Gas Chromatography:

The sample solutions were prepared by pipetting 1 ml each in 10 ml volumetric flask and 5 ml of THF followed by sonication for 2 min. and finally maintain the volume by dilution with THF.¹¹

RESULT AND DISCUSSION:

The aqueous KS solutionwas successively extracted using variety of solvents including petroleum ether, ethyl ether, chloroform, ethyl acetate, and benzeneand further utilized for the qualitative examination.³

Physico-chemical Investigation:

Standardization of KS formulation following pharmacopoeia was performed based on the results recorded from physicochemical evaluation.⁹The prepared KS formulation was complied with all pharmacopoeial examinations (Table 2).

Table 2: Physicochemical investigation of KS formulation

Sr. No.	Parameters	Specification as per API	Values ± SD* (Fd1)	Values ± SD* (Fd2)
1.	Organoleptic evaluation	Clear aromaticliquid and bitter taste	Light brown colored, bitter and contain traces of sediment	Light brown, bitter taste and traces of sediment
2.	pH	3.40-4.20	3.62 ± 0.017	3.62 ± 0.017
3.	Specific gravity	1.01-1.10	1.1243 ± 0.014	1.09 ± 0.02
4.	Total solid content (% w/v)	NLT 13	34.68 ± 1.990	33.10 ± 0.37
5.	Total alcohol content(% w/v)	5-10	7.33 ± 0.8141	6.18 ± 0.12
6.	Reducing sugars (% w/v)	NLT 7.5	9.07±0.11	10.22±0.16
7.	Non- reducing sugars(% w/v)	NMT 0.80	0.27 ± 0.010	0.69 ± 0.17
8.	Total phenolic content (% w/v)	-	0.63 ± 0.001	0.77 ± 0.03

Table 3. TLC screening III, IV and V fraction of chloroform extract

Isolated fraction (Chloroform)	Rfvalue	Color parameters			Indication
		Visible	Fluorescencein UV light
		Visible	Individual	With NH₃
III	0.720	Colorless	Purple	Light purple	Flavones
IV	0.811	Colorless	Light blue	Intense blue	Isoflavones
V	0.847	Yellow	Orange reddish	No change	Anthraquinone

2. Preparation of Extracts and its Qualitative Estimation:

A TLC analysis was performed on each extract after an aqueous solution of KS was sequentially extracted using a variety of solvents, including petroleum ether, ethyl ether, chloroform, ethyl acetate, andbenzene.¹⁰ The recorded results revealed that chloroform extract exhibited maximum about five spot amongst other solvents. However, extracts of benzene and petroleum ether showed three and one spot respectively. The extracts of other solvents including ethyl acetate, ethyl ether, and water no any spot recorded. Following initial chemical evaluation of all the extracts in various solvent, it was determined to concentrate on the extract in chloroform solvent because it had the highest number of spots. For the chloroform extract, TLC profiles and HPTLC fingerprints were created.¹⁴

TLC Studies:

The study identified five well-resolved fluorescent components in the chloroform extract TLC profile. These components' Rf values were 0.350, 0.510, 0.728, 0.811, and 0.847. The same mobile phase and preparative TLC effectively separated three amongst five (fractions III–V). The Rf values of fractions III–V were discovered to be 0.720, 0.811, and 0.847, respectively. Spectral and chemical methods were used to evaluate fractions III–V and investigate their composition.

It was discovered that fraction III included flavones and/or flavonol, while fraction IV contained isoflavones based on colour features in both visible and UV light. It was discovered that fraction V contained anthraquinones (Table 3). The findings of TLC investigations were validated by spectral studies. The fraction's UV and IR spectra revealed distinctive peaks that suggested quinones were present. For routine KS standardisation, the UV Visible spectrophotometry, FTIR, fluorescence spectra, and HPTLC fingerprints of fractions III–V can be utilised and mentioned in Table 3.^10,12Results recorded for TLC fraction III, IV, V were shown in Fig. 1.

It was discovered that the chloroform extract's fraction V was crystalline solid, orange-red in colour, and somewhat sticky. Modified Borntrager's test came back optimistic. The spot with Rf 0.847 turned purple after being sprayed with methanolic KOH solution (10%) on TLC plates. It had previously been yellow in colour. Its ethanolic solution produced two absorbance maxima in the UV area at 214 and 257 nm, and a maximum absorbance in the visible range at 431 nm.

(a) (b) (c)

Fig. 1 TLC analysis of fraction: a) III (b) IV c) V

The documented literature states that anthraquinones can be identified in UV and visible light by their yellow-brown hue. But when methanolic KOH (10%) was sprayed, the initial yellow and yellow-brown hues become red, violet, green, or purple. Further investigation was conducted since the chemical examinations, colour reactions, and spectrum data clearly suggested that an anthraquinone present in fraction V (Table 4).^12,15

Screening of Fraction V:

Chemical evaluation of Fraction V of Chloroform Extract:

One of the main ingredients of aloes is aloin, which was extracted from the aloe vera. The modified Borntrager's test was positive for isolated aloin that confirmed the existence of anthaquinone C-glycosides. The colour properties of fraction V and aloin were the identical at 366 nm. The modified Borntrager's test results for both were positive, suggesting the occurrence of anthraquinone C-glycosides.¹³ The comparative chromatographic investigation of Fraction V of chloroform extract and standard aloinhas been depicted in Table 5.

Table 5 Comparative analysis of standard aloin extract and fraction V of chloroform extract.

Test	Standard Aloin extract	Fraction V
Modified Borntrager’s Test	Positive	Positive
TLC	1 spot (Rf: 0.846)	2 spot, (Rf: 0.793, 0.846)
Color at 366 nm	Reddish	Reddish
10%methanolic KOH Spray	Purple	Purple
HPLC	1 peak, RT: 3.532 min	RT: 3.532 and 6.45 min
HPTLC	1 spot, Rf:0.847	2 spot, Rf : 0.057, 0.847

HPLC Analysis: Fraction V:

Using the same parameters as previously stated, the HPLC investigationfraction V produced two peaks with RT 5.52 and 6.45min, respectively (Fig. 2). Aloin HPLC analysis produced a lone peak with RT 3.532min under the identical conditions as described for fraction V (Fig. 5).¹⁰

TLC and HPTLC Analysis:

On a silica gel plate, TLC examination of aloin produced only reddish spot at 366nm with Rf 0.846 Table 5 indicates that a component of fraction V exhibited the same retention time (3.5min) in HPLC profiles and the same Rf value (0.847 and 0.81, respectively) by TLC and HPTLC as aloin as shown in Fig. 3. As a result, it is possible that fraction V contains aloin. It might serve as an analytical marker for Kumariasava standardisation. Specifications might be declared following the quantitative estimation of aloin. Then, Kumariasavas should be able to use this as a routine, accurate, and straight forward way of analysis.^{12, 13}

Fig.2: HPLC profile of isolated chloroform Fraction V

(a) (b) (c)

Fig. 3: HPTLC results of fraction V and standard aloin marker at wavelength: (a) 254 nm (b) 366 nm (c) 366 nm

FTIR Analysis:

The FTIR result displays many absorption spectra, each of which represents a functional group—basically, the compounds' functional group. The FTIR spectrum of standard aloin and aloin extracted from developed formulation were recorded as shown in Fig. 4. The IR spectrum fraction V absorption peaks at 3223, 1674, 1603, 1449, 1282 and 1175 cm^-1. Similar absorption is seen in this infrared region of the normal aloin spectrum, which suggests absorption by the OH functional group.^13,16The two compounds share the same absorption peaks at 1609, which correspond to carboxylic carbonyl absorption rather than aldehyde or ketone absorption. Additionally, the absorption at 1449.79 –1378.55 cm^-1 represents C–H bending vibrations, and those at 2914.91–2851.30 cm-1 represent C–H stretching vibrations. As illustrated in Fig 4 High biological activities are expected to result from crude extract, a combination of molecules, having more OH groups than the separated pure compound (s).¹⁷

Fig. 4: FTIR spectrum of isolated fraction-V of Kumaryasava formulation

NMR Study:

Comprehensive chromatography and NMR analysis revealed that aromatic protons have a chemical shift between δ 6.5-7.8, while an acid's hydroxyl proton (OH-COOH) has a chemical shift at δ 12.08. The whole leaf extract of A. barbadensis contains compound combinations that are unstable, making it difficult to fully elucidate the compounds. This instability is likely caused by insufficient fractions and other issues such contaminants in the compound that are reflected in the spectra, such as 13C, 1H, and 2D NMR. Resumé 129 of approximately δ 210 in the 13C-NMR spectrum corresponds to the carbon of a non-shielded carboxyl (C=O).^{18, 19} This was depicted in Fig. 5. The found antioxidant and anticancer properties examined in this investigation may be attributed to these substances.^{20, 21}

Fig. 5: 13C NMR spectra of isolated fraction V of KS formulation

CONCLUSION:

Addressing standardization of polyherbal formulations is a vital aspect that needs broader consideration. It is linked with the quality of formulations and must be executed for large scale commercial production. We also have standardised the Kumaryasava formulations chromatographically for qualitative and quantitative estimation of aloin, all the phytocompounds were found in appreciable amount.The study revealed that results acquired from spectroscopic analysis and HPTLC fingerprint of chloroform extract of Kumariasava exhibited significant performance in standardization of Kumariasavaso as to alleviate the variations occurrence in batch formulation. The overall work it can be concluded in Kumaryasava standardization aloin can be used as desired analytical marker.

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Received on 23.03.2024 Revised on 13.07.2024

Accepted on 20.09.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1176-1181.

DOI: 10.52711/0974-360X.2025.00170

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Fraction (Chloroform)	UV Visible (methanol)	FTIR (cm⁻¹)	Fluorescence
Fraction (Chloroform)	UV Visible (methanol)	FTIR (cm⁻¹)	Excitation maximum(nm)	Emission maximum (nm)
III	219, 274	2914.91	296	333
IV	215, 275	2850.78	338	395
V	213, 257, 429	1603.93	394	492