1. INTRODUCTION:

The growing concern over the adverse effects of synthetic surfactants on human health has led to an increasing focus on studies aimed at obtaining natural surfactants from plant sources and industrial waste. Natural surfactants have become increasingly important due to their economic viability and their positive impact on health and the environment, as opposed to synthetic surfactants. Initial studies in this field have primarily focused on obtaining natural surfactants from plants such as S. mukorossi, soybean (Glycine max L. Merrill), soapwort (Saponaria officinalis), bracken (Pteridium aquilinum), the horse chestnut (Aesculus hippocastanum), soap lily (Chlorogalum pomeridianum), and yucca¹.

1.1 Sapindus mukorossi:

Soapnuts, belonging to the Sapindaceae family, are widely recognized for their medicinal properties. They are used as an expectorant, emetic, contraceptive, and to treat various conditions such as excessive salivation, epilepsy, chlorosis, and migraines. Sapindus mukorossi, commonly known as soapnut, is a popular ingredient in Ayurvedic shampoos and cleansers. In Ayurvedic medicine, soapnuts are used to treat eczema, psoriasis, and to remove freckles. They also possess mild insecticidal properties and are traditionally used to eliminate lice from the scalp. In North India, soapnuts are commonly referred to as "areetha" and are considered one of the most important trees in the tropical and subtropical regions of Asia. Typically, soapnut trees are large deciduous trees, reaching heights of up to 12 meters, with some specimens growing as tall as 20 meters and having a girth of 1.8 meters. They have a rounded crown and fine, leathery foliage².

Bark: dark to light yellow, relatively smooth, features numerous vertical lines of lenticels, fine fissures and peels off in uneven, woody scales.

Blaze: 0.8-1.3cm, hard, non-fibrous, light orange-brown, brittle, and granular.

Leaves: 30-50cm long, alternate, paripinnate, common petiole with very narrow borders, smooth.

Leaflets: 5-10 pairs, opposite or alternate, 5-18 by 2.5-5 cm, lanceolate, acuminate, entire, glabrous, often slightly falcate or oblique; petioles 2-5m long.

Inflorescence: a compound terminal panicle, 30 cm or more in length, with pubescent branches.

Flowers: about 5 mm across, polygamous, greenish white, subsessile, numerous, mostly bisexual.

Sepals: 5, each with a woolly scale on both sides above the claw.

Fruit: a spherical, fleshy, 1-seeded drupe, occasionally with 2 drupes together, approximately 1.8-2.5 cm in diameter.

Seed: 0.8-1.3 cm in diameter, globose, smooth, black, loose in dry fruit³.

1.2 Phytochemistry:

Many scientists have extensively studied the phytochemical composition of Sapindus mukorossi, particularly focusing on its triterpenoidal saponins, which mainly belong to three types: oleanane, dammarane, and tirucullane. Recent research has unveiled several pharmacological properties of this plant, including antimicrobial, hepatoprotective, insecticidal, and pesticidal activities. One of the most notable findings is the contraceptive effect of the saponins extracted from the fruit pericarp. The seeds of Sapindus mukorossi contain approximately 23% oil, with 92% of it being triglycerides. The triglyceride fraction consists of various glycerides, such as oleo-palmito-arachidin glyceride, oleo-diarachidin glyceride, and di-olein type glycerides like dioleo-palmitin, dioleo-stearin, and dioleo-arachidin. Sengupta et al. isolated two lipid fractions, A and B, from the seed oil using preparative TLC. Fraction A, a normal triglyceride, constitutes 70.4% of the oil, while fraction B (29.6%) contains nitrogenous constituents, specifically a cyanolipid (1-cyano-2-hydroxymethyl prop-1-ene-3-ol)⁴.

Sapindus mukorossi fruits are reported to contain sesquiterpenoidal glycosides and six different fatty esters of tetracyclic triterpenoids. The leaf extract of this plant contains various flavonoids, including quercetin, apigenin, kaempferol, and rutin, isolated using column chromatography on a polyamide sorbent. Additionally, various triterpene saponins of oleanane, dammarane, and tirucullane types have been isolated from different parts of Sapindus mukorossi, including the galls, fruits, and roots. For instance, oleanane-type triterpenoid saponins named Sapindoside were isolated from the fruits, along with dammarane-type saponins named Sapinmusaponin, and tirucallane-type saponins named sapinmusaponins. These saponins have been identified and their structures elucidated through spectroscopic analysis, including 1D and 2D NMR techniques. Saponins, a class of phytochemicals found primarily in plants, exhibit foaming characteristics and consist of polycyclic aglycones attached to one or more sugar side chains. They are characterized by a hydrophobic sapogenin and a hydrophilic sugar part, which give them their foaming ability. Saponins are known for their bitter taste and some can be toxic. They vary in the number and length of saccharide chains attached to the sapogenins, with chain lengths ranging from 1 to 11 sugar residues, most commonly 2–5, and can have both linear and branched chains^{5, 6}.

1.3 Problems Associated with Extraction and Isolation of Saponins:

The extraction and isolation of saponins present a challenge to researchers due to the structural variety arising from different substituents such as OH, CH3, or COOH in the aglycone moiety. This complexity is further compounded by the number, arrangement, and orientation of sugar units, as well as the number and types of sugar chains attached to the aglycone moiety. Generally, saponins are highly polar, chemically and thermally labile, non-volatile, and are typically found in low concentrations in plants. Therefore, special care must be taken during extraction and preliminary treatment, applying relatively mild conditions since some saponins can undergo enzymic hydrolysis during water extractions, while esterification of acidic saponins and transacylation reactions may also occur during alcoholic extractions⁷.

Traditionally, saponins were extracted from plant material using hot aqueous alcoholic solutions, followed by the evaporation of alcohol and subsequent extraction of saponins into butanol through liquid–liquid extraction. However, hot extractions can cause unstable components, like acylated forms, to break down, resulting in artifacts instead of authentic saponins. Additionally, extraction with methanol (MeOH), especially for steroidal saponins, may result in the formation of methyl derivatives not originally present in the plant. To obtain the real composition of saponins, cold extractions with ethanol–water solutions are preferred. However, in liquid–liquid extractions, highly polar saponins, such as bidesmosides and tridesmosides, may remain in the aqueous layer, or the extraction may not be quantitative⁸.

Separating saponins is challenging because they exist in plants as mixtures of structurally similar compounds with similar polarity. Therefore, in the isolation of these compounds, various separation techniques such as thin-layer chromatography (TLC), column chromatography (CC), low-pressure liquid chromatography (LPLC), medium-pressure liquid chromatography (MPLC), and high-performance liquid chromatography (HPLC) are employed to effect complete separation and isolation of pure individual components. TLC is increasingly being used as a supporting technique for the analysis of saponin fractions from CC, allowing for the confirmation of purity and identity of isolated compounds⁹.

HPLC is one of the most versatile separation techniques, but the absence of a chromophore in most saponins makes their detection under ultraviolet light challenging, allowing only non-specific detection at 200–210 nm. However, this challenge has been addressed recently by evaporative light scattering detection (ELSD), which allows for the detection of scattered light generated by the non-volatile particles of analytes produced by nebulization into droplets of the effluent. ELSD is a universal, non-specific detection method that can provide a stable baseline even with gradient elution¹⁰.

High-speed counter-current chromatography (HSCCC) is an all-liquid chromatographic system that minimizes irreversible adsorbing effects and artifact formation. Its application in natural products chemistry is steadily increasing due to its superior separation abilities and excellent recovery rates.

1.4 Extraction of Saponins:

The initial step in processing saponins is extracting them from the plant matrix. The extraction solvent, extraction conditions (e.g., temperature, pH, and solvent to feed ratio), and the properties of the feed material (e.g., composition and particle size) are the main factors that determine process efficiency¹¹.

1.5 Isolation of Saponins:

Alternative methods for isolating saponins have been described elsewhere. While traditional techniques such as solvent extraction, column chromatography (CC), and preparative thin-layer chromatography (TLC) can yield pure substances, isolating individual saponins can be challenging. Typically, the following procedure has been adopted by various researchers. Defatted powdered material is first treated with petroleum ether and then extracted with methanol (MeOH) either in a Soxhlet extractor for 72 hours or through maceration at room temperature. The methanol extract is then concentrated under reduced pressure and sequentially partitioned using n-hexane, ethyl acetate (or chloroform), and n-butanol (n-BuOH). The n-BuOH soluble fraction and the aqueous part yield the major saponin triterpene fraction. The crude extracts are then separately applied to Diaion HP-20 columns, washed with water-MeOH mixtures at various ratios (0, 50, 85, and 100), and finally with acetone. Fractions with similar patterns are combined and further separated using silica gel column chromatography with a mixture of chloroform-methanol-water (40:10:1 v/v/v). Finally, the saponin compounds are separated by high-performance liquid chromatography (HPLC) using an ODS column with a MeOH-water eluent. Saponin compounds can be detected on TLC by spraying with a 10% (v/v) solution of sulfuric acid in ethanol and the Lieberman–Burchard reagent, or a mixture of p-anisaldehyde-sulfuric acid and glacial acetic acid (1:2:100 v/v/v), where triterpene saponins produce blue-violet spots upon heating^12-14.

1.6 Isolation and Characterization of constituents from ethyl acetate extract by Flash chromatography:

Flash chromatography is a quick and cost-effective method for purifying products of organic synthesis, such as e.g. in drug discovery or from natural extracts. Flash chromatography is a favored alternative when other separation methods are impractical or too complex. It offers a fast and affordable general technique for preparative separation of mixtures needing only moderate resolution. Suitable for both normal-phase and reversed-phase separation, flash chromatography can handle relatively high flow rates at low pressure, achieving good separation quickly under appropriate conditions. It uses disposable plastic cartridge columns, which save time and enhance reproducibility. Different cartridge sizes can be chosen based on the sample volume. Now a day's readily prepared cartridges are available based on particle size and stationary phase volume. Flash chromatography offers cost-effectiveness and requires minimal maintenance. It is particularly advantageous when the target compound is present in high concentration, enabling the isolation of the compound with high purity. When samples contain multiple chemical constituents with unknown concentrations, preparative chromatography is favored^15-17.

Advantages of Flash Chromatography:

1) Maximum Quantities of the sample can be separated (0.5-2.0 g).

2) Separation time is 10-15 min.

3) Elaborate equipment and the purchase of expensive equipment are not necessary.

4) Reusing cartridges is much more economical than using preparative columns, saving nearly eight times the cost.

5) No requirement for the sample to be soluble in the mobile phase.

6) Flash chromatography is highly beneficial for separating a variety of substances including antibiotics, impurities, and peptides.

7) This technique saves time and solvents.

8) Reliable and cost effective.

General Process of Flash Chromatography:

· In traditional column chromatography a sample to be purified is placed on the top of a column containing some solid support, often silica gel.

· The rest of the column is then filled with a solvent (or mixture of solvents) which then runs through the solid support under the force of gravity.

· The different constituents to be separated move through the column at varying speeds and can subsequently be collected individually as they exit from the bottom of the column. Unfortunately, the rate at which the solvent percolates through the column is slow.

· In flash chromatography, air pressure is utilized to accelerate the solvent flow, significantly reducing the purification time required for the sample. As a result, both column setup and the separation process could be completed in less than 10 - 15 minutes.

2. MATERIALS AND EQUIPMENTS:^18-22

2.1 Collection and identification of plant material:

The plant material used in this study was collected during month of august in Nashik District, India and authentication was done from botanical survey of India, Pune. A voucher specimen has been deposited.

2.2 Preparation of the Extract:

The dried fruits of Sapindus mukorossi were extracted sequentially using the Soxhlet method with petroleum ether, chloroform, and ethyl acetate. Water extraction was carried out at room temperature through maceration, followed by concentration over water bath and evaporated under reduced pressure. The yields of extract were calculated

2.3 Drying and grinding of plant materials:

The gathered fruits of Sapindus mukorossi were air-dried in shade and then crushed into a coarse powder.

Preliminary phytochemicals screening

2.4 Preliminary phytochemicals screening of extracts of fruits of Sapindus mukorossi.

· The plants may be considered as a biosynthetic laboratory for a multitude of compounds like alkaloids, glycosides, tannins, saponins, flavonoids and sugars, etc. that exert physiological effects. These compounds are responsible for therapeutic effects, usually the secondary metabolites. All the extracts of the plant material were subjected to preliminary phytochemicals screening for the detection of various plant constituents and their results are shown in Table:

A. Tests for Steroids:

· Salkowaski Test

A small amount of residue from each extract was placed in 2ml of chloroform, followed by the addition of 2ml of concentrated sulfuric acid, poured into the test tube from the side. The test tube was shaken for few min. The appearance of a red color in the chloroform layer indicated the existence of sterols.

B. Test for Alkaloids:

Few mg of the residue to each extract was taken separately in 5ml of 1.5% hydrochloric acid and filtered. Filtrate was used for testing of alkaloids.

a) Dragendorff’s Reagent:

· It was prepared by mixing solution a (17gm of bismuth sub nitrate + 200gm of tartaric acid + 800ml distilled water) and Solution B (160gm potassium iodide + 400ml distilled water) in 1:1 proportion. From this solution, working standard was prepared by taking 50 ml of this solution and adding 100gm of tartaric acid and made volume up to 500ml with distilled water.

· The above Dragendorff's reagent was sprayed on Whatmann filter paper and the paper was dried. After the test filtrate was basified using dilute ammonia, it was subjected to extraction with chloroform. The resulting chloroform extract was then applied onto filter paper that had been treated with Dragendorff's reagent, using a capillary tube. The appearance of an orange-red color on the paper indicated the presence of alkaloids.

b) Mayer’s Reagent:

· The Mayer's reagent was prepared, 1.36 gm of mercuric chloride was dissolved in 60 ml of distilled water, to this solution 5 gm potassium iodide was added and volume was made up to 100 ml.

· To a little of the test filtrate solution taken in a watch glass, a few drops of the above reagent was added. Formation of cream colored precipitate showed presence of alkaloids.

c) Wagner’s Reagent (Iodine-potassium iodide):

1.27 gm of iodine and 2 gm of potassium iodide were dissolved in 5 ml of water and solution was diluted to 100 ml with distilled water. Then few drops of this reagent were added to the test filtrate, a brown precipitate was formed indicating the presence of alkaloids.

d) Hager’s Reagent

Saturated aqueous solution of picric acid was employed for this test. Upon treating the test filtrate with this reagent, an orange-yellow precipitate formed, suggesting the presence of alkaloids.

C. Test for Saponins:

a) Foam test:

A few mg of the test residue was taken in a test tube and shaken vigorously with a small amount of sodium bicarbonate and water. If a stable, characteristic honeycomb like forth is obtained saponins are present.

b) Hemolysis test:

A little of test residue was dissolved in normal saline in such a way that 5 ml of the solution represented 1 gm of the crude drug. In a series of 5 test tubes, doses of 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1 ml were added and volume was made up to 1 ml in each case with normal saline. 1 ml of diluted blood (0.5 ml of rabbit's blood diluted to 25 ml with normal saline) was added to each tube and changes observed. The presence of saponins can be indicated by the occurrence of blood hemolysis.

D. Test for Tannins and Phenolic compounds:

The test residue of extract was taken separately in water, warmed and filtered. Tests were carried out with the filtrate using following reagents.

a) Ferric chloride test:

A 5% w/v solution of ferric chloride in 95% alcohol was prepared. Few drops of this solution were added to a little of the above filtrate solution. If dark green or deep blue colour is obtained, tannins are present.

b) Lead acetate test:

A 10% w/v solution of basic lead acetate in distilled water was added to the test filtrate. If precipitate is obtained, tannins or phenolic compounds are present.

c) Potassium dichromate test:

If on an addition of a solution of potassium dichromate in test filtrate solution, dark colour is developed indicates tannins or phenolic compounds are present.

d) Gelatin solution test:

1% w/v solution of gelatin in water, containing 10% sodium chloride was prepared. A little of this solution was added to the filtrate, if white precipitate is obtained, tannins or phenolic compounds are present.

e) Bromine water test:

Bromine solution was added to the test filtrate. If depolarization of bromine water occurs, tannins or phenolic compounds are present.

E. Test for Flavonoids (Shinoda test):

A small quantity of test residue was dissolved in 5 ml Alcohol (95%) and treated with few drops of concentrated hydrochloric acid and 0.5gm of magnesium metal. The pink colour is developed within a minute, if flavonoids are present.

F. Test for Proteins:

a) Biuret test:

A few mg of the extract was taken in water and 1ml of 4 % of copper sulphate was added to it, violet or pink colour is formed, if proteins are present.

b) Xanthoproteic test:

A little residue was taken with 2 ml of water and 0.5 ml of concentrated nitric acid was added to it, yellow colour is obtained, if proteins are present.

c) Millon's test (Mercury nitrate solution):

Million's reagent was prepared by dissolving 3ml of mercury in 27ml of fuming nitric acid, keeping the mixture well cooled, this solution was diluted with equal quantity of distilled water. Aqueous solution of the residue was taken and to it, 2 to 3ml of Million's reagent was added. The white precipitate slowly turns to pink, if proteins are present.

G. Test for amino acids:

a) Ninhydrin test:

The Ninhydrin reagent is 0.1% w/v solution of Ninhydrin in n-butanol. A small amount of this substance was introduced into the sample extract. A violet or purple colour is developed, if amino acids are present.

H. Test for Carbohydrates:

a) Molisch's test:

· The Molisch's reagent was prepared by dissolving 10 gm of alpha-naphthol in 100ml of 95% alcohol. A few mg of the test residue was placed in a test tube containing 0.5ml of water, and it was mixed with 2 drops of Molisch's reagent. Into this solution, 1 milliliter of concentrated sulfuric acid was poured gently down the side of the tilted test tube, ensuring that the acid settled as a separate layer below the aqueous solution without mixing. If a red brown ring appears at the common surface of the liquids, carbohydrates are present.

b) Barfoed's test:

· This reagent was prepared by dissolving 13.3gm of crystalline neutral copper acetate in 200ml of 1 % acetic acid solution. The test residue dissolved in water and heated with a little of the reagent. If a red precipitate of cuprous oxide is formed within two min., carbohydrates are present.

2.5 Preparation of Fraction:

· For trial prepared a fraction 5 of conc. of 20μg/ml. In 10ml volumetric flask pipette out 2ml of working standard solution of drug from 100 ppm was mixed with 3ml of 0.1N HCL and kept for 30 min for heating on water bath. After 30 min solution was diluted up to 10 ml with methanol.

· The (200mg) fraction 5solution was adsorbed over silica gel (# 60 – 120) in the ratio 1:4 (drug to silica gel) and finally dried under vacuum below 60⁰ C. A column of 5litres capacity was first loaded with 1 to 2 g of silica gel (# 60-120) with chloroform as solvent (dry packing).

· The adsorbed material (200 mg) was charged and eluted with chloroform: methanol gradient (100:0--- 90:10---80:20---70:30---60:40---50:50---40:60--- 30:70---20:80---0:100). Fractions of 100ml were collected. The fractions collected were concentrated by distillation under vacuum using rota vapour and weighed.

Isolated fractions of acid fraction 5 were collected in sample vials. There was 6 fraction of fraction 5 from that fraction 5 was shown good isolation of fraction 5. So these fractions 1 continue for further process for characterization by UV, HPLC, IR, MS, and NMR.

3. OBSERVATION AND RESULTS:

Table: 1. Preliminary Phytochemicals Screening + ve -- present; -- ve absent

Sr no	Plant constituents	Test /reagent	PTE	CHL	ETA	AQE
1	Steroids	Salkovaski	--	--	--	--
2	Alkaloids	Dragendroff’s test	--	--	--	--
		Hager’s test	++	++	++	++
		Mayer’s test	++	++	++	++
		Wagner’s test	++	++	++	++
3	Saponins	Foam test	--	--	++	++
3	Saponins	Haemolysis test	--	--	++	++
4	Fats and oils	Filter paper test	++	++	++	++
5	Tannins and Phenolic	Ferric chloride test	--	--	++	++
		Lead acetate test	--	++	++	++
		Pot. Dichromate	--	++	++	++
		Bromine water	--	--	++	++
6	Flavonoids	Shinoda test	--	++	++	++
6	Flavonoids	Lead acetate test	--	++	++	--
7	Carbohydrates	Molisch test	--	--	--	--
		Fehling’s test	--	--	--	--
		Barfoed’s test	--	--	--	--
8	Proteins	Millon’s test	--	++	++	++
8	Proteins	Biuret test	--	++	++	++
9	Amino acid test	Ninhydrine test	--	++	++	++

PTE -- Petroleum ether extract CHL – Chloroform

EAT – Ethyl acetate extract AQE -- Water extract

Chromatogram of API drug (20 μg/ml)at mobile phase Benzene: ethyl acetate: water (3:4:3)

Fig. 1: Chromatogram of ethyl acetate extract at mobile phase benzene: ethyl acetate: water (3:4:3)

Peak (ethyl acetate extract):

Prepared a conc. of 2000μg/ml from above working standard solution (10,000 ppm).In 10 ml of volumetric flask pipette out 0.2 ml of working standard solution was mixed with 5 ml of 0.5N HCL and kept for 30 min for heating on water bath. After 30 min solution was diluted up to 10 ml with methanol.

The prepared (2000 ppm) fraction 5solution was adsorbed over silica gel (# 60 – 120) in the ratio 1:4 (drug to silica gel) and finally dried under vacuum below 60⁰ C. Initially, a column with a capacity of 5 liters was filled with 1 to 2 grams of silica gel (#60-120) using chloroform as the solvent (dry packing). The adsorbed material (200 mg) was charged and eluted with chloroform: methanol gradient (100:0--- 90:10---80:20---70:30---60:40---50:50---40:60--- 30:70---20:80---0:100). Fractions of 100 ml were collected. The fractions collected were concentrated by distillation under vacuum using rota vapour and weighed.

Fig. 2: Chromatogram of fraction 1

Fig. 3: Chromatogram of fraction 2

Fig. 4: Chromatogram of fraction 3

Fig. 5: Chromatogram of fraction 4

Fig. 6: Chromatogram of fraction 5

Fig. 7: Chromatogram of Fraction 6

Fig. 8: Chromatogram of Fraction 7

Fig. 9: Chromatogram of Fraction 8

Fig. 10: Chromatogram of Fraction 9

Fig. 11: Chromatogram of Fraction 10

Fig. 12: Chromatogram of Fraction 11

Characterization of acid fraction 5 fraction:

The structures of isolated fraction of degradation products were characterized by UV, NMR, Mass spectra and functional groups were identified by IR spectra.

1. UV Spectra:

To analyse the collected fraction of ethyl acetate from flash chromatography samples was scanned under UV in the range of wavelength 200-400 nm. In followed UV spectra it shows change in wavelength that was at 242nm. From this result it was conclude that the fraction 5 was isolated successfully.

Fig 13. UV spectra of fraction 5

2. FT-IR:

Preparation of sample for IR:

The collected fraction of fraction 5 adsorbed on sufficient Qty. of silica gel. This residue was then mixed with KBr in the ratio 1:300 and this sample was analyzed. The observed frequencies are shown in table no.2

Fig.14: FT-IR Spectrum of acid degrading

Table No. 2: IR Observed Frequency of Fraction 5

Peak no	Observed frequency (cm^-1)
1	1072.24
2	1187.24
3	1384.74
4	1804.27
5	2898.54
6	3287.27

3. LC-MS:

Preparation of Sample for MS:

The collected fraction of acid fraction 5fraction 5 further diluted with solvent methanol and analyzed under ESI- ionization mass spectra. Calibrate the spectra on software Bruker Compass Data Analysis 4.2. Mass spectra of Fraction 5 were obtained.

Fig 15: MS spectra of Fraction 5

Sr No	Peak no	R_f value
1	1	0.73

Sr No	Peak no	m/z value
1	1	968.17

4. NMR:

Preparation of sample for NMR

The collected fraction of ethyl acetate fraction 5 was placed in petri plate to evaporate all the solvent at RT. The obtained residue was collected and dissolved in DMSO solvent and loading on Bruker advance III HD and analysed on software Topspin 3.2 for proton NMR. The spectral assignment for proton and carbon signals chemical shift values.

Fig 16: Sample for NMR

5. HPTLC:

HPTLC analysis of Pet ether extract of Sapindus mukorossi fruits.

Under UV 254 nm Under UV 366 nm

Fig. 17: HPTLC profile of Pet ether extract of Sapindus mukorossi fruits. Solvent system: Methanol: Methanol: Water (7:1:1)

Fig. 18. HPTLC densitometry chromatogram scan at 360 nm of Pet ether extract of Sapindus mukorossi fruits

Table No 3: Rf value and Area of % of Pet ether extract

Sr No	Pet ether
Sr No	Rf value	% Area
1.	0.07	0.28
2.	0.12	1.05
3.	0.21	28.32
4.	0.39	0.85
5.	0.57	10.21
6.	0.63	0.80
7.	0.67	0.40
8.	0.83	43.54
9.	0.92	0.16

HPTLC analysis of methanol extract of Sapindus mukorossi fruits.

Under UV 254 nm Under UV 366 nm

Fig 19: HPTLC profile of methanol extract of Sapindus mukorossi fruits Solvent system B Methanol: Acetone: (3:7)

Fig. 20: HPTLC densitometry chromatogram scan at 360 nm of methanol extract of Sapindus mukorossi fruits

4. CONCLUSION:

Phytochemical screening confirmed the presence of various secondary metabolites in Sapindus mukorossi fruits. Flash chromatography facilitated the isolation of Fraction 5 from the ethyl acetate extract. Fraction 5 was characterized using UV, FT-IR, LC-MS, NMR, and HPTLC techniques. The results suggest that Fraction 5 contains compounds with specific functional groups and molecular weights, which could be further investigated for their pharmacological activities and potential applications, especially as natural surfactants. This study provides valuable information for the potential utilization of Sapindus mukorossi as a source of natural surfactants, with broader implications for pharmaceutical and industrial applications.

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Received on 17.06.2024 Revised on 15.10.2024

Accepted on 22.12.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1357-1367.

DOI: 10.52711/0974-360X.2025.00196

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