INTRODUCTION:

Hygrophila auriculata, also known as Asteracantha longifolia and Hygrophila spinosa, is an erect, annual plant that grows throughout India, especially in marshy areas on the plains. The plant was reported as ikshura, ikshagandha, and kokilasha, which denotes that it resembles the eyes of an Indian cuckoo. It contains phytosterols, alkaloids, tannins, flavonoids, and other compounds. This plant belongs to the Ayurvedic class of Vrishya which is used for enhancing sexual performance. The weed can be used to boost the immune system, in insomnia, to treat asthma, in icterus, liver ailments, and in swelling. It can also be used for blood-related diseases.^1-4

EXPERIMENTAL DETAIL:

Selection and Identification of Plant Material:

Flowers and fruits are visible from October to November. In October 2022, glowing plants of Hygrophila auriculata (Schumach) Heine were gathered from the field area of Markol Chokadi, Narsarovar Road, Sanand, India. The plant specimen received an authentication number, GU/BOT/AH1, after certification by Dr. Hitesh Solanki, a professor at the Gujarat University, School of Science. The plants were dehydrated at normal temperature and pressure, and then ground into moderately fine particles separately.

Figure 1: View of Plant Hygrophila Auriculata

Physicochemical Analysis:

Drugs are identified based on their sensory characteristics like color, odor, and taste. This process is known as organoleptic characterization, which involves studying specific parameters to determine the drug's sensory properties. Ash values, Foaming index, Swelling index, and Moisture content are determined using standard methods as outlined in the Ayurveda Pharmacopoeia, Indian Pharmacopoeia, and WHO guidelines. The result shown in Table-1.Determination of various extractive values like Petroleum ether, n-hexane, ethyl acetate, isopropyl alcohol, methanol, and water are evaluated which shown in Table-2. Extraction was carried out by cold process.^5-10

Table-1: Physicochemical Analysis Of leaf and seed part of H.auriculata

Physicochemical Parameter	Leaf	SSEED
Color	Green	Brown
Odour	Characteristic	Odorless
Taste	Tasteless	Bitter
Total Ash	11%	28%
Water soluble Ash	1.7%	11.32%
Acid insoluble Ash	8.3%	18.18%
Sulphated Ash	10.9%	28%
Swelling Index	NA	2ml
Foaming Index	< than 100	NA
Moisture Content	4%	3.5%

Preparation of Plant Extract (Successive Solvent Extraction):

100g of dried leaves were extracted in a Soxhlet extractor at 80°C using a hydro-alcoholic solvent. The extract was fractionated in order of increasing polarity to obtain hexane, ethyl acetate, and n-butanol fractions.

The process began with extracting seeds using petroleum ether. This was done continuously for 48 hours at 60°C. The extract was then reduced to 1/4^th of its original volume. After that, it was mixed with a 10% w/w aq. KOH solution and saponified at 60°C for 8 hours. Once the saponification process was complete, the solution was cooled and partitioned with solvent ether 3 times. The organic layer was collected and concentrated afterwards.

Phytochemical Screening:

An analysis was conducted to identify chemical compounds in H. auriculata leaves and seeds. These compounds may have potential healing properties for various ailments. The analysis revealed the presence of saponins, terpenoids, alkaloids, flavonoids, sterols, and other compounds according to standard methods in published work. Table 3 displays the secondary metabolites identified in H. auriculata^11-15.

Quantitative Estimation:^16-19

Total Phenolic content:

To prepare the standard solution, dissolve 10 mg of Gallic acid in 10ml of methanol to obtain a concentration of 1000µg/ml. Dilute to get a concentration range of 10 – 70µg/ml. For the sample solution, dissolve 20mg of extract in 10ml of methanol to obtain 2000µg/ml. Mix 0.5ml of this solution with 2 ml of Folin's phenol reagent and 4ml of Na₂CO₃solution. Incubate the mixture at room temperature for 30minutes and measure the absorbance at 765nm using a UV-VIS spectrophotometer.

Total Flavonoid Content:

To prepare the standard solution, dissolve 50mg of RE (Rutin) in 50ml of methanol. Among that, prepare working standard solutions with concentrations ranging from 100-900µg/ml. For testing, mix 0.6ml of the sample with 6.8ml of methanol, add NaNO₂ and AlCl₃ solutions (10%) and NaOH (1%), then measure the absorbance at 506nm using a UV-VIS spectrophotometer. For the sample solution, dissolve 20 mg of extract in 10ml of methanol to attain a concentration of 2000µg/ml. Then follow the same process as for the standard solution to test it.

Table-3: The detail of Phytochemical Analysis.

Sr. No	Phyto constituent	Hydro alcoholic extract (leaf)	Ethyl acetate fraction (leaf)	N-butanol fraction (leaf)	Hexane fraction (leaf)	Pet-ether extract (seed)	Unsaponifiable fraction (seed)
1	Alkaloid	+	+	-	-	+	-
2	Flavonoid	+	+	+	-	-	-
3	Steroid	+	+	+	+	+	+
4	Terpenoid	+	+	+	+	+	+
5	Tannin	+	+	-	-	-	-
6	Saponin	+	+	-	-	-	-
7	Phenolic compound	+	+	+	+	+	-

(+)=POSITIVE, (-) =ABSENCE

Table-4: Quantitative determination of Flavonoid and Phenolic content in H. auriculata leaf.

Extract	Total Flavonoid content (mg per gm)	Total Phenolic content(mg per gm)
Ethyl acetate	124.44 mg	31.32 mg
N-butanol	182.77 mg	34.23 mg
Hexane	44.44 mg	24.00 mg
Hydro alcoholic	257.22 mg	60.42 mg

HEAVY METAL ANALYSIS BY ICP-MS:

Inductive couple plasma mass spectroscopy (ICP-MS) was used to identify heavy metals like arsenic, lead, cadmium and mercuric in the leaf and seed powder of H. auriculata.

Standard Solutions:

10ppm working standard solution: 1mL of mother solution (1000 ppm) in 100mL graduated flasks; fill with Type-1 water. 2 ppb standard solution: 2µL of working standard (10ppm) in 10mL volumetric flask; fill with Type-1 water. 5 ppb standard solution: 5µL of working standard (10ppm) in 10mL volumetric flask; fill with Type-1 water. 1ppm standard solution: 1mL of working standard (10ppm) in 10mL volumetric flask; fill with Type-1 water.

Sample Solution:

Weigh 0.4g of sample in PFA Teflon vessels. Add 5mL of HNO₃ along the sides of the vessel to remove adhering matter. Keep vessel open for 30min in fumigation chamber if effervescence forms. Close the vessels tightly and keep on the turner. Set the method parameters as follows: Power should be Maximum800w at 100%, Ramp-30, tempreture-180^.c and Hold time-10min.

After digestion, fumigate vessels for 10min. Release pressure slowly and pass on the solution to 50mL measuring flasks with distilled water. Filter with 0.22µ filter paper. Use as test solution with blank solution. Plot calibration curve on ICP-MS to detect metals in samples which shown in Figure-2.

The concentrations of heavy metals were determined by ICP-MS and showed the Trace amounts of arsenic, lead, cadmium and Mercuric were detected which were all within the acceptable limit set by the WHO for herbal materials.^20-24The result shown in Table-5.

Table-5: The Determination of Heavy Metals in the Leaf and Seed Parts of H. Auriculata.

Heavy Metal Analysis	RESULT Leaves	RESULT Seed	LIMIT AS PER API
Arsenic	0.886ppm	1.038 ppm	NMT 3 ppm
Lead	1.671ppm	1.763ppm	NMT 10 ppm
Cadmium	0.078ppm	0.028ppm	NMT 0.3 ppm
Mercuric	0.148ppm	0.246 ppm	NMT 1 ppm

MICROBIAL LIMIT TEST:^25-27

Analysis for the microbial load of leaf and seed of H. auriculata were determined using Soyabean casein digest broth, MacConkey agar, Sabouraud agar, Salmonella agar, Cetrimide agar and Mannitol salt agar which were bought from Vasu research center. Samples were analyzed in duplicate and the average was used.

Preparation of cultural media:

Take 1 gm/mL samples in 10mL/100mL sterile soya bean casein digest broth. Mixing the samples by using cyclo mixture. The Pour –plate method is used for Microbial limit test.

Total microbial plate count (TPC):

Pipette 1mL of a prepared sample into two sterile Petri plates. Label them and pour 25mL of cooled Soya bean casein digest agar medium in each. Incubate at 30° to 35°C for 3-5 days, and then count colonies from two plates with less than 250 colonies. Find the mean and calculate the colony forming unit.

No. of colonies x dilution

Colony forming unit (cfu): -----------------------------------

Weight of sample

Total yeast and mould count (TYMC):

To test for microbial contamination, pipette 1 mL of the prepared sample onto two sterile Petri plates. Label them and add Sabouraud dextrose agar medium, rotating them slowly for proper mixing. After solidification, incubate the plates for 5 days at 20-25°C and count the colonies from each plate to calculate the colony-forming unit. Results indicate in table-6 that the contamination in the leaf and seed samples was below the maximum limit.

Table-6: Microbial load of H. auriculata leaf and seed.

Microbiological Analysis	Result Seed	Result Leaves	Limit as per API
TPC	1642 cfu/g	1666cfu/g	10⁵ cfu/g
TYMC	505 cfu/g	485 cfu/g	10³ cfu/g
Staphylococcus aureus	×	×	Absent/g
Salmonella sp.	×	×	Absent/g
Pseudomonas aeruginosa	×	×	Absent/g
Escherichia coli	×	×	Absent/g

API – Ayurvedic Pharmacopoeia of India; ppm - Parts per million, cfu/g – Colony forming unit per gram,×-Absent

DETECTION OF LUPEOL AND BETASITOSTEROL COMPOUND:

Different fractions such as unsaponifiable fraction, Pet-etherextract from seed and Hexane, N-butanol, and Hydroalcoholic extract from leaves were analyzed to check the presence of Lupeol and Betasitosterol compounds using Thin Layer Chromatography (TLC). Each sample, weighing 10mg was dissolved in 10ml of methanol. After separating the compounds using chromatography, Liebermann-Burchard reagent was used for post-derivatization. To separate these compounds, we used chromatography TLC plates (silica gel G 60 F254, Merck) using Toluol: Ethylethanoate: Ethanoic acid (10:1:0.5 v/v/v) as an eluent. The developed plates were air-dried and viewed under UV light to identify the fluorescent spots that correlate with the standard mark. These plates were then separately sprayed with 50% sulfuric acid/ 4-Anisaldehyde reagent, heated at 110^oC for 10 minutes, and analyzed.^28-31

Figure: 3 Sequence of spots on a thin layer chromatography plate are as follows: Unsaponifiable fraction, Lupeol, β-Sitosterol, Pet-ether, Hexane, N-butanol, Hydro alcoholic extract.

RESULT AND DISCUSSION:

A physico-chemical investigation was conducted to identify the characteristics of an herbal substance. The studies showed that the material has extractive properties, and measured the ash value, moisture content, swelling index, and foaming index. The ash value test checks for the presence of inorganic residue, such as phosphate, carbonate, and silicate, in herbal materials to ensure that no toxic minerals are present. The foaming index of H. auriculata leaves, which was less than 100, indicates the presence of saponins. On the other hand, the seed of H. auriculata did not show any foaming index, which indicates the absence of saponin. Moreover, the swelling index of H. auriculata seed was found to be 2, which could indicate the presence of constituents such as adhering mucilage. Foaming and swelling index are important parameters that help in determining the quality of plant material. The qualitative estimation of secondary metabolites confirmed the presence of numerous compounds, including alkaloids, flavonoids, saponins, sterols, tannins, and terpenoids. The concentrations of heavy metals were also determined using ICP-MS, and trace amounts of arsenic, lead, cadmium, and mercury were detected, all of which were within the acceptable limits set by the WHO for herbal materials. The quantitative analysis revealed that the different extracts had varying total phenolic and flavonoid contents. The hydro-alcoholic extract had the highest total phenolic content at 60.24 mg/g, followed by N-butanol, Ethyl acetate and Hexane fractions at 34.23 mg/g, 31.32 mg/g, and 24.00 mg/g, respectively. In terms of total flavonoid content, the hydro-alcoholic extract had the highest value at 257.22 mg/g, followed by N-butanol at 182.77 mg/g, Ethyl acetate at 124.44 mg/g, and Hexane fraction at 44.44 mg/g. Medicinal plants contain antioxidants like polyphenols and flavonoids that inhibit oxidative stress, reducing the risk of various diseases. Microbiological Analysis shows that S.aureus, Salmonella sp, P.aeruginosa and E.coli is absent in the leaf and seed. Plant terpenoids and phytosterols were identified by analyzing extracts and fractions of seeds and leaves using a new mobile phase called Toluol: Ethyl ethanoate: Ethanoic acid (10:1:0.5 v/v/v). The analysis showed good results. Standard Lupeol and β-Sitosterol have retention factors of 0.5 (3.2/6.4) and 0.35 (2.3/6.4) respectively. Lupeol was clearly observed in unsaponifiable fraction, pet ether extract, n-butanol fraction, and hydroalcoholic extract. Beta-sitosterol was found in both parts of the plant. These two compounds are the main constituents of the plant with high concentration.

CONCLUSION:

The aim of this study was to evaluate the macroscopic, physicochemical, phytochemical, heavy metal, and microbiological properties of H. auriculata leaves and seeds using simple and available methods. The purpose of this evaluation is to standardize the medicinal plant and ensure its safe and effective use.

CONFLICTS OF INTEREST:

There are no conflicts of interest declared by the authors.

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Received on 08.01.2024 Modified on 11.05.2024

Research J. Pharm. and Tech 2024; 17(11):5196-5200.

DOI: 10.52711/0974-360X.2024.00795

Sr. no	Solvent	Colour and physical state Leaves	% of Extractive (%w/w)	Colour and physical state of seed	% of Extractive (%w/w)
1	Petroleum Ether(60-80)	Yellowish pasty mass	5.78%	Lemon yellow colour oil	16%
2	n-hexane	Yellow mass	6.05%	Yellow colour oil	37%
3	Ethyl acetate	Green colour mass	12.09%	Yellow colour mass	36%
4	Iso propyl alcohol	Yellowish green colour mass	9%	Yellow colour mass	8%
5	Methanol	Dark green mass	23%	Brown colour mass	29%
6	Water	Brown colour mass	34%	Brown colour mass	45%


Figure-2: calibration curve on ICP-MS to detect metals in samples