INTRODUCTION:

Recently, herbal remedies have attracted a lot more interest as an alternate source of effective pharmaceuticals for both treating and preventing numerous types of diseases It has been discovered that Holoptelea integrifolia (Roxb) Planch is a member of the Ulmaceae family. Known locally as Chilbil or papri in Hindi, Holoptelea integrifolia, a synonym of Ulmus integrifolia, is also called the Indian elm or Jungle cork tree.

The majority of the most important phytochemicals, which are often used to cure a variety skin of diseases in both humans and animals, are present in Holoptelea integrifolia. Ayurvedic method of medicine mostly treats allergic problems with the bark and leaves of plants, but it can also treat edema, diabetes, and intestinal disorders. Ringworms and scabies are treated using seeds and stem bark paste. Since the beginning of time, therapeutic compounds have been abundantly found in herbal plants. They contain the core elements of many traditional medical systems, It includes Chinese, Unani, Siddha, Ayurvedic, and so on. The plant varieties continue to be valuably mentioned as cost-effective anti-contagious medicines with few side effects¹. When applied topically, the juice extract made from this plant's boiling bark has reportedly proven to be particularly effective in the treatment of rheumatism. In addition, bark paste is used to treat rheumatic swellings². The growth of ringworm, eczema, and other cutaneous disorders of the skin are treated topically by applying a paste made of seeds, bark, and stem. The external use of stem bark paste is used to cure ringworms, scabies, and enlargement of the lymph nodes. For the treatment of leucoderma types, an external application of bark and leaf paste is used^3,4,5. Additionally, H. integrifolia possesses a wide range of antifungal potential. The alcoholic extract revealed antitubercular and moderate-to-good antimicrobial activity⁶. Numerous other phytochemicals, including proteins, and carbohydrates are present in this plantspecies^7,8. Additionally, H. integrifolia contains phytoconstituents of quinines, coumarins, cardiac glycosides, phenols, and flavonoids⁹. Holoptelea integrifolia's stem bark has also been shown to contain phytochemical components such as 2-Aminonaphthoquinone, -sitosterol, and -D-glucose¹⁰.

Herbal remedies have long been used to cure a variety of illnesses, and this plant is one of India's top pollen allergens and its ethanolic leaf extract plays a vital part in antidiarrheal action.^11,12.

The phytochemical tests have shown some secondary metabolites, including tannins, cardiac glycosides, anthracenes, saponins, and alkaloids.^13,14,15In addition to its curative properties, this medicinal plant has been shown in studies in pharmacy and medicine to have anti-inflammatory, wound-healing, antibacterial, anticancer, anti-diabetic, and antioxidant properties.^16-19Stem bark paste employed directly has anti-inflammatory effects, particularly for the eyes. This plant's outer bark and leaf paste can be applied externally on the white areas, or a skin disease called.^20-22Thin-layer chromatography (TLC), also known as planar chromatography (PC), remains one of the most conventional methods in analytical chemistry.^23-24Extracted and formulated product from this plant leaves is used topically to treat inflammatory illnesses of the eyes, ears, and nose as well as a range of skin conditions.²⁵The present work describes the HPTLC method for the simultaneous estimation of gallic acid and quercetin by utilizing a methanolic solvent. By ICH criteria, the approach was further verified.²⁶The HPTLC method shows a broad peak with the complicated mobile phase. We developed and verified the HPTLC technique for the estimation of compounds exhibiting narrow peaks with the simplest mobile phase composition in the current study.²⁷The present paper describes a precise, accurate, sensitive HPTLC method to extract standard components from the extracted products.²⁸Different parts of this plant have been reported to have several medicinal qualities, including anthelmintic, antibacterial, antioxidant, and analgesic effects.^29-30Ethnomedically used to cure wounds, vitiligo, leprosy, filariasis, diabetes, hemorrhoids, dysmenorrhea, rheumatism, intestinal worms, dyspepsia, flatulence, colic, and inflammation. About 10% of people with atopic dermatitis are sensitized to it, making it a significant pollen allergen in India.^31-33The bark and leaves are bitter, repelling, carminative, laxative, anthelmintic, astringent, thermogenic, anti-inflammatory, and digestive. This plant has been investigated for current studies of anti-inflammatory, antidiarrheal, anthelmintic, wound healing, antioxidant, antibacterial, anticancer, and antidiabetic properties.^34-40

THE RESOURCES AND METHODS:

Plant Authentication:

Holoptelea integrifolia leaves collected in Lucknow’s Arjun ganj Sultanpur road from May to June in the year 2022. Freshed and cleaned leaves were scorched well by placing them in between a nonstick paper of the book thus, prepared a herbarium file (sample). This herbarium file was sent for identification and authentication to the Science Communication and Policy Research, National Institute of Science, CSIR, New Delhi 110012. The authentication number is NIScPR/RHMD/consult/

2022/4174-75-2.

Developing Plant Extract Solutions:

Leaves were washed and cleaned before being allowed to dry at room temperature for one month in the shade. The dried plant leaves have already been ground using a grinder into a coarse powder. 15 grams of coarse powder leaves were taken from the crude medication powder. This coarse powder was placed in a muslin cloth thimble and extracted as well as in n-hexane and hydroalcoholic solvent in the ratio of 50:50 V/V by extracted successively with a Soxhlet for 24 hours in each of the two solvents (n-hexane, a highly nonpolar solvent, and hydro-alcohol, a polar solvent).After the extraction process was finished, a steam bath with a temperature range between 50°C to 100°C was used to evaporate the extra solvent to obtain a concentrated product, then packed or stored in airtight containers made of polypropylene test tubes before being sent to NBRI, Lucknow, for chemical testing (HPTLC) and receiving an analysis report.

Table 2: Track 1, ID: Quercetin (Standard)

Peak	Start Position (Rf)	Start Height (AU)	Max. Position (Rf)	Max. Height (AU)	Max %	End Position (Rf)	End Height (AU)	Area (AU)	Area %	Assigned substance
1.	-0.02	0.6	-0.01	112.7	6.63	0.02	1.8	1480.1	1.72	Unknown
2.	0.04	5.0	0.07	12.7	0.75	0.07	11.7	276.0	0.32	Unknown
3.	0.10	15.3	0.13	27.0	1.59	0.14	24.6	793.8	0.92	Gallic Acid
4.	0.18	32.1	0.23	64.7	3.80	0.24	61.7	2366.9	2.76	Unknown
5.	0.24	62.0	0.37	727.7	42.76	0.40	60.2	47178.5	54.95	Quercetin
6.	0.41	68.1	0.45	247.7	14.58	0.48	77.5	7806.9	9.09	Unknown
7.	0.50	81.8	0.52	93.0	5.47	0.53	31.0	2548.8	2.97	Unknown
8.	0.55	95.4	0.58	101.0	6.11	0.59	33.4	3697.9	4.31	Unknown
9.	0.64	110.7	0.69	132.3	7.77	0.75	39.9	10610.8	12.36	Unknown
10.	0.84	54.7	0.92	109.3	6.42	0.95	32.3	8413.9	9.80	Unknown
11.	0.95	61.8	0.96	70.5	4.14	0.98	9.1	666.3	0.80	Unknown

Table 3: Track 2, ID: Gallic Acid (Standard)

Peak	Start Position (Rf)	Start Height (AU)	Max. Position (Rf)	Max. Height (AU)	Max %	End Position (Rf)	End Height (AU)	Area (AU)	Area %	Assigned substance
1.	-0.02	6.5	-0.00	31.1	2.30	0.02	0.6	379.8	0.68	Unknown
2.	0.07	12.4	0.14	708.5	52.48	0.18	15.8	26620.8	51.03	Gallic Acid
3.	0.39	46.9	0.49	87.0	6.45	0.49	35.0	6821.9	12.16	Unknown
4.	0.51	90.6	0.53	95.4	7.07	0.54	33.5	2223.2	3.96	Unknown
5.	0.55	97.8	0.61	109.2	8.09	0.61	38.3	5897.1	10.52	Unknown
6.	0.66	118.5	0.67	120.3	8.91	0.73	36.7	5043.5	8.99	Unknown
7.	0.82	40.1	0.88	61.1	4.52	0.89	57.6	3269.0	5.83	Unknown
8.	0.90	60.8	0.92	78.6	5.82	0.95	42.4	3238.1	5.77	Unknown
9.	0.95	42.7	0.96	58.8	4.35	0.98	8.1	588.3	1.05	Unknown

Table 4: Track 3, ID: Sample 1

Peak	Start Position (Rf)	Start Height (AU)	Max. Position (Rf)	Max. Height (AU)	Max %	End Position (Rf)	End Height (AU)	Area (AU)	Area %	Assigned substance
1.	-0.03	3.9	-0.03	24.6	1.79	-0.02	1.0	142.0	0.26	Unknown
2.	-0.02	1.0	-0.01	21.0	1.53	0.00	3.5	272.9	0.50	Unknown
3.	0.05	8.8	0.09	19.1	1.39	0.10	17.8	634.6	1.16	Unknown
4.	0.16	27.2	0.21	41.2	3.00	0.22	36.4	1737.3	3.18	Unknown
5.	0.22	37.0	0.22	52.4	3.81	0.23	41.0	574.0	1.05	Unknown
6.	0.23	41.4	0.25	66.3	4.82	0.27	46.1	1838.3	3.37	Unknown
7.	0.32	54.1	0.37	107.9	7.84	0.39	30.7	5254.1	9.63	Quercetin
8.	0.40	90.9	0.43	135.8	9.87	0.44	15.5	4531.7	8.30	Unknown
9	0.46	111.4	0.49	136.7	9.93	0.51	18.8	6294.3	11.53	Unknown
10.	0.53	120.3	0.59	141.5	10.28	0.60	33.1	8128.3	14.89	Unknown
11.	0.64	130.4	0.64	132.6	9.64	0.71	71.7	7280.8	13.34	Unknown
12.	0.73	69.9	0.76	102.4	7.44	0.77	30.8	3075.3	5.63	Unknown
13.	0.77	101.0	0.78	113.7	8.26	0.80	39.5	2503.0	4059	Unknown
14.	0.80	108.9	0.81	115.2	8.37	0.83	33.3	3217.6	5.90	Unknown
15.	0.85	87.4	0.91	165.8	12.04	0.95	5.6	9097.2	16.67	Unknown

Table 5: Track 4, ID: Sample 2

Peak	Start Position (Rf)	Start Height (AU)	Max. Position (Rf)	Max. Height (AU)	Max %	End Position (Rf)	End Height (AU)	Area (AU)	Area %	Assigned substance
1.	-0.03	24.3	-0.01	620.3	45.15	0.03	29.1	10369.0	28.98	Unknown
2.	0.03	29.6	0.04	47.3	3.44	0.06	20.3	978.1	2.73	Unknown
3.	0.06	20.4	0.07	38.0	2.77	0.09	18.1	728.9	2.04	Unknown
4.	0.23	32.6	0.25	50.4	3.67	0.27	36.4	1638.4	4.58	Unknown
5.	0.34	47.5	0.39	107.5	7.83	0.40	73.1	3828.1	10.70	Unknown
6.	0.41	73.1	0.44	88.9	6.47	0.45	36.2	3344.1	9.35	Unknown
7.	0.45	90.7	0.46	97.3	7.08	0.47	79.6	1733.7	4.85	Unknown
8.	0.49	99.2	0.51	104.5	7.60	0.52	33.5	2661.9	7.44	Unknown
9.	0.61	110.9	0.64	121.6	8.85	0.70	58.5	8382.2	23.43	Unknown
10.	0.89	48.6	0.90	52.8	3.84	0.93	27.0	1778.6	4.97	Unknown
11.	0.93	28.8	0.94	45.2	3.29	0.96	2.4	337.2	0.94	Unknown

Table 6: Quantification by HPTLC

Sample Name	Quercetin	Gallic acid
H. Integrifolia I	0.264%	ND
H. Integrifolia II	ND	ND

RESULT AND DISCUSSION:

Pre-saturated Camag Twin TLC plates were developed through the chamber.

The solvent system utilized was 6:4:0.3 it contains toluene, ethyl acetate, and formic acid.

Scan wavelength: 254nm and 366nm

Standard: Quercetin and Gallic acid

HPTLC:

HPTLC assessment is done to determine phytochemicals found in the process of leaf extraction of this plant. In the HPTLC analysis study, numerous solvent systems were used for the estimation of polyphenolics and flavonoids. The Analysis of solvent systems through a mobile phase that is composed of a 6:4:0.3 v/v/v ratio of toluene, ethyl acetate, and formic acid [Fig.1 and 2]. The recognition of quercetin (Q) and gallic acid(GA) was confirmed by bands obtained from the test sample and standards. The peaks of Quercetin in the test samples were very near to the standard value. The Rf value of Quercetin was to be found for the test sample1, The Quercetin value is 0.39Rf (Table4, Track3) against the standard value, 0.40Rf (Table2, Track1) while in sample 2, Quercetin is not detected (ND) (Table5, Track4). The Standard Quercetin value is 0.40 Rf (Table 5, Track 4) which has nearly the same value of 0.39 Rf (Table 4, Track 3). Therefore, test sample 1 has quercetin 0.264% and sample 2 has not been reported but the value of Gallic acid (GA) was not found in test sample 1 and sample 2 (Table 6).

CONCLUSION:

Through the HPTLC analysis study, it was compared the compounds' Rf values with the reference standards. Quantification by HPTLC indicates the existence of active ingredients in this plant will be very beneficial for the discovery of new bioactive substances and indicators.The results of the present investigation confirmed that Holoptelia integrifolia (Roxb.)Planch has a number of phytochemicals that may be useful for the plant's therapeutic characteristics, which supports its use as a treatment for skin conditions.

ACKNOWLEDGMENTS:

The authors would like to thank expert Prof. (Dr.) Shikhar Verma, Maharishi School of Pharmaceutical Sciences, Maharishi University of Information Technology (MUIT), Lucknow, IIM Road near Maharishi Vidya Mandir, Lucknow. We are grateful to Dr. Pritt Verma (Associate Professor)for providing us with all the tools required to carry out our research work. From Goel Institute of Pharmacy and Sciences, located on Faizabad Road, Lucknow.

CONFLICT OF INTEREST:

There is no conflict of interest, according to the authors.

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Received on 05.12.2023 Modified on 12.04.2024

Research J. Pharm. and Tech 2024; 17(11):5247-5253.

DOI: 10.52711/0974-360X.2024.00803

Parameters	Values
The parameters for Linomat 5 applications
Mist gas Spray	Nitrogen gas
Typical solvent used	Methanol
Rate of dosage	150 nl/s
Volume for predosage	6 µl
Syringes dimension 50°C to 100°C	100 µl
Position for application	5.0 mm
Length of band	6.0 mm
Solvent for the front position	98.0 mm
The adjustment factors
Mode for calibration	Multiple level
Mode for statistics	CV
Mode for evaluation	Peak height and Peak Area
CAMAG detection forTLC scanner
Tracks number	04
The place of tracks X	5 mm
Track distances of separation	09.4mm
The starting point for scanning Y	5 mm
Scanning start end position Y	98 mm
Dimension of slit	06.00x 0.30mm, micro
Upgrade the optical system	Light
Scanning speed	20 mm/s
Resolution of data	At 100 µm/step
Integrity refers to Qualities
Adjustment of baseline	Lowest inclination
Highest peak threshold minimal slope	05
Peak threshold minimum inclination	27.0 AU
Peak threshold at area of minimum	276.0 AU
Level of peal threshold at maximum height	727.7 AU
Track commencement point	05 mm
End position of the track	98.mm
Graphic scaling	Autonomous mode
The analysis measurement
At wavelength	254 nm and 366 nm
Light of lamp	D2/Hg
Type of measurement	Remitted
Mode of measurement	Fluorescence or absorption
Optic filtration	K400/second order
Mode of detection	Autonomous mode
High voltage PM	181V