VALIDATING THE PROPERTIES OF NYMPHAEA PUBESCENS WILLD. BY PERFORMING UV-VIS, TLC, FTIR AND HPLC ANALYSES

VALIDATING THE PROPERTIES OF NYMPHAEA PUBESCENS WILLD. BY PERFORMING UV-VIS, TLC, FTIR AND HPLC ANALYSES

Madhulika ^{* 1}, Sanjay Kumar ², K. N. Dwivedi ² and Sangeeta Gehlot ¹

Department of Kriya Sharir ¹, IMS, BHU, Varanasi - 221005, Uttar Pradesh, India.

Department of Dravyaguna ², IMS, BHU, Varanasi - 221005, Uttar Pradesh, India.

ABSTRACT: Introduction: Nymphaea pubescens Willd. is one of the well-known medicinal plants, being used from ancient times in India and commonly called “Kumuda”. It is an aquatic perennial herb. It has leaves having a smooth upper side, a strong root, and a branched rhizome. According to Bentham and Hooker classification “Genera Plantarum”, this plant belongs to the division Polypetale of Dicotyledones, which has been divided into 15 order and 84 families. Nymphaea comes under the Araceae family, which itself contains 8 genera and 100 species. Method: Morphological characters have been examined through a transverse section cut by microtome. Physiochemical studies and other screenings were performed accordingly. In addition to this, UV-VIS, TLC, FTIR, and HPLC tests were also carried out for further validation. Result: The morphological and physiochemical examination revealed the presence of saponins, proteins, and various other tests in Nymphaea pubescens Willd. Conclusion: The microscopic, UV-VIS, TLC, FTIR, and HPLC validation can be helpful for using it for medicinal and commercial purposes.

Pharmacognostic studies, Water lily, Thin layer chromatography, Organoleptic property, Kumuda

INTRODUCTION: Nymphaea pubescens Willd.  is also known as water lily; it is grown in ponds, lakes, and water bodies, having simple heart-shaped leaves, branched rhizome, striated seed, and globular fruit ¹. The whole plant is used as a medicine in Type 2 diabetes mellitus and urinary tract disease ². Although it is one of the types of lotus species, a very brief illustration is found in different botanical and Ayurvedic treatises. The scarcity of information regarding the characteristics and its uses motivates the author to review it extensively in various research journals and other related literature.

And to standardized the parameter after performing Organoleptic properties, powder microscopy, physiochemical, UV-VIS, TLC, FTIR, and HPLC studies. Such exploration and validation help society in the treatment of various diseases and for medicinal and commercial purposes.

MATERIALS AND METHODS:

Collection: Nymphaea pubescens Willd. was collected from its natural habitat near Banaras Hindu University, Varanasi, in July 2018.

Authentication of Plants: Sample (Voucher specimen no. Nymphaea.2018/1) was authenticated by the expert from the Department of Botany, Institute of Science, Banaras Hindu University, Varanasi. Plant specimen was deposited in the museum of the Department of Dravyaguna, Faculty of Ayurveda, for future reference.

Chemicals: All analytical grade chemicals used in the study were purchased through Advanced Quality traders, E. Merk, Germany.

Organoleptic Evaluation: Organoleptic evaluation of the plant sample was done with the help of sensory organs ³.

Powder Microscopy: The coarse powder of the sample was boiled with chloral hydrate and collected in a Petri dish, and stained with the different reagent ⁴.

Physiochemical Study: Coarse powder of the sample was used for an analytical test which is shown in table number ³.

Loss on Drying: About 10 gm of the sample was placed in a tarred evaporating dish, and the sample was dried at 105 ºC for 5 h and weighed. After that, it was cooled in desiccators for 30 min and weighed.

Determination of Total Ash: About 2.0 g of the sample was incinerated in a tarred silica dish at a temperature not exceeding 450 ºC until free carbon was left, cooled, and finally weighed.

Acid Insoluble Ash: Ash was dissolved in 25 ml of dilute HCL and boiled for 5 min; then, insoluble matter was collected on an ash-less filter paper, washed with hot water, and ignited to constant weight.

Water Soluble Ash: The sample was dissolved in 25 ml of diluted HCl and made up of 50 ml with water and boiled. Then filtered with Whatmann filter paper and residue was collected.

It was weighed and maintained in a muffle furnace for 6 h at 450-650 ºC. The crucible was taken out and cooled at room temperature and weighed.

UV-Vis (Ultraviolet-Visible Spectroscopy): 1 gram of plant extracts added in 10 ml of distill water then filtered with the help of cartilage (0.2 µm) afterward, it was scanned under ultraviolet, visible spectrophotometer (Perkin Elmer) at a range of 200-900 nm to measuring the size of biomolecules and uncertainty source that may arise from nature of the compound of plant extract.

TLC (Thin Layer Chromatography): Thin layer chromatography was detected by observation of spots for identical R_f values and to determine the purity of a sample.

Stationary Phase:  Pre-coated plate with silica gel G (Merck) was used as the stationary phase.

Solvent System: Toluene: Ethyl acetate: Formic acid: Methanol was used as a solvent system in the respective ratio of 4.3: 4.3: 1.2: 0.3.

Spraying Reagent: Methanol: sulphuric acid (9:1) was used for drying plates in a hot air oven.

Procedure: Extract was applied 2 cm on the lower edge of the plate with the help of a microcapillary tube. And then extracts were loaded in a small volume spot on each plate, the plate was taken out, the solvent front was marked, and the plate was dried at room temperature.

FTIR (Fourier- Transform Infrared Spectro-scopy): A pinch of powder drug was taken and placed over the crystal present on stage. The IR spectrum (Perkin Elmer, Spectrum-2) was scanned between 4000 to 400-1, and transmittance was recorded.

Before scanning the sample, the background signal was also recorded. The peaks thus obtained were matched against the IR interpretation chart, and the functional groups were noted.

HPLC (High-Performance Liquid Chromato-graphy): 1 g of plant extract is added in 10 ml of methanol then sonicator in the sonicator machine (Labman), afterward filtered with the help of cartilage (0.2 µm) and inject with micro syringe (20 µl) and finally scanned with HPLC machine to detect the flavonoids and Phenol.

Standard: (Flavonoids and Phenol) Catechin hydrate, Myricetin, Rutin, Quercetin, Caffeic acid, Kaempferol and Gallic acid, all solution is prepared in methanol (1 mg/ml).

Mobil Phase A: methanol: acetonitrile: water: acetic acid (50 ml : 25 ml : 425 ml : 5 ml) For 0-20 min.

Mobil Phase B:  methanol: acetonitrile: acetic acid (300 ml : 200 ml : 5 ml) for 20- 25 min.

RESULTS:

Organoleptic Properties: Organoleptic properties of N. pubescens Willd. show is shown in Table 1.

TABLE 1: ORGANOLEPTIC PROPERTIES OF N. PUBESCENS WILLD.

Powder Microscopy: Behaviour of N. pubescens Willd. powder with different reagents.

Physiochemical Study: Result of physiochemical analysis of N. pubescens Willd. is shown in Table 3. The method of loss on drying has actually calculated the presence of moister of N. pubescence (11.98 %) Less number of moister prevent from bacteria and fungus ⁵.

The total Ash value of N. pubescence (6.15%), indicates the presence of minerals in seed ⁶. Acid insoluble ash of N. pubescence (11.8%) indicates the presence of the earth mineral and silica. 6.3% Water-soluble Ash presence in N. pubescence Table 3.

TABLE 2: POWDER MICROSCOPY OF N. PUBESCENS

TABLE 3: PHYSIOCHEMICAL STUDY OF N. PUBESCENS WILLD.

The Ultraviolet, visible spectroscopy profile of graph 1 of the extract was observed at 200-900 nm wavelength range and 266 nm recorded band. Phenolic and flavonoid components generally absorb at 230-290 nm ⁷. Hence, it conform phenolic and flavonoids are present in the extract of N. pubescens Willd.

GRAPH 1: ULTRAVIOLET VISIBLE SPECTROSCOPY OF DISTIL WATER EXTRACT OF N. PUBESCENS WILLD.

Thin Layer Chromatography: Thin layer chromatography was detected by the observation of spots for identical R_f values and to determine the purity of a sample. In petroleum ether extract showed six spot, Chloroform indicate five spot, Acetone and Benzene identified seven spots, Ethanol has three spots and lastly Methanol and distill water separate two spot Table 4. Hence, the TLC profile shows compounds are smoothly separated with the help of mobile phase 8 9 10 11.

TABLE 4: TLC OF N. PUBESCENS WILLD.     

Extract	Solvent Front (cm)	TLC plates	Peaks Obtained (cm)	Rf Value (cm)
Petroleum Ether	7.3		S1 1.4	0.30
			S2        2.7	0.36
			S3      3.1	0.42
			S4      3.9	0.53
			S5      5.6	0.76
			S6         6.7	0.91
Chloroform	7.5		S1      2.9	0.38
			S2       3.6	0.48
			S3      4.6	0.61
			S4      5	0.66
			S5       7.1	0.94
Acetone	7.5		S1     2	0.26
			S2    2.8	0.37
			S3    4	0.53
			S4   4.3	0.55
			S5    4.6	0.61
			S6     6.4	0.85
			S7      7.4	0.98
Benzene	6.7		S1      3.5	0.52
			S2    4.4	0.65
			S3    4.6	0.68
			S4    4.8	0.71
			S5    5.5	0.82
			S6    5.8	0.86
			S7    6.5	0.97
Ethanol	5.6		S1     1.4	0.25
			S2     2.3	0.41
			S3    3.6	0.64
Methanol	7.5		S1     2.2	0.29
			S2   3.3	0.44
Distil Water	7		S1    1.2	0.17
			S2        2	0.28

FTIR (Fourier- Transform Infrared Spectro-scopy): FTIR is a characterization method who give the vibration energy based on peak value, compressing act of the functional group that is available on the extract of N. pubescence. The major bands were observed at 3282, 1633, 1317, 1227, 1015 and 630 to 609, cm^-1. The peak indicates Table 5 OH stretching might be alcohol, carboxylic acid. CH stretching is alkyne. NH bending is an amine. C=C Stretching is Con. And conjugate alkene. OH, bending is Phenol. S=O Stretching is Sulfone. C-F Stretching is Fluro compound. C-N Stretching is Amine and Aromatic Amine. C-O Stretching is Alkyle arile ether, and C-X (X=Cl or Br) is halo compound ^{12, 13}.

GRAPH 2: FTIR OF CRUDE EXTRACT OF N. PUBESCENS WILLD. Fourier transform infrared spectroscopy is a technique to measure the wavelength of the sample (N. pubescens Willd.) and find out the probable functional group

TABLE 5: FTIR OF N. PUBESCENS WILLD

HPLC (High Performance Liquid Chromato-graphy): High-performance liquid chromatography of methanol extract of N. Pubescens Willd is shown in Graph 3.

GRAPH 3: HPLC CHROMATOGRAM OF STANDARD PHENOLIC ACID (Ca = Caffeic acid, K = Kaempferol, Ga = Gallic acid), Flavonoids (Ch = Catechin hydrate, Q = Quercetin, R = Rutin and M= Myricetin) and Np = methanol extract of Nymphaea pubescence

TABLE 6: ANALYTICAL CONDITIONS

TABLE 7: RETENTION TIME (RT), WAVELENGTH (NM), AREA AND HEIGHT OF METHANOL EXTRACT OF N. PUBESCENCE AND STANDARD OF PHENOLIC ACID AND FLAVONOIDS FOR HPLC METHOD VALIDATION

The HPLC machine details shown in Table 6 and in Table 7. The plant extract was evaluated with seven standards (gallic acid, quercotin, catechin, rutin, caffic acid, myricetin) of phenolic and flavonoids phytoconstituents to detect their capability. Caffeic acid (3.536 RT), Gallic acid (3.575), and Rutin (3.567) are present in both extracts of methanol and distill water of N. pubescence. Rutin and Caffeic acid are both possessing in previous studies on another plant of R. arvensis ¹⁴.

CONCLUSION: The present study evaluates macroscopic, microscopic, powder microscopy, physiochemical, UV-VIS, FTIR, TLC, and HPLC of the whole plant of N. Pubescens Willd. for correct identification and standardization. Powder microscopy shows the presence of calcium oxalate crystal, lignified Cells, and cuticle. Physiochemical test showing 11.98% loss of the crude drug on drying, 6.15% of total ash, 11.8% of acid in soluble, 6.3% of water-insoluble ash.

The TLC result indicates that the mobile phase successfully separates the compound, and the R_f value shows the medicinal value of N. Pubescens Willd. FTIR shows the presence of different functional groups such as Carboxylic acid, Alcohol, Alkene, Amine, Conjugated alkene, Phenol, Sulfone, Aromatic Amine, Alkyl aryl ether, Fluoro compound, and Halo compound.

All of these functional groups have an anti-diabetic effect, especially, Tannin, flavonoids, and phenol are showing antioxidant properties as an explanation for anti-diabetic effects of the Nymphaea pubescence.

ACKNOWLEDGEMENT: The corresponding author expresses her sincere gratitude to her supervisor and co-supervisor for their guidance and providing basic facilities during this study. Grateful thanks Head department of zoology, Dr. Sonam Agrawal, Dr. Jasmeet Singh, Sanjay Kumar, Shalvi Agrawal, Veena, Imran Khan, Suraj and Arun Srivastava to providing support. Also, thankful to Dr. Anurag Mishra for the valuable advice and Dr. Anam Aftab for the required editing in the manuscript. I acknowledge sincere thanks to DST for my studies done in the Department of Kriya Sharir and Dravyaguna Laboratory.

CONFLICTS OF INTEREST: The corresponding author declares no conflicts of interest.

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    How to cite this article:

    Madhulika, kumar S, Dwivedi KN and Gehlot S: Validating the properties of Nymphaea pubescens willd. by performing UV-Vis, TLC, FTIR and HPLC analyses. Int J Pharm Sci & Res 2021; 12(1): 615-21. doi: 10.13040/IJPSR.0975-8232.12(1).615-21.

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