INTRODUCTION:

In our ancient period, the plants play vital role in healthcare for our medicinal purposes. Traditionally whole plant parts are used as analgesic, antipyretic, anti-inflammatory, immunosuppressant agents¹. Arecaceae family plants are commonly known as palm trees. They have highly medicinal valued plants with various therapeutic activities and are used for health care in a long-time human being². In recent culture medicinal plants have a worldwide impact in our healthcare system which has played a crucial role in our lifestyle. In 19th century 80% of medicine was formulated for plant parts³. Literature shows that biologically active compound like flavonol, phenolic acid, vitamins which are found in germinated sprout⁴. In future medicinal practices, herbal medicine is also going to influence the pharmaceutical industries and affect the use of synthetic medications. Variety of Phytochemicals have a significant role as therapeutic agents for and nutritional properties. B. flabellifer endosperm (germinated sprout) is an edible part that is taken from the seed part for 2-3 months (Figure 1). The other edible parts of the plant are pulp, tuber, and tender palm. It belongs to the family Aracaceae, commonly known as “TAL”, “Palmyra palm”⁵. It is cultivated from native tropical Africa, Asia, Indonesia, India, Jawa, Laos, Malaya, Myanmar, Socotra, parts of China, Sri Lanka, Sulawesi, Thailand, Vietnam, Philippines, South and Southeast Asia throughout of India. The parts of the fruit that are pulp and germinated sprouts are used as sweet dishes in their seasonal times^6,7.

Figure 1: Borassus flabellifer (L) endosperm (germinated sprout)

B. flabellifer is used for its different nutritive and medicinal properties worldwide. September-October is the season of the fruits when the fruits are available⁸. In India, it is mainly cultivated in Tamil Nadu and some other parts of the country for its delicious when fruits and to produce palm sugar, alcoholic beverages, oil. But in other side it contains rich sources of carbohydrates, fat, amino acids, protein, fresh pulp has plenty of vitamin A and B complex. Also, it contains macro and microelements like sodium, potassium, calcium, magnesium, zinc, and iron⁹.

B. flabellifer fruit parts are found to be used traditionally in various tribal regions. The plant extracts have shown different pharmacological activities like antidiabetic, anti-inflammatory, antiarthritic activity, antipyretic activity, analgesic activity, cytotoxic effects. Flower sap is used like tonic, sugar made from the sap and used for liver disorder. Sap or toddy of the plant is used as alcoholic beverage by Maharashtrian villagers and also is used in antifungal activity (Candida albicans and Aspergillus niger). Villagers used the face pack for sensitive skin from palm sugar. The pulp portion also traditionally used in inflammation activity. (Figure 2).^10,11,12

Figure 2: Different Pharmacological activity of Borassus flabellifer

In B. flabellifer has different bioactive compounds like Flavonoids, glycosides, and anthraquinone. The common structure of these compounds is phenyl benzopyrone ring (C6-C3-C6) (Figure 3). They are Commonly found in different plant parts like fruits, stems, cereals, nuts, vegetables, flowers, and seeds. Flavonoids have a wide range of biological activities (10) Here, Quercetin is a flavonol glycoside that is categorized as a bioflavonoid and it has been documented for its diverse range of health-promoting attributes. These include demonstrated anti-inflammatory, antidiabetic, and anticancer activities, alongside another bioactive compound that enhances physical performance ^13,14.

Figure 3: Phenyl benzopyrone ring

Thin-layer chromatography (TLC) holds a preeminent position in phytochemistry due to its extensive applicability, covering nearly every class of compounds, except those highly volatile. It proves particularly useful in the preliminary detection of the presence of various compounds in crude plant extracts. High-performance thin-layer chromatography (HPTLC) represents an efficient, sophisticated, and automated evolution of the conventional TLC method, boasting improved separation efficiency and lower detection limits highly sensitive and entirely reliable method suitable for both qualitative and quantitative analysis. Consequently, in the present study, the HPTLC technique was employed to confirm the presence of major classes of secondary metabolites, like quercetin.

The existing literature review discloses that in spite of being an interesting fruit part having medicinal potency the endosperm of B. flabellifer remains unrevealed till date.

Therefore, the aim of the present study was to establish presence the quercetin and its quantification in B. flabellifer using.

MATERIALS AND METHODS:

Plant material:

B. flabellifer endosperm (germinated sprout) was collected from Kanchrapara, West Bengal October 2023. The plant part was authenticated from Central National Herbarium, Botanical Survey of India, Shibpur, West Bengal, India (Specimen no-GNIPST/2024/24).

Chemicals:

Chemicals and solvents used which are used in analytical grade and they purchased from E. Merck in Darmstadt, Germany. Quercetin (standard flavonoid) was bought from Lobo Chemie in Mumbai, India (Purity > 97%). TLC aluminum plates pre-coated with silica gel 60 F254 (10x10cm, 0.2mm thick) were purchased from E. Merck Ltd (Mumbai, India).

Extraction:

The plant parts were shed dried and coarsely powdered. The powder was extracted with methanol by maceration process for 7 days¹⁵.

QUALITATIVE AND QUANTITATIVE STUDY OF FLAVONOID:

Qualitative Phytochemical test for flavonoids:

The phytochemical test for flavonoids was carried out in methanol extract of B. flabellifer using standard method. ^16,17

QUANTITATIVE DETERMINATION OF TOTAL FLAVONOID CONTENT:

Preparation of Standard Quercetin for Calibration Curve:

The plant part extract for Total Flavonoid Content (TFC) was determined by using a colorimetric aluminum chloride method. Standard solution (100mg/ml) was Prepared with 100milligrams of quercetin dissolved in 1ml of methanol. This standard solution was diluted and prepared in different concentrations of 100, 200, 400, 600, 800, and 1000μg/ml. Then 4 ml of distilled water and 0.3ml of 5% NaNO₂ were added in a test tube. After 5minutes later, 0.3ml of 10% AlCl3 was added with the solution. After 6 min 1M NaOH in 2ml was added to the mixture. Then the mixture was diluted with distilled water and fill the volume up to 10ml. A UV-vis spectrophotometry was employed to determine the absorbance at 510nm^18,19.

Preparation of sample for total flavonoid content:

The extract was prepared with 100mg/ml stock solution mixed up with methanol, which was diluted for different concentrations of solution 0.3mg/ml. Then extract was prepared with same method which used to describe for quercetin solution, and UV-vis spectrophotometry was employed to determine the absorbance at 510nm. The Total Flavonoid Content found in the plant part which was used to from three different absorbance values. And using Plotted linear equation which was based on the standard calibration curve, and determined the flavonoid content was respected as quercetin equivalent (mg QE/100g).

QUALITATIVE AND QUANTITATIVE STUDY OF QUERCETIN:

Thin layer chromatography analysis:

The analytical method of TLC (Thin-layer chromatography) was performed according to standard method. Plates were coated with Silica gel G. Used to separate the developed the investigated substances. The plate was developed by using quercetin solvent system that is toluene: ethyl acetate: formic acid (5:4:0.2) in a chamber. The chamber was saturated with the mobile phase for 5 minutes at room temperature. After running the solvent system dried the plates and analysed using the ultraviolet light 254nm^20,21.

QUANTITATIVE STUDY OF QUERCETIN USING HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY:

Standard Preparation HPTLC:

A reference solution (standard solution) of quercetin was prepared in methanol to final concentration 100mg/ml. The mixture required a sonication before use.

Sample preparation HPTLC:

The edible part endosperm (germinated sprout) of Borassus flabellifer is dried, then ground into a powder form. Then extracted it with methanol by maceration process and this process till in 7 days. Then filter it out and collect the filtrate.

HPTLC Instrumentation and Experimental Conditions:

Thin-layer chromatography (TLC) plates precoated with silica gel (E. Merck, Darmstadt, Germany) were used for this experiment. These plates (60F254, 10cm x 10cm, 0.2mm thickness) were first cleaned by thoroughly dipping them in methanol and air-drying them in a fume hood.

Chromatography was performed in a 10cm x 10cm twin trough glass chamber (Camag, Muttenz, Switzerland) filled with 10mL of mobile phase. The mobile phase consisted of a mixture of toluene: ethyl acetate: formic acid (5:4:0.2, v/v/v). The chamber was saturated with the mobile phase for 25 minutes at room temperature²².

After development, the TLC plates were air-dried using a hot air oven in normal mode for 5minutes. Densitometric analysis was performed using a scanner III with WinCATS software (Camag) in UV mode with the deuterium source set at 275nm. Quercetin was identified by its spots at Rf = 0.55.

METHOD VALIDATION:

Linearity range- The linearity range was validated through the analysis of standard solutions (100 mg mL/1) of quercetin by five different concentration levels (50-200 L). Linear least-squares regression was used to verify linearity and produce a calibration curve. The regression equation, including slope, intercept, and coefficient of correlation (R2), was obtained (Table 1)²³.

Limit of Detection and Limit of Quantification:

The compound was identified using values and UV-visible spectral overlays with reference substances. Diluted standards were added to HPTLC plates to plot calibration curves. The LOD was based on the lowest concentration identified by the instrument in each of the two standards, while the LOQ was based on the lowest concentration quantified in the samples (Table 1).

Accuracy: To determine the method's accuracy, pre-examined samples were spiked with standard quercetin solution and analysed again using HPTLC. Spiking was done at three concentration levels, and the average percent recovery was calculated (Table 2). The experiment was carried out in triplicate.

Specificity: The specificity was done by analysing with standard quercetin and methanolic extract of the sample. The sharp peak was obtained and compared by the Rf value of standard quercetin and sample.

Intra and Inter-day Precession Analysis- The developed methodology was evaluated for intra-assay precision by analyzing three replicates of the substance at three concentration levels of quercetin (100, 150, and 200 µl per band) on the same day. Inter-day precision was evaluated at the same levels on three different laboratory days (Table 3).

Figure 4: HPTLC plate of quercetin (QE) and sample (B1 and B2) of B. flabellifer

QE = Quercetin, B1 and B2= Borassus flabellifer

Figure 5: Densiometric spectra of quercetin acquired by proposed method from standard and Borassus flabellifer extract

Quantification of quercetin of methanolic extract of Borassus flabellifer:

The extract (test sample) was distributed on plate and visible on the chromatogram under the same condition that was maintained for the standard quercetin. The area of the peak corresponding to the Rf value of the quercetin standard was recorded and how much amount was present in the part that was calculated from the graph.

RESULT:

QUALITATIVE AND QUANTITATIVE STUDY OF FLAVONOID:

Phytochemical test for flavonoid- Qualitative analysis of flavonoids test discovered the presence of flavonoids.

Total flavonoid content (TFC)- A colorimetric assay based on aluminium chloride method was used to determine the Total Flavonoid Content of B. flabellifer endosperm (germinated sprout) extract. Quercetin equivalent (QE)/g of weight of methanolic extract was calculated by using the quercetin calibration curve. the calibration curve shows y=0.0005x + 0.029, R2= 0.9994 (Figure 5). Total Flavonoid Content of the extract of Borassus flabellifer (1.43mg QE/g).

Figure 6: Calibration curve of standard quercetin

QUALITATIVE AND QUANTITATIVE STUDY OF QUERCETIN:

Thin layer chromatography- Analytical TLC profiling confirmed that quercetin is present with specific Rf in methanolic extract of Borassus flabellifer. The reference Rf of quercetin is 0.55 and here the Rf shows 0.54. This solvent system was chosen as the final mobile phase for HPTLC.

Quantitative study of quercetin using high-performance thin-layer chromatography:

HPTLC analysis- High-performance thin-layer chromatography (HPTLC) analysis revealed that the mobile phase consists of toluene- ethyl acetate- formic acid in a (5:4:0.2, v/v/v) volume ratio effectively separated the flavonoid, quercetin in the sample. This is evident in (Figure 3), which depicts the thin-layer chromatography profiles of both samples observed under visible light at wavelengths of 245 nm and 366 nm ²⁴.

METHOD VALIDATION:

Linearity range- The method's linearity was validated through the analysis of standard solutions (100 mg mL−1) of quercetin at five different concentration levels (50-200ml). Linear least-squares regression was used to verify linearity and produce a calibration curve. The regression equation, including slope, intercept, and coefficient of correlation (R2), was obtained (Table 1) ^25,26.

Limit of Detection and Limit of Quantification- The proposed method demonstrated a limit of detection (LOD) of 0.0051µg/spot and a limit of quantification (LOQ) of 0.0156µg/spot for quercetin. These sensitive detection limits indicate the method's broad applicability for effectively detecting and quantifying quercetin in the sample.

Accuracy- The recovery studies presented in Table 2 yielded results within acceptable limits, achieving a value of 105.8%. This indicates that the method demonstrates good accuracy in quantifying the target analyte.

Calibration curve- As illustrated in (Figure 6) the calibration plot demonstrates a direct proportional relationship between the response and the concentration of quercetin within the range of 50-200 µg/ml. The strength of this linear association is confirmed by a high correlation coefficient (R2) of 0.9989. Additionally, the equation for this line is represented by a slope of 0.0004 and an intercept of 0.0002. For further details regarding the specific data points, refer to Table 1.

Figure 7: Standard curve of standard quercetin

Table 1: Linear Regression data for standard Quercetin

	Linear Regression Parameter	Data
	Linearity range (µg/spot)	50-200
	Regression equation	0.0002x + 0.0004
	Correlation equation (R2)	0.9989
	SLOPE	0.0002197
	INTERCEPT	0.0004
	SE OF INTERCEPT	0.000700973
	SD OF INTERCEPT	0.001567422
	LOD	0.005172493
	LOQ	0.015674222

Table 2: Recovery study for proposed method (n=3)

Concentration	Repeatability (Intra-day precision)			Repeatability (Intra-day precision)
Concentration	Area±SD	Standard error	%RSD	Area±SD	Standard Error	%RSD
100	0.0112±0.63	0.00015	0.84	0.0121±0.4	0.00018	0.61
100	0.0117±0.21	0.00012	0.97	0.0141±0.6	0.00015	0.94
100	0.0210±0.52	0.00016	1.40	0.0210±0.3	0.00012	2.10

Recovery Studies: The recovery studies presented in Table 2 yielded results within acceptable limits, achieving a value of 105.80%. This indicates that the method demonstrates good accuracy in quantifying the target analyte.

Intra and Inter-day Precession- The findings for repeatability and intermediate precision, presented as standard deviation (SD) percentages, are detailed in Table 3. The relative standard deviation (RSD) ranged from for 0.84- 1.40 repeatability and from for 0.61-2.10 inter-day precision. These low values demonstrate the method’s high level of precision.

Table 3: Precision of the proposed method (n=3)

Excess drug added to analyte (%)	Concentration found (µg±SD)	%Recovery	%RSD
50	53.71±0.34	91.98	0.84
100	91.68±0.77	91.66	0.84
150	158.7±0.61	105.80	0.38

Figure 8: HPTLC chromatogram of standard quercetin

Figure 9: HPTLC chromatogram of methanolic extract of Borassus flabellifer (germinated sprout)

Quantitative estimation of quercetin in the Methanolic Extract of Borassus flabellifer:

Quercetin peak from the methanolic extract of Borassus flabellifer was identified by their single spot at Rf = 0.55 (Figure 8) which was obtained by chromatography of the standard under the same conditions. The quercetin content in methanolic extracts of endosperm (germinated sprout) was quantified by using the standard concentration and indicated the AUC which was presented. Quercetin content in methanolic extract of Borassus flabellifer is 0.98mg/100g ²⁷.

DISCUSSION:

HPTLC method is a useful tool for identification and quantification secondary metabolites of plant. The methanolic extract of B. flabellifer (L.) has positive result for the phytochemical test of flavonoids, and rich total flavonoid content was found in the extract. Further TLC Study with the mobile phase toluene- ethyl acetate- formic acid in a (5:4:0.2, v/v/v) shows prominent Rf at 0.54. Therefore, HPTLC analysis carried out to identify and quantify the flavonoid, quercetin in germinated sprout of Borassus flabellifer. In the present study, The HPTLC method demonstrated good linearity, accuracy, precision, specificity, and sensitivity, making it suitable for routine analysis. HPTLC is one of the best methods for ensuring product quality, stability as well as identification or validation of plant parts extracts. Quercetin was identified with a single peak at Rf = 0.55, and estimated to be 0.98mg/100g in the methanolic extract of germinated sprout in B. flabellifer. In the plant, germinated sprout was found to be one of the potent sources of Quercetin and the present study detected good amount of quercetin in it which is beneficial for nutritional value and for management of a number of diseases and disorders.

CONCLUSION:

Borassus flabellifer (L) is one of the common fruits of Aracaceae family with potent therapeutic values. Endosperm of the fruit although a delicious part of food, but is available only during a particular time of the year and usually requires specific processing before intake. In spite of its therapeutic potential and a good choice for consumption, this part remains scientifically unexplored totally.

Preliminary phytochemical analysis showed its flavonoid content which in present study was extended to detect and establish the Quercetin content in the endosperm of Tal. And thus, the present study established tal endosperm as one of the promising sources of quercetin. In similar way present study can be extended to explore the quantitative and qualitative aspects of quercetin in the pulp of the fruit, which is still remained unexplored. Further the outcome of the present study shows that it may be useful to establish the mechanism of action of its different parts for their medicinal properties as well as to prepare its herbal formulation in the future.

ACKNOWLEDGEMENT:

The authors would like to acknowledge Guru Nanak Institute of Pharmaceutical Science and Technology for providing the support for this research work.

REFERENCE:

1. Paschapur MS, Patil S, Patil SR, Kumar R, Patil MB. Evaluation of the analgesic and antipyretic activities of ethanolic extract of male flowers (inflorescences) of borassus flabellifer L. (arecaceae).

2. Sharma P, Dwivedee BP, Bisht D, Dash AK, Kumar D. The chemical constituents and diverse pharmacological importance of tinospora cordifolia. Vol. 5, Heliyon. Elsevier ltd; 2019.

3. Ullah R, Alqahtani AS, Noman OMA, Alqahtani AM, Ibenmoussa S, Bourhia M. A review on ethno-medicinal plants used in traditional medicine in the kingdom of Saudi Arabia. Vol. 27, Saudi Journal of Biological Sciences. Elsevier b.v.; 2020. P. 2706–18.

4. Khan Shinwari Z, Qaiser M. (Medicinal plants: conservation and sustainable use) efforts on conservation and sustainable use of medicinal plants of Pakistan. 2011; 43.

5. Gummadi VP, Rao Battu G, Diyya K, Manda K. A review on Palmyra palm (Borassus flabellifer) [internet]. 2016. Available from: http://eol.org/pages/1123573/names/common_names.

6. Shrestha R. A review on nutritional properties of Palmyra palm (Borassus flabellifer L.). International Journal of Science and Research [internet]. Available from: www.researchgate

7. Shinde PK, Kokate RH, Gawade GS. Physicochemical, phytochemical, biological and chromatographic evaluation of Polyalthia longifolia plant leaves-A review. Research Journal of Science and Technology. 2023; 15(1): 41-8.

8. Arunachalam K, Saravanan S, Parimelazhagan T. Nutritional analysis and antioxidant activity of Palmyrah (Borassus flabellifer L.) Seed embryo for potential use as food source. Food Sci Biotechnol. 2011; 20(1):143–9.

9. Jerry A. A comprehensive review on the medicinal properties of Borassus flabellifer. Journal of Academia and Industrial Research (JAIR). 2018;7(7).

10. Ullah A, Munir S, Badshah SL, Khan N, Ghani L, Poulson BG, et al. Important flavonoids and their role as a therapeutic agent. Vol. 25, molecules. MDPI ag; 2020.

11. Jan R, Mohi-ud-din R, Nisa KU, Mir RH. Edible Medicinal Plants from Chhattisgarh (India) and their Economic Significance. InEdible Plants in Health and Diseases: Volume 1: Cultural, Practical and Economic Value 2022 Jan 13 (pp. 235-257). Singapore: Springer Nature Singapore.

12. Kavya Kulkarni, Govindaiah. Evaluation of Anti-oxidant properties of some medicinal plant products by ABTS Radical Scavenging Assay. Research Journal of Science and Technology. 2022; 14(4): 213-8.

13. Anand David AV, Arulmoli R, Parasuraman S. Overviews of biological importance of quercetin: a bioactive flavonoid. Vol. 10, Pharmacognosy Reviews. Medknow publications; 2016. P. 84–9.

14. Selvakumar S, Gangatharan S, Rao MR. Preliminary Phytochemical Screening of Root Extracts of Crossandra infundibuliformis. Research Journal of Pharmacy and Technology. 2016; 9(2):131-4.

15. Pant DR, Pant ND, Saru DB, Yadav UN, Khanal DP. Phytochemical screening and study of antioxidant, antimicrobial, antidiabetic, anti-inflammatory and analgesic activities of extracts from stem wood of Pterocarpus marsupium roxburgh. J Intercult Ethnopharmacol. 2017; 6(2): 170–6.

16. Doshi G, Une H. Quantification of quercetin and rutin from Benincasa hispida seeds and Carissa congesta roots by high-performance thin layer chromatography and high-performance liquid chromatography. Pharmacognosy Res. 2016; 8(1): 37–42.

17. Soni J, Bhardwaj SK. Preliminary Phytochemical screening of root extract of Martynia annua. Research Journal of Science and Technology. 2024; 16(1): 39-42.

18. Kamtekar S, Keer V, Patil V. Estimation of phenolic content, flavonoid content, antioxidant and alpha amylase inhibitory activity of marketed polyherbal formulation. J Appl Pharm Sci. 2014; 4(9): 61–5.

19. Doshi GM, Chaskar PK, Une HD. Elucidation of β-sitosterol from Benincasa hispida seeds, Carissa congesta roots and Polyalthia longifolia leaves by high performance thin layer chromatography. Pharmacognosy Journal. 2015; 7(4): 221–7.

20. Doshi G, Une H, Shanbhag P. Rasayans and Non-rasayans herbs: future immunodrug - targets. Pharmacognosy Reviews. 2013;7: 92–6.

21. Swamini A Dighe, Suhas S Siddheshwar, Ganesh S Shinde. A Review on Analytical Method for Determination of Venlafaxine HCl in Bulk and Pharmaceutical dosage form. Research Journal of Science and Technology. 2021; 13(3):208-2.

22. Doshi g, une h. Quantification of quercetin and rutin from benincasa hispida seeds and carissa congesta roots by high-performance thin layer chromatography and high-performance liquid chromatography. Pharmacognosy res. 2016 jan 1;8(1):37–42.

23. Alzeer HS, Alzaid SF, Aldawsari FS, Alshehri YM. Development and validation of a simple method for the determination of triamcinolone acetonide in nasal spray. Saudi Pharmaceutical Journal. 2023 oct 1; 31(10).

24. Soni J, Bhardwaj SK. Preliminary Phytochemical screening of root extract of Martynia annua. Research Journal of Science and Technology. 2024 Mar 8; 16(1):39-42.

25. Tentu Nageswara Rao, T.B. Patrudu, Suneel Kumar. A, N. Krishna Rao, Karri Apparao. A New Analytical Method Validation and Quantification of Residual Solvents in Telmisartan Bulk Drug Product by Headspace gas Chromatographic Method. Research J. Science and Tech. 2018; 10(2):98-104.

26. Purohit PJ, Kapupara PP, Shah KV. Development and validation of analytical method for simultaneous estimation of curcumin and gallic acid in different polyherbal formulations by HPLC. Research Journal of Pharmacy and Technology. 2014; 7(7):749-53.

27. Umadevi K, Hoque M, Ramya SS. Quantitative estimation of roxithromycin and ambroxol in bulk and tablet dosage forms by RP-HPLC method. Research Journal of Science and Technology. 2023; 15(1): 1-7.

Received on 25.04.2024 Modified on 01.06.2024

Research J. Science and Tech. 2024; 16(3):219-228.

DOI: 10.52711/2349-2988.2024.00032