INTRODUCTION:

Medicinal plants have bioactive phytochemicals are used for treatment of diseases since long ago. A number of antibiotics with established mechanisms have been used for the treatment of many infections but now a days search for newer antibiotics is a worldwide challenge, since many pathogens are becoming resistant to synthetic drugs.¹

Starting from the human civilization a long history of a number of plants known to treat various diseases as specifically to Prosopis species antidiabetic², anti-inflammatory³, antimicrobial and antioxidant⁴, effects have been observed through in vitro and in vivo studies. ⁵ It is always beneficial to treat any disease with multiple approaches and in the consequence to the same recent time plants derived drugs have gained attention in the form of Complementary and Alternative Medicine (CAM).⁶ Prosopis juliflora is native to Mexico, south America and Caribbean, is widespread hyperaccumulating shrubs or tree. It has now become invasive weed which is a thorny, deciduous, large crowned, and deep-rooted bush that grows up to a height of about 2-10m depending on the climatic condition in asia, australia and other places.⁷It has been proven that Prosopis juliflora have alkaloids, flavonoids, phenolic derivatives, tannins and terpenoids and coumarins in their different parts like Leaves, gum, whole plant, flower and stem. In addition to Pakistan, in western madya Pradesh bark of Prosopis juliflora is used to treat asthma.⁸ Many techniques are available for qualitative and quantitative estimation of plant constituents for their identification, isolation and screening.⁹ The HPTLC is inexpensive and fast method which has been accepted around the globe as powerful analytical technique by the research laboratories for new drug discovery.¹⁰ WHO also suggest HPTLC a fast and standardized technique for further research.^11,12,13,14 Phytochemical analysis play an important role for fixing quality in the research study.^{15,16,17,18,19,20} The present study was designed and performed for phytochemical profiling along with quantification of quercetin and gallic acid to magnify the therapeutic use of Prosopis juliflora technique through HPTLC analysis.

MATERIAL AND METHODS:

Collection, identification, and authentication of plant:

Fruiting aerial part of Prosopis juliflora (Sw.) DC commonly known as vilayati or Musquite or jangli babool or Vilayati kikkar (Family: Fabacea ) were collected in the month of july 2022 from Faizullaganj, Lucknow. The plant authentication was done from CSIR-NISPR under authentication no. NIScPR-RHMD/Consult/2022/4173-74-3.

Extraction:

The authenticated part of the plant was washed and air dried at 30-37°C using tray drier for 5 days. The plant material was not exposed to direct sunlight during the entire process of drying. The air dried sample was ground into fine powder with the help of mechanical grinder and passed through 60# and stored at room temperature in air tight containers. On the basis of increasing order of polarity, powdered samples (20g.) were successively extracted with n-hexane (69°C) and hydro-alcoholic [Ethanol and water in 1:1 v/v] (60°C) solvents. Initially extraction was performed from n-hexane using soxhlet for 24hrs. Powder removed from thimble was air dried for approx. 1hour till whole solvent evaporated. Now dried powder was again packed into thimble and exposed to next solvent. The excess solvent then evaporated in water bath till semisolid mass obtain and stored in air tight containers. The semisolid mass was then dissolved in required amount of methanol for HPTLC purpose.

Preparation of standard solution:

Initially prepared 1mg/1ml of quercetin and gallic acid diluted with methanol upto 0.5mg/ml were prepared.

Preparation of plant samples:

Dried extract (10mg) of Prosopis juliflora fruiting aerial part was dissolved in 1ml HPTLC grade methyl alcohol then filtered through 0.22µm filters. This solution was used as a test solution for HPTLC study.

Instrumentation:

A CAMAG HPTLC system consisted of LINOMAT 5 auto sprayer connected to a nitrogen cylinder fitted with 100µl syringes, winCATS software and CAMAG TLC scanner was used.

Chemicals and Solvents:

All the solvents and chemicals used were of chromatographic and analytical grade respectively. Reference quercetin and gallic acid were gifted by the National botanical research laboratory (NBRI, Lucknow).

Chromatographic Conditions:

The HPTLC was performed on 20 X10cm pre coated with 0.2mm silica gel 60 F 254 HPTLC plate (E-Merck KGaA). No pre-washing but manual modification by cutting the plate at required size and activation in an oven at 40^oC prior to analysis was done. In the form of bands aliquots of samples (10, 20µl) and standards solution were applied to the plate, 5mm from the bottom with CAMAG Linomat-5 applicator fitted with 100µl syringe at a constant application rate of 150nl/s. Plates were developed using mobile phase consisted of toluene: ethyl acetate: formic acid in 6:4:0.3 (v/v/v) ratio. After development plate was air dried for 5 minutes and scanned immediately at 254nm and 366nm using CAMAG scanner III equipped with winCATS software. Images were captured across the bands at 254nm and 366nm with the help of CAMAG visualizer for photo-documentation (Table 1).

Table 1: Parameters used for HPTLC

Parameters	Values
Calibration Parameter
Calibration mode	Multiple level
Statistics mode	CV
Evaluation mode	Peak height and peak area
Linomat 5 application parameters
Spray gas	Nitrogen
Sample solvent type	Methanol
Dosage Speed	150 nl/s
Predosage volume	6 µl
Syringe size	100 µl
Application position	5.0 mm
Band length	6 mm
Solvent front position	98.0 mm
Detection CAMAG TLC scanner
Number of track	4
Position of track	5.0 mm
Distance between track	9.4 mm
Scan start position Y	5.0 mm
Scan end position Y	98.0 mm
Slit dimension	6.00 x 0.30 mm, micro
Optimize optical system	Light
Scanning speed	20 mm/sec
Data resolution	100 µm/step
Integration: properties
Baseline correction	Lowest slope
Peak threshold min. slope	5
Peak threshold min. height	10 AU
Peak threshold min. area	50
Peak threshold max. height	990 AU
Track start position	5.0 mm
Track end position	98.0 mm
Display scaling	Automatic
Measurement
Wavelength	254 nm and 366 nm
Lamp	D2/Hg
Measurement type	Remission
Measurement mode	Absorption/fluorescence
Optical fibre	Second order/K400
Detector mode	Automatic
PM high voltage	181V

UV active compounds present on TLC plate will appear as dark spot on a bright background due to fluorescence quenching at 254nm while at 366nm appears as bright spot on a dark background²⁰.

Calibration curve of quercetin and gallic acid:

The quercetin and gallic acid were determined by using calibration curve established with a standard concentration ranging from 2 to 10µg/spot. A stock solution was prepared in methanol with concentration 0.5mg/ml. The different volume of stock solution injected like, 4µl, 8µl, 12µl, 16µl, 20µl to were spotted on HPTLC plate to obtained standards 2µg, 4µg, 6µg, 8 µg, 10µg/spot respectively. Calibration curves were obtained by plotting absorbance unit against concentration of standards (Fig. 4 and Fig. 6).

RESULTS AND DISCUSSION:

This research study reveals the phytochemical profiling of bioactive compounds along with percentage of querecetin and gallic acid for the first time in Prosopis juliflora as illustrated in the figures and tables below. The chromatograms were obtained at short (Fig. 1) and long UV wavelength (Fig. 2). The Rf values, peak area, peak height and percentage area of unknown substance along with quercetin and gallic acid were obtained as depicted in Figs. 3, 5, 7, 8, 9 tables 2, 3, 4, 5.

Q GA Q + GA Sample1 Sample 2

Fig. 1 Image of TLC plate at 254 nm

Q GA Q + GA Sample1 Sample 2

Fig. 2 Image of TLC plate at 366 nm

Fig. 3: HPTLC chromatograms of stand. Quercetin

Fig. 4: Calibration curve of standard quercetin

Fig. 5: HPTLC chromatograms of stand. Gallic acid

Fig. 6: Calibration curve of standard gallic acid

Fig. 7: HPTLC chromatograms of standard Q. and GA.

Fig. 8: Chromatogram of n-hexane extract of plant

Fig. 9: Chromatogram of hydro-alcoholic extract of plant

Table 2: HPTLC peak table of standard quercetin

Track 1, ID: Standard 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.	-0.02	0.5	-0.01	33.5	5.52	0.02	0.1	628.8	3.75	Unknown
2.	0.43	25.9	0.48	294.5	48.59	0.50	22.5	7340.2	43.81	Q
3.	0.51	22.9	0.53	34.9	5.76	0.55	19.2	991.6	5.92	Unknown
4.	0.63	23.4	0.64	27.1	4.47	0.66	22.0	618.6	3.69	Unknown
5.	0.78	33.4	0.86	89.8	14.82	0.86	36.8	4713.5	28.13	Unknown
6.	0.86	86.9	0.87	90.9	15.00	0.90	0.7	2086.8	12.46	Unknown
7.	0.95	0.4	0.96	23.6	3.90	0.97	5.8	229.9	1.37	Unknown
8.	0.97	6.2	0.98	11.7	1.94	0.99	0.0	144.9	0.87	Unknown

Table 3: HPTLC peak table of standard Gallic acid

Track 2, ID: Standard 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.	-0.02	0.5	-0.01	38.0	3.86	0.01	3.2	471.0	1.60	Unknown
2.	0.21	18.5	0.25	499.5	50.76	0.29	20.0	16358.1	55.64	GA
3.	0.42	29.7	0.45	176.0	17.88	0.48	26.0	3926.3	13.36	Unknown
4.	0.49	29.9	0.51	45.5	4.62	0.53	35.6	1245.5	4.24	Unknown
5.	0.74	36.1	0.77	48.4	4.92	0.77	46.2	1536.1	5.23	Unknown
6.	0.79	53.7	0.83	87.8	8.92	0.84	34.4	3525.3	11.99	Unknown
7.	0.86	84.9	0.87	88.9	9.03	0.89	35.4	2335.1	7.94	Unknown

Table 4: HPTLC peak table of n-hexane extract of plant

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.	-0.030	0.5	-0.01	97.0	8.27	0.01	0.6	1296.4	3.54	Unknown
2.	0.11	4.1	0.12	15.0	1.28	0.13	4.2	138.1	0.38	Unknown
3.	0.23	9.2	0.26	27.1	2.31	0.27	17.4	593.4	1.62	GA
4.	0.28	17.3	0.32	35.3	3.01	0.33	34.7	1155.9	3.16	Unknown
5.	0.33	32.6	0.35	47.3	4.03	0.36	36.2	1181.4	3.23	Unknown
6.	0.37	36.4	0.39	58.4	4.98	0.40	55.4	1458.5	3.99	Unknown
7.	0.40	60.3	0.43	107.1	9.13	0.44	35.3	3366.4	9.20	Unknown
8.	0.44	95.7	0.46	128.2	10.94	0.49	52.0	4028.6	11.01	Q
9.	0.60	55.1	0.66	100.5	8.58	0.69	59.8	6809.6	18.61	Unknown
10.	0.71	60.4	0.75	93.1	7.94	0.76	38.0	3344.5	9.14	Unknown
11.	0.76	88.1	0.79	123.7	10.56	0.80	16.8	4639.7	12.68	Unknown
12.	0.80	117.4	0.82	149.1	12.72	0.84	37.0	4539.4	12.40	Unknown
13.	0.84	137.6	0.84	140.4	11.98	0.87	78.0	2858.8	7.81	Unknown
14.	0.96	46.1	0.97	50.0	4.27	0.99	20.0	1187.0	3.24	Unknown

Table 5: HPTLC peak table of hydro-alcoholic extract of plant

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	0.6	-0.01	613.5	64.52	0.05	8.5	11959.1	47.68	Unknown
2.	0.39	11.9	0.42	25.5	2.68	0.42	21.6	512.2	2.04	Unknown
3.	0.44	24.6	0.46	34.9	3.67	0.47	16.2	769.4	3.07	Q
4.	0.61	16.5	0.63	30.1	3.17	0.63	25.4	515.2	2.05	Unknown
5.	0.65	32.7	0.66	43.2	4.54	0.69	24.5	1558.9	6.22	Unknown
6.	0.71	24.6	0.74	40.0	4.20	0.74	37.4	867.3	3.46	Unknown
7.	0.77	47.9	0.86	110.0	11.56	0.89	9.4	7556.1	30.13	Unknown
8.	0.92	11.5	0.94	22.7	2.39	0.95	15.7	423.4	1.69	Unknown
9.	0.95	18.3	0.98	31.0 AU	3.26	0.99 Rf	4.9 AU	918.7	3.66	Unknown

Quantification of quercetin and gallic acid along with presence of numbers of bioactive compounds in the methanolic extract of P. julliflora was determined as depicted in figures and tables. Quercetin and gallic acid were detected in different percentage in test sample I (extracted with n-hexane) and sample II (extracted with hydro-alcohol). The chromatograms obtained from the reference quercetin (Fig. 3) and gallic acid (Fig. 5) was reported with their Rf values in track 1 and track 2. The number of peaks denotes the no. of bioactive compounds present on the samples. Standard quercetin chromatogram (Fig. 3) represents 8 peaks denotes impurities and presence of quercetin at Rf 0.50 (table. 2 or track 1). Standard gallic acid chromatogram (Fig. 4) represents 7 peaks defines various impurities and presence of gallic acid at Rf 0.29 (table. 3 or track 2).Chromatogram representing quercetin and gallic acid together (Fig. 7) showed their respective Rf as depicted in Fig. 3 and Fig. 4. Sample I chromatogram (Fig. 8) represent peaks resolving at Rf 0.49 (Track 3) and Rf 0.27 (Track 3) nearly superimposing with peaks respective to standard quercetin and gallic acid (Track 1 and Track 2) confirms presence of quercetin and gallic acid.

Sample II chromatogram (Fig. 9) represent peaks resolving at Rf 0.47 (Track 4) nearly superimposing with peak respective to standard quercetin (Track 1) confirms presence of quercetin only. The content of quercetin and gallic acid in samples were calculated on the basis of peak area and peak height, was found to be 1.364% and 0.088% in sample I while in sample II only quercetin was present and found 0.27%. The finding of HPTLC analysis reveals the presence of various bioactive compounds along with percentage of Quercetin and gallic acid in the test samples of Prosopis juliflora, However identification of compounds other than quercetin and gallic acid was not done.

CONCLUSION:

The present study revealed the presence of quercetin and gallic acid along with several other phytochemicals whose importance might be beneficial and relevant towards future therapeutic application of Prosopis juliflora for curing various ailments.

ACKNOWLEDGEMENTS:

The author is thankful to National botanical research institute (NBRI), Lucknow (UP) for their technical facilities and assistance.

COMPETING INTEREST:

Authors declare no competing interest at all.

REFERENCES:

1. Patil J. J KI. Kumar K. KG K. Prosopis juliflora. Res. J. Pharmacology and Pharmacodynamics. 2016; 8(4): 175-180. doi:10.5958/2321-5836.2016.00032.X

2. Mary Rose DL. Arockianathan PM. Lawrance A. Felicita AS. In-Vitro Study of Anti-diabetic, Anti-cancer and Anti-urolithiatic Activity of Different Extracts of Prosopis Juliflora Seeds. International Journal of Pharmaceutical Sciences and Research. 2021; 13(5): 2101-08. doi: 10.13040/IJPSR. 0975-8232.13(5).2101-08

3. Hassan SM. Taha AM. Eldahshan OA. Sayed AA. Salem AM. Modulatory effect of Prosopis juliflora leaves on hepatic fibrogenic and fibrolytic alterations induced in rats by thioacetamide. Biomedicine and Pharmacotherapy. 2019; 115: 108788. doi: 10.1016/j.biopha.2019.108788

4. Tajbaksh S. Barmak A. Vakhshiteh F. Gharibi M. Invitro Antibacterial Activity of the Prosopis Juliflora Seed Pods on Some Common Pathogens. J Clin Diagn Res. 2015; 9(8): 13-15. doi: 10.7860/JCDR/2015/13549.6370

5. Rad SJ. Kobarfard F. Martins N. Prosopis plant chemical composition and pharmacological attributes: Targeting clinical studies from preclinical evidence. Biomolecules. 2019; 9(12): 777. doi: 10.3390/biom9120777

6. Sagar K. Shivashankar PN. Amarappa S. Phytochemical Studies and Quantification of Total Phenols,Tannins and Flavanoids in Less Known Semi-arid Plants of Ballari, Karnataka. International Journal of Pharmaceutical Sciences and Research. 2021; 13(5): 2182-87. doi: 10.13040/IJPSR.0975-8232.13(5).2182-87

7. Ukande DM. Shaikh S. Murthy KS. A Review on pharmacological potentials of Prosopis juliflora. Journal of Drug Delivery and Therapeutics. 2019; 9(4-s): 755-760. doi: https://doi.org/10.22270/jddt.v9i4-s.3372

8. Mehari ZH. The invasion of Prosopis juliflora and Afar pastoral livelihoods in the Middle Awash area of Ethiopia. Ecol Process. 2015; 13(4): 0039-8. doi:https://doi.org/10.1186/s13717-015-0039-8

9. Khandelwal P. Sharma RA. Agarwal M. Isolation and identification of steroids from different parts of Prosopis juliflora. Int. J. Curr. Pharm. Res. 2017; 9(3): 54-57. doi: http://dx.doi.org/10.22159/ijcpr.2017v9i3.18896

10. Tadros MJ, Al-Assaf A, Othman YA, Makhamreh Z, Taifour H. Evaluating the Effect of Prosopis juliflora, an Alien Invasive Species, on Land Cover Change Using Remote Sensing Approach. Sustainability. 2020; 12(15): 5887. https://doi.org/10.3390/su12155887

11. Jain V. Jain T. Saraf S. Saraf S. HPTLC method for routine quality control of Ayurvedic formulation Drakshadi gutika. Asian J. Pharm. Ana. 2013; 3(4): 111-114. https://ajpaonline.com/AbstractView.aspx?PID=2013-3-4-1

12. Krishna K.V.V.S, Sankar KG, Vardhan MS, Krishna SR, Tamizhamani T. Simultaneous Estimation of Gallic acid and Rutin in Kaishore guggulu and Vatari guggulu by HPTLC. Res. J. Pharmacognosy and Phytochem. 2018; 10(3): 221-225. doi-10.5958/0975-4385.2018.00036.5

13. Prajapati LM. Patel A. Patel JR. Joshi AK. Kharodiya M. High-performance Thin Layer Chromatography method for simultaneous estimation of cafepime hydrochloride and sulbactum sodium. Asian J. Pharm. Ana. 2016; 6(4): 207-212. doi:10.5958/2231-5675.2016.00031.4

14. Jain V, Jain T, Saraf S. HPTLC method for routine quality control of ayurvedic formulation drakshadi gutka. Asian J. Pharm Ana. 2013; 3(4): 111-114. doi: 10.1016/j.jaim.2017.07.006

15. Goswami S. Singh RP. Gilhotra RM. HPTLC Fingerprinting of Quercetin and comparative assessment of Anthelmintic potency against Eudrilus eugeniae of Schleichera oleosa (Lour) Oken and Tagetes eretca Linn. Research J. Pharm. and Tech. 2020; 13(12): 5699-5704. doi: 10.5958/0974-360X.2020.00992.0

16. Ramasamy Aiyalu Rajasekaran. Detection and Quantification of anti-oxidant markers like rutin, catechin, quercetin, gallic acid, ellagic acid, ferulic acid, vitexin and mangiferin in herbal raw materials available in the market belong to rutaceae family by HPTLC technique. Asian Journal of Pharmaceutical Analysis. 2021; 11(2): 151-5. doi: 10.52711/2231-5675.2021.00026

17. Nikam PH. Kareparamban J. Jadhav A. Kadam V. Future Trends in Standardization of Herbal Drugs. Journal of Applied Pharmaceutical Science. 2012;2(6). doi:10.7324/JAPS.2012.2631

18. Singh V. Rizvi A. Sara UV. Standardization and Phytochemical Screening of Carica papaya Seeds. Res. J. Pharm. and Tech. 2021; 14(9): 4540-6. doi:10.52711/0974-360X.2021.00790

19. Sasikala M. Mohan S. Nandha K. Kumar Sathish. Varshini N. HPTLC and Antioxidant activity of Ethanolic Extract of Ipomoea obscura (L.) Ker Gawl Stem. Research J. Pharm. and Tech. 2020; 13(10): 4661-4667. doi:10.5958/0974-360X.2020.00820.3

20. Rode VP, Tajne MR. A Validated Stability-Indicating High-Performance Thin-Layer Chromatographic Method for the Analysis of Pitvastatin in Bulk Drug and Tablet Formulation. Asian J. Pharm. Ana. 2018; 8(1): 49-52. doi-10.5958/2231-5675.2018.0009.1

Received on 12.05.2023 Modified on 13.08.2023

Research J. Pharm. and Tech 2024; 17(8):3801-3806.

DOI: 10.52711/0974-360X.2024.00590