INTRODUCTION:

Traditional drugs from medicinal plants have key barrier that they do not have quality measures and documentation to produce their role in treatment of various diseases and this requires standardization of practices to assure their qualities and justify its safety and efficacy.^1-3 Neolamarckia cadamba (F: Rubiaceae) is a very important plant in the management of several diseases like eyes infections, skin diseases, stomach disorders, gum related problems, blood related issues and urinary disorders.⁴ The ethnomedicinal application and nutritive value of N. cadamba reveals that its bark have antioxidant, antigenotoxic and cytotoxic activity while its nutritious fruits have antioxidant, anthelmintic, membrane stabilizing and glucose level controlling properties.

The immune modulation activity of leaf extract utilized against cervical cancer also studied.^5-9 Apart from above studies ethanolic extracts of dried leaves of Neolamarckia cadamba is also used in inflammation, anti-microbial activity, anti-fungal activity, wound healing activities. However a very few plant chemicals like sapogenins: cadambagenic acid, quinovic acid, and beta-sitosterol from bark, alkaloids like cadambine, 3 alpha-dihydrocadambine and isohydrocadambine have been reported.^10-15

There are many techniques available for screening of plants products^16-19 in which HPTLC is one of the which is worldwide accepted and very fast and inexpensive.^20-24 Present study is designed to investigate the phytochemical profiling of aerial parts of the N. cadamba along with quantification of quercetin and gallic acid.

MATERIALS AND METHODS:

Collection, identification, and authentication of plant:

The aerial parts of Neolamarckia cadamba (Roxb.) Bosser syn. Anthocephalus cadamba (Roxb.) Miq. commonly known as kadamb was collected in the month of July 2022 from faizullaganj, ghaila road, Lucknow. The identification has been done on the basis of macroscopic studies of the sample followed by in-depth scrutiny of literature and identical the sample with authentic samples deposited in the RHMD (Raw Material Herbarium and Museum, Delhi). The plant authentication was done from CSIR-NIScPR under authentication no. NIScPR-RHMD/Consult/2022/4173-74-1.

Extraction:

The plant materials were washed, shed dried, powdered and subjected to successive extraction initially from n-hexane (69^oC). After completion of 10 cycles of continuous extraction thimble was removed and powder was air dried. Again this dried powder was extracted with hydroalcohol (1:1, v/v) till at least 10 cycles completed. To obtain dry product excess solvent evaporated by vacuum rotary evaporator. The dried extract was stored in air tight container till the experiments were carried out.

Preparation of standard solution:

0.5mg/ml of quercetin and gallic acid prepared with HPTLC grade methanol were prepared.

Preparation of plant samples:

Test samples were prepared by dissolving 10grams of dried extract into 1ml of HPTLC grade methanol and filtered with 0.22µm filter.

Instrumentation:

CAMAG TLC scanner III, winCATS software and LINOMAT 5 auto sprayer fitted with nitrogen cylinder and 100µl syringe IN CAMAG HPTLC system were used.

Chemicals and Solvents:

All chemicals and solvents were of analytical and chromatographic grade. Reference markers were gifted by the National Botanical Research Institute (NBRI), Lucknow.

Chromatographic Conditions:

No pre washing but manual modification of plate (E-Merck KGaA) (20x10cm) pre coated with 0.2mm silica gel was used. Activation of plate at 40^oC before analysis was done. Test samples were applied in 10µl and 20µl, 5mm above from bottom of the plate at constant rate 150nl/s. Mobile phase consisted of toluene: ethyl acetate: formic acid in 6:4:0.3 (v/v/v) were used in the developing chamber and after 5 minutes scanning at short 254nm and long 366nm wavelength through CAMAG scanner III was performed (Table 1).

UV active compounds present on TLC plate will appear as dark spot on a bright background due to fluorescence quenching at 254nm while at 366 nm appears as bright spot on a dark background¹¹.

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

Calibration curve of quercetin and gallic acid:

Standard concentration ranging from 2 to 10µg/spot prepared from 0.5mg/ml stock solution applied in 4µl, 8µl, 12µl, 16µl, 20µl on HPTLC plate to obtained standards 2µg, 4µg, 6µg, 8µg, 10µg/spot respectively. Calibration curves were obtained by plotting absorbance unit against concentration of standards (Fig. 6 and Fig. 7).

RESULTS AND DISCUSSION:

HPTLC chromatograms of test samples reveal that the phytochemical profiling and quantification of quercetin and gallic acid as illustrated in the figures and tables below. The chromatograms were obtained at short (254nm) (Fig. 1) and long (366nm) UV wavelength (Fig. 2). The Rf values, peak area, peak height and percentage area of unknown bioactive compounds depicted in tables 2, 3, 4, 5. Images of the TLC plate were taken at 254nm and 366nm represents their tracks for standard quercetin (track 1), gallic acid (track 2) and test samples (track 3 and track 4).

The HPTLC chromatogram obtained by standard quercetin (fig. 3) and gallic acid (fig. 4) showing their Rf values for quercetin at Rf 0.50 in track 1 and gallic acid at Rf 0.29 in track 2.

Chromatograms representing standard quercetin and gallic acid showed more than one peak representing that there were few impurities along with standards.

There were two test samples (sample 1 and sample 2). Sample 1 was initially extracted with n-hexane while sample 2 with hydro-alcoholic solvent. Calibration curve of standard quercetin and gallic acid was shown in fig. 6 and fig. 7 respectively. Chromatogram of sample 1 and sample 2 represents 16 peaks and 10 peaks respectively representing number of bioactive compounds bioactive compounds. The number of peaks denotes the number of bioactive compounds in the samples.

Sample 1 and sample 2 chromatogram represents peak at Rf 0.52 (track 3 and track 4) when compared to standard quercetin chromatogram quercetin peak Rf 0.50 ( track 1) was found that quercetin was present in sample 1 and sample 2 as there were approximately similar Rf values (0.50) of the compound when compared to standard quercetin Rf value (0.50).

Chromatogram of sample 1 and sample 2 when compared with standard gallic acid reveals that no Rf value of standard gallic acid is matching with sample Rf value denoting absence of gallic acid in both samples. Thus study concludes quercetin was detected in sample 1 and sample 2 while gallic acid was not detected in both samples. Quantification was done by using evaluation parameters peak area and peak height and quercetin was found to be 1.29% in sample 1 and 0.77% in sample 2.

Fig 1: Image of TLC Plate at 254 nm

Fig 2: Image of TLC Plate at 366 nm

Fig. 3: HPTLC Chromatogram of standard quercetin

Fig. 4: HPTLC Chromatogram of standard gallic acid

Fig. 5: HPTLC Chromatogram of standard Q and GA

Fig. 6: Calibration curve of quercetin

Fig. 7: Calibration curve of GA

Fig. 8: HPTLC Chromatogram of sample 1

Table 2 or Track 1: 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 or Track 2: 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 or Track 3: HPTLC Peak table of sample 1

Table 4, Track 3 ID: HPTLC Peak table of 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	1.0	-0.01	131.5	7.51	0.01	27.8	1725.6	2.83	Unknown
2.	0.01	28.1	0.01	30.0	1.71	0.03	10.3	579.4	0.95	Unknown
3.	0.03	10.5	0.06	41.8	2.39	0.08	1.3	816.4	1.34	Unknown
4.	0.09	1.3	0.12	43.0	2.46	0.16	21.7	1604.5	2.63	Unknown
5.	0.22	4.7	0.23	13.8	0.79	0.24	5.4	199.1	0.33	Unknown
6.	0.53	8.1	0.36	18.5	1.06	0.36	16.0	362.2	0.59	Unknown
7.	0.37	15.7	0.43	90.6	5.18	0.43	37.1	3118.3	5.11	Unknown
8.	0.44	87.4	0.45	93.6	5.34	0.46	32.4	1956.3	3.20	Unknown
9.	0.47	90.3	0.50	182.8	10.44	0.52	35.3	5428.7	6.89	Q
10.	0.52	65.4	0.53	87.0	4.97	0.55	36.0	1939.8	3.26	Unknown
11.	0.55	67.1	0.56	77.9	4.45	0.58	56.7	1969.2	3.23	Unknown
12.	0.59	55.9	0.67	111.3	6.35	0.71	34.5	9224.2	15.11	Unknown
13.	0.71	64.7	0.76	343.1	19.60	0.79	47.0	14704.3	24.09	Unknown
14.	0.79	147.4	0.80	161.9	9.24	0.81	55.6	3284.2	5.38	Unknown
15.	0.81	156.2	0.84	288.2	16.46	0.88	28.1	13142.3	21.53	Unknown
16.	0.95	30.3	0.97	36.2	2.07	0.98	30.3	937.4	1.54	Unknown

Table 5 or Track 4 ID: HPTLC Peak table of sample 2

Table 5, Track 4 ID: HPTLC Peak table of 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	5.2	-0.01	695.8	69.36	0.03	43.2	13623.1	50.87	Unknown
2.	0.03	44.1	0.04	67.1	6.69	0.07	5.4	1330.4	4.97	GA
3.	0.38	6.4	0.41	33.1	3.30	0.42	22.7	745.4	2.78	Unknown
4.	0.47	15.1	0.49	57.3	5.71	0.52	16.9	1817.2	6.79	Unknown
5.	0.62	27.2	0.66	74.5	7.42	0.70	17.3	3219.2	12.02	Unknown
6.	0.76	28.3	0.85	75.4	7.51	0.88	3.9	8046.6	22.58	Unknown

CONCLUSIONS:

This study reveals phytochemical profiling of Neolamarckia cadamba along with quantification of quercetin and gallic acid to set its therapeutic role for further studies in various diseases.

ACKNOWLEDGEMENTS:

Author is thankful to NBRI, Lucknow for assistance in research work.

CONFLICT OF INTEREST:

Authors declare no competing interest at all.

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Received on 25.03.2023 Modified on 03.07.2023

Research J. Pharm. and Tech 2024; 17(1):271-276.

DOI: 10.52711/0974-360X.2024.00042