INTRODUCTION:

The management of poor health through medications has entered an era of rapid growth. The plant kingdom yet holds distinct unrevealed species, containing phytoconstituents of pharmacological and nutraceutical value that have yet to be revealed. Extraction of active constituents and production of drug formulations is a futuristic technology and asset intensive with attractive consideration¹. Custard apple (Annona squamosa L.) is an important tropical fruit which is cultivated in many countries like West Indies, Ecuador, Southern and central part of America Brazi, Peru, Mexico, India Bahamas Egypt and Bermuda and the fruit belongs to the family Annonaceae.

Leaves of custard apple plants have been studied for their health benefits, which are attributed to a considerable diversity of chemical constituents. These compounds include phenol-based compounds, e.g., proanthocyanidins, comprising 18 different phenolic compounds, mainly alkaloids and flavonoids². Extracts from Annona squamosa leaves (ASLs) have been studied for their therapeutic activities, like anticancer, antidiabetic, antioxidant, antimicrobial, antiobesity, lipid-lowering, and hepatoprotective functions^1,
2. The leaves are traditionally used as a vermicide, for treating cancerous tumors, also applied to abscesses, insect bites and other skin complaints. To overcome the hysteria and fainting spells the crushed leaves were sniffed and they were also applied on wounds and ulcers.The leaf decoction is also employed in baths to alleviate rheumatic pain¹.

The ethanolic extract Annona squamosa leafs contain tannins, alkaloids, flavonoids, phenols, and carbohydrates. A Total of seven individual phenolic compounds, viz., gallic acid, chlorogenic acid, salicylic acid, ferulic acid, caffeic Acid, cinnamic acid, and quercetin, responsible for antioxidant and antidiabetic activities, were quantified from the methanolic leaf extract of custard apple⁴. Five main flavonoids with two pairs of epimers, including quercetin-3-O-robinobioside, rutin, quercetin-3-O-β-D-glucoside, kaempferol-3-O-robinobioside, and kaempferol-3-O-rutinoside, were successfully separated by high-speed counter-current chromatography⁵. Among all phytoconstituents, analysis mainly focuses on gallic acid and quercetin. Both phytoconstituents are important active principles that have been used for quality control and standardization of plants and their formulation as a marker compound.

Qualitative and quantitative analysis of a mixture of phytochemicals is one of the biggest challenges faced by a researcher. Although the availability of advanced techniques such as ¹³C NMR, ¹H NMR, mass spectrometry, capillary electrophoresis, and HPLC has helped to make deep inroads into the accurate determination of the complex structure and molecular weight of phytochemicals, there still remain numerous scarcity. Apart from the outrageous cost of these sophisticated techniques, there is also an imperative for high analytical skills for sample and data handling that together make them unsuitable as techniques of choice for routine analysis.

Considering the widespread use of plant based products for their therapeutic and preventive action in disease and health, we have deemed it essential to standardize them and confirm their quality to ensure they are appropriate for public utilization. For routine qualitative and quantitative assessment and standardization of phytochemical‑based products, high‑performance thin‑layer chromatography (HPTLC) has emerged as a cheap, fast, reproducible, economic, simple, and accurate technique that is finding wide acceptance⁶.

A comprehensive survey of the literature reveals that researchers have not reported any analytical methods for the simultaneous estimation of gallic acid and Quercetin in Annoa squamous leaf plant extract. Therefore, the aim of the present study was to develop a simple, inexpensive, selective, specific, reproducible and robust high-performance thin-layer chromatography (HPTLC) method for the estimation of gallic acid and Quercetin in plant extracts. Using the International Conference on Harmonization (ICH) guidelinesthe proposed method was validated.

MATERIALS AND METHODS:

Materials:

Fresh leaves of A. squamosa, was collected from Chondhe farm, Pune district (Maharashtra) during October month. The plant was authenticated by Agharkar Research Institute, Department of Science and Technology, Govt. of India, Pune, with report no (AUTH 22-88). The leaves of the plant were shade dried, coarsely powdered, and passed through 40 mesh sieves. The powdered material is stored in an airtight container for future use. All the utilized chemicals were acquired from Merck Ltd. (Darmstadt, Germany) and were of analytical grade. Gallic Acid (CAS 149-91-7; purity >98% w/w) and Quercetin (CAS 117-39-5; purity >98% w/w) was purchased from YUCCA Chemicals (Mumbai, India). Merck aluminium-backed silica gel TLC plates coated with fluorescent indicator F₂₅₄ were used for the study.

Extraction:

Around an amount of 10g of air-dried leaf sample was accurately weighed and taken in a 500ml conical flask with 120mL of ethanol and 80ml water (hydroalcoholic extract) for around 24h, shake frequently for up to 6hr, keep it aside for 18hr .Using a rotary evaporator the subsequent solutions were filtered and concentrated.

Preparation of sample and standard solution:

A 1000μg/mL solution of plant extract was prepared in water: alcohol (40:60), vortexed, and sonicated for 20 min at room temperature to prepare the stock as a sample solution. The solutions were filtered through a sterile membrane filter (0.45µm, Millipore, USA) before application on TLC plate. A 100μg/mL stock solution of Quercetin and gallic acid reference standard were prepared the same as a sample solution.

Instrumentation and Chromatography condition:

A CAMAG (Muttenz, Switzerland) HPTLC system outfitted with a specimen tool Linomat V with a CAMAG test syringe 100μL, twin-trough plate development chamber (20 × 10cm, 10 x 10cm), TLC Scanner 3 and computer programming winCATS 1.4.3, relative humidity 40%, HPTLC plates 20 × 10cm TLC plate, precoated with silica gel 60 F₂₅₄; 0.2mm thickness (Merck, Darmstadt, Germany), Experimental condition temperature 25±2^OC, Solvent system Toluene: Ethyl acetate : Formic Acid (5.4:3.8: 0.8 v/v), Scanner speed 10mm/s, Detection wavelength 280 nm for gallic acid and 366 nm for Quercetin, Rate of spotting 0.1μL/s, Band width 6mm, Distance between adjacent bands- 4mm., source of radiation Deuterium lamp was used for analysis. Sample solutions, standard solutions of Quercetin and Gallic acid were used for the simultaneous quantification of Quercetin and Gallic acid in plant extract.

Preparation of calibration curve and simultaneous quantification (Estimation) of Quercetin and Gallic acid in plant extract:

The different volumes of standard stock solution 2, 4, 6, 8, 10 and 12μL were spotted on HPTLC plate (10 × 10 cm) both for Quercetin and Gallic acid followed by spotting of 10μL of sample stock solutions (plant extract) in triplicate. Horizontal elution of 20 min was followed in the chromatography process. The plates were dried and observed in CAMAG TLC Visualiser at 254nm, after the development. The developed plate was then scanned.

Quercetin: Molecular Formula=C₁₅H₁₀O₇

Gallic Acid: Molecular Formula=C₇H₆O₅

Method validation:

The method was validated as per the ICH guidelines and earlier cited literature.^7-10

Linearity and range:

Different volumes (2–12μl) of standard solutions of both markers were applied to the HPTLC plate to obtain a concentration range of 200–1200ng/band Gallic acid and quercetin in five replicate measurements were conducted. The measured peak areas versus the corresponding concentrations of both drugs were evaluated with an ordinary linear regression analysis.

Instrumental precision (TLC densitometer scanner):

The instrumental precision of the TLC densitometer scanner was checked by repeatedly scanning the same spot of Quercetin (0.6μg/spot) and Gallic acid (0.6 μg/spot), respectively, and was communicated as the percent relative standard deviation (%RSD).

Repeatability:

The %RSD was communicated after analyzing six spots on same TLC plates and finding the repeatability of the strategy with Quercetin at 0.6 μg/spot and Gallic acid at 0.6 μg/spot.

Precision (Interday and intraday):

The amounts of Quercetin and Gallic acid, represented by 0.4, 0.6 and 0.8 , were analyzed to assess their variability, each being applied as μg/spot for two consecutive hours (intraday precision) and the %RSDs were communicated. As the results of the interday precision study conducted on three different days using the specified concentrations of both markers.

Sensitivity (Limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were calculated based on the standard deviation of the response and slope of the calibration curve of markers using the formula set forth in the ICH guidelines:

limit of detection = 3.3 × σ/S

limit of quantification = 10 × σ/S

Where “σ” is the standard deviation of the y intercepts of the regression lines, and

“S” is the slope of the calibration curve.

Specificity:

By analyzing standard compounds and samples the specificity was ascertained. The bands for Quercetin and Gallic acid from sample solution were confirmed by comparing the R_f and spectra of the bands to those of the standard. By comparing the ultraviolet (UV) spectra of the sample and standard Quercetin and Gallic acid the peak purity of the compound was examined.

Robustness:

The evaluation of influence of small but deliberate variations in chromatographic conditions on Robustness was carried out for the determination of Quercetin and Gallic acid. By changing the mobile phase composition, mobile phase volume, duration of mobile phase saturation and scan wavelength Robustness of the method was determined.

Accuracy:

The accuracy of the method was measured by performing recovery experiments at three different levels (80%, 100% and 120% addition of Gallic acid and Quercetin, respectively) utilizing the standard addition method. The known measures of Gallic acid and Quercetin standards (0.5 and 0.5 μg/spot, respectively) were added by spiking. The recovery percentages and average recovery percentages for Quercetin and Gallic acid were calculated.

System suitability:

We determined system suitability by applying freshly prepared standard solutions of both Quercitin and Gallic acid with a concentration of 0.6 μg/spot, 6 times under the same chromatographic conditions. Following this, we scanned and recorded the densitograms.

RESULT:

Method development:

Optimization of a suitable mobile phase plays an important role in chromatographic method development. As far as the individual estimation of gallic acid and Quercetin by chromatographic method, is concerned, several solvent systems have been reported. However in a single solvent system there has not been cited a single report for the separation of these compounds simultaneously. Therefore, in this study, several solvent systems used for the simultaneous estimation of these phenolics and flavonoids were investigated to evaluate the combinatorial separation of these compounds in a single solvent system and between different components of the extract. Among the different solvents systems investigated, the mobile phase consisting of toluene-ethyl acetate-formic acid in the ratio of 5.4:3.8:0.8 v/v demonstrated compact spots with a good resolution between other peaks of the extract. The developed HPTLC method resolved the standard compound at R_f value of nearly about 0.45 for Quercetin and 0.28 for Gallic Acid confirming the presence of quercetin and gallic acid in plant extract by visualizing the band parallel to standards (quercetin and Gallic acid) spot along with other resolved components in the developed TLC plate.

Selection of wavelength:

The use of an appropriate wavelength determines the sensitivity of the HPTLC method with ultraviolet detection. The developed plate was subjected to densitometric measurements in scanning mode in the UV–visible region of 200–700nm, and the overlapped spectrum was recorded on a CAMAG TLC Scanner 1.4.3. Both markers Gallic acid and Quercetin gives maximum peak area appreciably absorbed the light at 280 and 366 nm respectively.

(a)

Figure: 1: Peak for Gallic acid and Quercetin at (a) 280 and (b) 366nm

Both marker shows the peak at both wavelength but maximum peak area for gallic acid at 280nm in (Figure 1 (a)) and for quercetin at 366nm as shown in (Figure 1(b))

Calibration curve:

The calibration curve was linear in the range of 0.2–1.2 μg/spot for Quercetin and Gallic Acid. The plot confirmed the good linear relationship for the Linear regression data. Calibration equations and coefficients of correlation are presented (Table No. 1and Fig. 2) The LOD and LOQ values indicate the adequate sensitivity of the method together. The linear regression of the Gallic Acid standard curve was determined with R²± SD = 0.979±49.91 with the regression line; y = 10908x+ 1520. The regression curve of Quercetin was determined with R² ±SD = 0.999±35.98 with the regression line; y = 9850x - 1049.

The markers Gallic acid and Quercetin were estimated in leaf hydroalcoholic extract using regression equations from the calibration plot and expressed as µg/mg of hydroalcoholic extract. [Table 1].

Table 1: Linearity data of developed HPTLC method for simultaneous separation of quercetin and gallic acid

Compound	Gallic acid	Quercetin
Solvent system	toluene-ethyl acetate-formic acid (5.4:3.8: 0.8 v/v)
Linearity (ng/spot)	200-1200
Equation	10908x + 1520	9850.x - 1049
Regression (r²)±SD	0.979±0.015	0.999±0.0010
Slop±SD	10908 ± 49.91	9850 ± 35.98
Intercept±SD	1520±2.75	-1049±1.89
LOD (ng/spot)	15	12
LOQ (ng/spot)	45	36
R_f Value	0.28±0.05	0.45±0.05
Scanning (nm)	280	366
% RSD	0.6738	0.8966
Hydroalcoholic leaf extract (µg/mg)	22.30	48.01

Method validation:

Linearity:

The ability to provide the results directly or via a mathematical transformation is the linearity of an analytical method, which is proportional to the concentration of the analyte within a given range. Gallic Acid and Quercetin show a good correlation coefficient (R² = 0.979 for Gallic Acid and R² = 0.999 for Quercetin) in the proposed concentration ranges of 200–1200ng/band for both markers (Table No. 1). Excellent new linearship was shown by the linear regression data for calibration curves (n = 3).

LOD and LOQ:

The limits of detection and limit of quantification were determined for gallic acid and quercetin, respectively. The LOD was found to be 15ng/spot and 45ng/spot, while the LOQ was 12ng/spot and 36ng/spot. (Table No. 1) indicating the sensitivity of the proposed method.

Instrumental precision (TLC densitometer scanner):

The percent relative standard deviation (%RSD) was communicated as the measure of instrumental precision for the TLC densitometer scanner, determined by scanning the same spot six times (Table 2).

Repeatability:

The strategy's repeatability was confirmed by analyzing the 0.6μg/spot of quercetin and gallic acid respectively. On the TLC plate (n = 6) and communicated as % RSD. (Table 2).

Table 2: instrumental Precision and Repeatability of the developed HPTLC method for the simultaneous determination of quercetin and gallic acid (n=6)

Con. (ng/spot)	Instrumental Precision (6 times scan of same spot)		Repeatability (6 times same con. spotting)
	Mean peak area±SD	%RSD	Mean peak area±SD	%RSD
Gallic acid
600	4658.5±10.2	0.55	4510.13± 2.4	0.55
	4653.5±16.8	0.38	4532.98±1.7	0.38
	4663.45±4.7	0.51	4539.43±2.3	0.51
	4658±10.8	0.96	4528.5±4.3	0.96
	4654±21.93	0.66	4516.5±3.0	0.66
	4662.16±6.9	0.70	4529.3± 3.2	0.70
Quercetin
600	1408±10.2	0.22	1465.5±10.2	0.22
	1455.2±16.8	0.36	1463.5±16.8	0.36
	1442.3±4.7	0.10	1466.45±4.7	0.10
	1489.2±10.8	0.23	1465.2±10.8	0.23
	1450.9±21.93	0.47	1454±21.93	0.47
	1492.8±6.9	0.14	1462.16±6.9	0.14

Precision:

The precision of the method was confirmed with intra-day and inter-day relative standard deviations (RSD) being less than 2% (Table 3).

Table 3: Precision of the developed HPTLC method for the simultaneous determination of Quercetin and Gallic acid(n=6)

Concentration (ng/spot)	Interday		Intraday
	Mean peak area±SD	% RSD	Mean peak area±SD	% RSD
Gallic acid
400	3355.9±10.63	0.7812	2086.1±27.91	0.87
600	4830.2±11.24	0.1396	4531.7±13.83	0.75
800	5949.1±10.17	0.3128	5569.4±12.83	0.85
Quercetin
400	897.8±3.87	0.8024	798.9±5.17	0.62
600	1649.8±16.40	0.7625	1364.6±4.39	0.73
800	2402.9±5.58	0.9025	1922.9±3.02	0.87

Specificity:

By analyzing the standard compounds and samples the specificity was ascertained. Comparing the ultraviolet (UV) sprectra of the sample and standard quercetin and gallic acid the peak purity of the compound was examined as well as by comparing the overlay spectra at the peak start, the peak apex (middle) and the peak End positions of the band and good correlation of Quercetin r (S,M) was 0.984916 and r (M,E) was 0.998562 and Gallic acid r (S,M) was 0.996005 and r (M,E) was 0.998562 with the sample was recorded.

Robustness:

The method demonstrated robustness with relative standard deviations of peak areas being less than 2% after deliberate changes in various parameters (Table 4). The peak area and R_f value were slightly affected by the change in parameter..

Table 4: Robustness study of developed HPTLC method.

Changing Volume of Mobile Phase. (10 ± 2 ml)
Marker	Mobile phase Volume	R_f	Area ± SD (ng/band)	% RSD
Gallic acid	12 ml	0.40	4292.59 ± 35.53	0.82
Gallic acid	8 ml	0.32	4257.19 ± 37.83	0.81
Quercetin	12 ml	0.55	1294.50 ± 12.06	0.96
Quercetin	8 ml	0.47	974.43 ± 8.25	0.92
Changing Scanning Wavelength (± 2 nm)
Marker	Scanning wavelength	R_f	Area ± SD (ng/band)	% RSD
Gallic acid	282	0.38	6473.98 ± 42.19	0.59
Gallic acid	278	0.38	6492.38 ± 43.29	0.65
Quercetin	368	0.52	2543.03 ± 23.58	0.92
Quercetin	364	0.53	3509.52 ± 29.42	0.87
Changing Saturation Time (± 5 min)
Marker	Time (in minute)	R_f	Area ± SD (ng/band)	% RSD
Gallic acid	20 min	0.28	2404.25 ± 14.51	0.63
Gallic acid	10 min	0.30	3989.24 ± 35.11	0.88
Quercetin	20 min	0.45	1824.57 ± 16.2	0.86
Quercetin	10 min	0.49	1564.41 ± 14.23	0.92
Changing Volume of Mobile ratio. (± 0.2 ml)
Marker	Mobile phase ratio	R_f	Area ± SD (ng/band)	% RSD
Gallic acid	5.6 : 4 : 1	0.32	3634.88 ± 28.70	0.82
Gallic acid	5.2 : 3.6 : 0.6	0.27	5617.18 ± 36.70	0.63
Quercetin	5.6 : 4 : 1	0.46	1099.51 ± 12.15	0.99
Quercetin	5.2 : 3.6 : 0.6	0.43	1590.15 ± 5.72	0.96

Accuracy:

When used to evaluate the recovery after spiking with three concentrations of standard , 80%, 100% and 120%,the proposed method showed percentage recovery rates between 100.89–101.72 for Quercetin and 100.51–101.27 for Gallic acid, which were within the acceptable range of 100 ± 2% . Which indicated that the developed method was accurate and satisfactory.

Table 5: Accuracy data of developed HPTLC method for simultaneous determination of quercetin and gallic acid

Marker	Amount added (%)	Amount recovered (%)	SD	% RSD
Gallic acid	80	101.26 ± 0.351	1.18	1.21
	100	100.51 ± 0.127	0.98	0.94
	120	101.27 ± 0.427	1.10	1.05
Quercetin	80	101.72 ± 0.283	0.92	0.90
	100	100.89 ± 1.219	0.89	0.84
	120	101.64 ± 0.196	1.10	1.02

DISCUSSION AND CONCLUSION:

Different constituents of plant extracts and fractions are successfully separated and provided with useful qualitative and quantitative data through the consistent use of HPTLC that are reliable, accurate, and economic for various applications including quality control and standardization of food and marketed herbal formulations ^11,12. Flavonoids are the present day panacea as they are established to exert protective action in oxidative stress‑related pathologies, such as cardiovascular diseases, cancer, and a variety of neurodegenerative disorders¹³. So higher constituents proportion of flavonoid and phenolic compound give preventive action against various disorder. The content of quercetin and Gallic acid in plant was calculated on the basis of peak area was found to be 0.023% and 0.048 % (w/w) respectively. These biomarkers have different therapeutic potentials and their identification and quantification in Annona Squamosa hydroalcoholic leaf extract can also increase the industrial value of this species, which may boost its farming and may create a quality mercantile option for our farmers.

REFERENCES:

1. Zahid M. Mujahid M. Singh PK. Farooqui S. Singh K. Parveen S. Arif M. Annona squamosa Linn. (custard apple): an aromatic medicinal plant fruit with immense nutraceutical and therapeutic potentials. International Journal of Pharmceutical Science and Research. 2018; 9(5): 1745-59. doi:10.13040/IJPSR. 0975-8232.9(5).1745-59..

2. Kumar M. Changan S. Tomar M. Prajapati U. Saurabh V. Hasan M. Sasi M. Maheshwari C. Singh S. Dhumal S. et al. Custard Apple (Annona squamosa L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health-Promoting Biological Activities. Biomolecules. 2021; 11: 614. https://doi.org/10.3390/biom11050614.

3. Madhuri Ashokrao Theng. Dr. Channawar Madhuri A. Dr. Kochar NI. Dr. Mohale DS. Dr. Chandewar AV. Phytochemical Investigation and HPTLC Fingerprinting of Annona Squamosa, Aegle Marmelos Leaves. European Chemical Bulletin. 2023; 12(1): 859-865. DOI: 10.31838/ecb/2023.12.s1.092

4. Yashwant Kumar, Chandra Ajay Kumar. Shruti and Gajera HP. Evaluation of antidiabetic and antioxidant potential of custard apple (Annona squamosa) Leaf extracts: A compositional study. International Journal of Chemical Studies. 2019; 7(2): 889-895.

5. Heng Zhu. Long Chen. Jinqian Yu. Li Cui. Iftikhar Ali. Xiangyun Song. Jeong Hill Park. Daijie Wang and Xiao Wang. Flavonoid epimers from custard apple leaves, a rapid screening and separation by HSCCC and their antioxidant and hypoglycaemic activities evaluation. Scientific Report. 2020; 10: 8819. doi: 10.1038/s41598-020-65769-5

6. Jayachandran Nair CV. Ahamad S. Khan W. Anjum V. Mathur R. Development and validation of high-performance thin-layer chromatography method for simultaneous determination of polyphenolic compounds in medicinal plants. Phcognosy Research. 2017; 9:S67-73.DOI: 10.4103/pr.pr_122_16

7. Guideline IHT Validation of analytical procedures: text and methodology Q2 (R1), International Conference on Harmonization, Geneva, 2005

8. Saurabh Satija. Murtaza M. Tambuwala. Kavita Pabreja. Bakshi Hamid A. Chellappan Dinesh Kumar. Alaa A. Aljabali Srinivas Nammi. Thakur Gurjeet Singh. Harish Dureja. Gupta Gaurav. Kamal Dua. Mehta Meenu. Garg Munish. Development of a novel HPTLC fingerprint method for simultaneous estimation of berberine and rutin in medicinal plants and their pharmaceutical preparations followed by its application in antioxidant assay. JPC – Journal of Planar Chromatography – Modern TLC. 2020; 33: 313–319. https://doi.org/10.1007/s00764-020-00035-y

9. Kilambi Pundarikakshudu . Anil Kumar Sharma. Bhatt Chaitanya J. Kanaki Niranjan S. Development and Validation of a High-Performance Thin-Layer Chromatographic (HPTLC) Method for Simultaneous Quantification of Reserpine, Atropine, and Piperine in Sarpagandha Ghanvati, a Classical Ayurvedic Preparation. Journal of AOAC International. 2019;102(4):1021-1026. doi: 10.5740/jaoacint.18-0382.

10. Verma Shikhar. Dwivedi Jyotsana . Srivastava Richa. Prakash Deep. Simultaneous estimation of four different biomarkers in Cinnamomum verum J. Presl bark using a validated high-performance thin-layer chromatography method. Journal of Planar Chromatography - Modern TLC. 2021; 34(4). DOI:10.1007/s00764-021-00082-z

11. Heyden YV. Extracting information from chromatographic herbal fingerprints. LCGC Europe 2008; 21:438‑43.

12. Loescher CM. Morton DW. Razic S. Agatonovic‑ Kustrin S. High performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) for the qualitative and quantitative analysis of Calendula officinalis‑advantages and limitations. Journal of Pharmaceutical and Biomedical Analysis. 2014; 98:52‑9. DOI: 10.1016/j.jpba.2014.04.023

13. Suen J. Thomas J. Kranz A. Vun S. Miller M. Effect of flavonoids on oxidative stress and inflammation in adults at risk of cardiovascular disease: A systematic review. Healthcare (Basel) 2016; 4: 69. Doi: 10.3390/healthcare4030069.

Received on 15.06.2023 Modified on 01.11.2023

Research J. Pharm. and Tech. 2024; 17(3):1200-1206.

DOI: 10.52711/0974-360X.2024.00187