JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70

https://doi.org/10.1007/s00764-023-00221-8

ORIGINAL RESEARCH PAPER

Analytical method development and validation for the simultaneous

estimation of quercetin, berberine, rutin and curcumin in a polyherbal
formulation using high‑performance thin‑layer chromatography
Akshada Khairnar1 · Sathiyanarayanan Lohidasan2 · Rahul Dubey2 · Sandeep Sankaran1

Received: 7 October 2022 / Accepted: 30 December 2022 / Published online: 28 January 2023
© Akadémiai Kiadó, Budapest, Hungary 2023

Abstract
People all over the world are affected by the chronic, disabling autoimmune illness known as rheumatoid arthritis (RA).
Herbal products are more commonly used in treating RA as of their safety and efficacy. The selected formulation for the
present study is ANTARTH capsules, which is a polyherbal formulation containing different herbs effective in the treat-
ment of RA. The purpose of this study was to develop and validate a precise, accurate, novel high-performance thin-layer
chromatography (HPTLC) method for the simultaneous estimation of quercetin, berberine, rutin, and curcumin and to deter-
mine the levels in a polyherbal formulation. A good chromatographic separation was accomplished using a mobile phase
consisting of toluene‒ethyl acetate‒methanol‒formic acid (5:3:2:0.5, V/V ) with wavelengths of 366 nm and 426 nm using
an ultraviolet‒visible (UV‒VIS) detector. The retention factors for quercetin, berberine, rutin, and curcumin were found
to be 0.57, 0.316, 0.093, and 0.663, respectively. A good linear regression relationship between peak areas and concentra-
tions was obtained with correlation coefficients of 0.9988, 0.9987, 0.9977, and 0.9984 for quercetin, rutin, berberine, and
curcumin, respectively. The method developed was found to be accurate, precise, and robust. The method was then applied in
a marketed formulation (ANTARTH capsule), and the percent content was found to be 5.26 ± 1.09, 4.76 ± 0.83, 3.80 ± 0.94
and 7.62 ± 1.58 for quercetin, rutin, berberine, and curcumin, respectively.

Keywords Rheumatoid arthritis · Quercetin · Rutin · Berberine · Curcumin · High-performance thin-layer chromatography

1 Introduction and joint deterioration during the course of the disease,

The autoimmune disease rheumatoid arthritis (RA), which
is characterized by the breakdown of cartilage and bone, is
a chronic, inflammatory condition [1]. RA is a type of sys-
temic inflammatory arthritis with the diarthrodial joint as its
primary site of involvement [2]. Factors like smoking, hered-
ity, and female gender all increase the risk of developing RA
[3]. Seropositive or seronegative RA is determined by the
presence or absence of antibodies. When compared to sero-
positive individuals who experience greater inflammation
* Sathiyanarayanan Lohidasan
l.satyanarayan@bharatividyapeeth.edu
1
Department of Quality Assurance Techniques, Poona
College of Pharmacy, Bharati Vidyapeeth Deemed to be
University, Pune 411038, Maharashtra, India
2 hence are considered as a gold mine for the treatment of
Department of Pharmaceutical Chemistry, Poona College
of Pharmacy, bharati Vidyapeeth Deemed to Be University,
Pune 411038, Maharashtra, India
seronegative patients have more inflammation at the time
of presentation. Seropositive cases or cases with advanced
illness may possess extra-articular symptoms. Anti-citrulli-
nated protein antibody (ACPA) is linked to bone erosions,
discomfort, and ongoing inflammation [4]. First, there is
persistent synovial membrane inflammation resulting in
the destruction of articular cartilage, then persistent mal-
formation and bone erosion. RA has a tendency to impact
the synovial-lined joints of the cervical spine as well as the
feet, wrists, knees, hips and shoulders. Cartilage and bone
degeneration are caused by the growth of synovial [5].
The current era has seen a shift in global trends from syn-
thetic to herbal treatment, or “return to nature”. A number
of natural products possessing diverse classes of flavonoids,
terpenes, quinones, catechins, alkaloids, anthocyanins, and
anthoxanthins modulate the inflammatory response and
RA [6].

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Quercetin is a flavone, which is a subclass of flavo-
noids, chemically it is 2-(3,4-dihydroxyphenyl)-3,5,7-
trihydroxychromen-4-one [7]. It can be found in a variety
of fruits and vegetables, including black currant, black
tea, onion (Allium cepa Linn.), and apple (Malus pumila
Miller) (Ribes nigrum Linn.). It plays a part in a variety
of processes including those that are antioxidative, anti-
carcinogenic, anti-inflammatory, anti-aggregatory, and
vasodilation [8, 9]. Buckwheat, asparagus, leaves and
petioles of rheum species contain rutin, a citrus flavonoid
glycoside [10]. Chemically it is 2-(3,4-dihydroxyphenyl)-
5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-
[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]
oxymethyl]oxan-2-yl]oxychromen-4-one [11]. It thins the
blood, inhibits platelet aggregation, reduces capillary per-
meability, thereby enhances circulation [12]. Berberine,
phytoconstituent present in daruhaldi [13], chemically is
16,17-dimethoxy-5,7-dioxa-13-azoniapentacycl-henic-
osa-1,2,4,9,14,16,18,20-octaene. It is a well-known anti-
inflammatory agent useful in a wide variety of disorders
including hypercholesterolemia, hypertension [14]. Cur-
cuma longa Linn., a common Indian spice plant, yields the
yellow pigment curcumin [15]. Chemically it is (1E,6E)-
1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-
3,5-dione. The antioxidant, anti-inflammatory, antiviral,
antibacterial, antifungal, and anticancer features are some
of the potent properties of curcumin. Researchers believe
that curcumin offers a great potential for treating a vari-
ety of illnesses, including allergies, diabetes, Alzheimer’s
disease, and arthritis [16].
The combination of the above four biomarkers is pre-
sent in the marketed formulation product ANTARTH
capsule used in the management of RA. So, in order to
ensure that quality is not compromised, numerous analyti-
cal techniques like high-performance liquid chromatog-
raphy (HPLC), liquid chromatography‒mass spectrom-
etry (LC‒MS), gas chromatography‒mass spectrometry
(GC‒MS) and high-performance thin-layer chromatogra-
phy (HPTLC) are used either alone or in combination for
product assessment.
A literature review found different analytical tech-
niques for estimating these chosen markers either alone
or in combination with other markers. So far, simultaneous
HPTLC estimation of these selected markers has not been
reported. Therefore, in the present study, an attempt was
made to develop a new HPTLC method for the simultane-
ous estimation of quercetin, rutin, berberine and curcumin.
In accordance with the International Council for Harmo-
nisation of Technical Requirements for Pharmaceuticals
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JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70
for Human Use (ICH) Q2 (R1) guideline, the method was
validated and the applicability of the method was tested
by analyzing the marketed formulation.
2 Experimental
2.1 Materials and method
2.1.1 Phytochemical standards
Curcumin was obtained as a gift sample from Omni Active
Health Technologies (Pune, India). Quercetin and rutin were
purchased from Yucca Enterprises (Mumbai, India) and
berberine was purchased from Yarrow Chemicals Pvt. Ltd.
(Mumbai, India).
2.1.2 Marketed formulation
ANTARTH capsules (Millennium Life Sciences; Delhi,
India) used for the study was procured from a local pharmacy.
Each 600-mg capsule contains the following herbs: Gug-
gul (Boswellia serrate), 15 mg; Methi (Trigonella foenum),
6.6 mg; Shuddha guggul (Commiphora mukul), 21.4 mg;
Nirgundi (Vitex negundo), 5.5 mg; Ashwagandha (Withania
somnifera), 12.5 mg; Punarnava (Boerhaavia diffusa), 5.8 mg;
Chopchini (Smilax china), 10 mg; Trikatu (Piper longum,
Piper nigrum, Zingiber officinale), 7.1 mg; Gokshura (Tribu-
lus terrestris); 5 mg; Triphala (Terminalia chebula, Terminalia
belerica, Emblica officinalis), 25 mg; Guduchi (Tinospora cor-
difolia), 8.3 mg; Shuddha shilajit (Asphaltum), 8.3 mg; Haridra
(Curcuma longa), 8.3 mg.
2.1.3 Chemicals
Toluene, ethyl acetate, methanol and formic acid of analytical
reagent grade were acquired from Merck Chemicals (Mumbai,
India).
2.2 Instrumentation
The employed HPTLC system included a CAMAG Lino-
mat 5 semi-automatic spotting device (CAMAG, Muttenz,
Switzerland), operated by visionCATS software (V 3.15,
CAMAG). On pre-coated silica gel 60 ­F254 aluminum plates
(20 cm × 20 cm, 250 μm thickness), the HPTLC system sample
application was carried out using a 100-μL Hamilton syringe
(Bonaduz, Switzerland). A CAMAG twin-trough glass cham-
ber (20 cm × 10 cm) with a stainless steel top was used for
plate development. In CAMAG TLC Visualizer 2, visual
JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70
image was captured at 254 and 366 nm and CAMAG TLC
Scanner 3 was used for densitometric analysis. As radiation
source, tungsten and deuterium lamps were employed.
2.3 Preparation of stock solutions
Accurately weighed 10 mg of quercetin, rutin, berberine,
and curcumin were dissolved in 10 mL of methanol sepa-
rately to get a concentration of 1000 μg/mL; then aliquots
of the above solution were accurately withdrawn and diluted
with methanol to get concentrations of 500 μg/mL, 100 μg/
mL, and 50 μg/mL for quercetin, berberine, and curcumin,
respectively, whereas the same stock solution of 1000 µg/
mL was used for rutin.
2.4 HPTLC method development and optimization
of chromatographic conditions
The plate used for HPTLC method development was cut of
6 cm × 10 cm and applied 6 mm sample band each, i.e., 2
μL of quercetin (500 μg/mL),5 μL of rutin (1000 μg/mL), 5
μL of berberine (100 μg/mL) and 3 µL of curcumin (50 μg/
mL) and at 10 mm distance between bands. The plates were
pre-saturated for 20 min with the mobile phase consisting
of toluene‒ethyl acetate‒methanol‒formic acid (5:3:2:0.5,
V/V) and then developed in ascending position. The length
of each run was 8 cm. The plate run took 15 min. The plate
was dried, visualized at 254 and 366 nm and then scanned
in an absorbance mode at 366 nm and 425 nm. The slit size
was set at 5 mm × 0.45 mm, and the scanning speed was
20 mm/s. Table 1 summarizes the optimized chromato-
graphic conditions.
Table 1  Optimized Parameters
chromatographic conditions
Stationary phase
Plate size
Sample band size
Sample distance
Sample volume
Development chamber
Mobile phase
Saturation time
Separation technique
Migration distance
Sample run time
Drying time
Drying temperature
Radiation source
Slit dimension
Detection wavelength
65
2.5 Validation of the method
The ICH recommendations were used to verify the devel-
oped HPTLC method for the simultaneous determination
of quercetin, rutin, berberine, and curcumin for linearity,
precision, robustness, limit of detection (LOD), limit of
quantification (LOQ), and recovery as per ICH Q2 (R1) [17].
2.5.1 Linearity
Different volumes in the range of 1–6 μL for quercetin and
rutin and 2–7 μL for berberine and curcumin were applied
on TLC plate to obtain the concentrations of 500–3000 ng/
spot, 1000–6000 ng/spot, 200–700 ng/spot and 100–350 ng/
spot, respectively. The calibration curves were constructed
by plotting respective peak areas vs. concentrations of the
four standards.
2.5.2 Precision
Both intra-day and inter-day method’s repeatability and
reproducibility were performed. The intra-day repeatability
was assessed by injecting six replicates of 1000 ng/spot for
quercetin, 2000 ng/spot for rutin, 400 ng/spot for berberine,
150 ng/spot for curcumin, on the same day. Repeating the
same process served to demonstrate inter-day repeatability
with the same concentrations for quercetin, rutin, berberine
and curcumin for three days. The percent relative standard
deviation (%RSD) was used to validate the repeatability and
reproducibility of the method.
Conditions
TLC silica gel 60 F­ 254 plates
6 × 10 cm
6 mm
10 mm
Quercetin: 2 µL, rutin: 5 µL, berberine: 5 µL, curcumin: 3 µL
Twin-trough glass chamber (10 × 10 cm) with stainless steel lid
Toluene–ethyl acetate–methanol–formic acid (5:3:2:0.5, V/V)
20 min
Ascending
8 cm
15 min
10 min
Room temperature
Tungsten and deuterium
5 × 0.45 mm
Quercetin, rutin, berberine: 366 nm, curcumin: 425 nm
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66 JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70

2.5.3 Robustness 2.5.5 Recovery

Small purposeful adjustments to the mobile phase composi- The accuracy of the method was determined from recovery
tion, saturation time, and development distance were made studies. Recovery studies were carried out by spiking 80%,
for robustness experiments. The mobile phase composition of 100%, and 120% of the standard markers onto the sample solu-
toluene‒ethyl acetate was changed by ± 0.2 (V/V); saturation tions. The experiment was carried out in triplicate.
time was changed by ± 5 min, and development distance was
changed by ± 5 cm. The %RSD of the peak area was subse- 2.5.6 Standardization of herbal formulation
quently calculated.
To determine the content of quercetin, rutin, berberine and
2.5.4 Sensitivity curcumin in the extract of the marketed formulation of
ANTARTH capsule, accurately weighed five capsules were
The LOD is the smallest amount of analyte that, under specific added in a 25-mL volumetric flask containing 15 mL metha-
experimental circumstances, can be found in a sample but is nol. Further, the mixture was sonicated for 30 min, cooled
not always measured. The lowest amount of an analyte that and the volume was made up to the mark using methanol.
can be detected and quantified with respectable accuracy, pre- This solution was centrifuged for 10 min at 10,000 rpm and
cision, is called LOQ. The standard error approach was used the supernatant was filtered and used for the study. Then 5 µL
to determine LOD and LOQ. They were calculated using the and 10 µL of extract sample were applied on the TLC plate
following equations utilizing the slope of the calibration (S) followed by development and scanning. Three repetitions were
curve and the standard error of the y-intercept (σ): performed, and the percent contents were determined using the
𝜎 formula as follows:
LOD = 3.3 ×
S Percent content in formulation (%)
Area of sample Weight of standard
𝜎 = ×
LOQ = 10 × Area of standard Weight of sample
S
× Dilution factor × 100

Fig. 1  HPTLC plate for the four markers, i.e., quercetin, rutin, berberine and curcumin along with sample on visualization at 366 nm (a). Densi-
togram of the mixed standard solution at optimized chromatographic condition (b)

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JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70 67

Table 2  Linear regression data for the four marker standards

Parameter Values
Quercetin Rutin Berberine Curcumin

Linearity range (ng/spot) 500–3000 1000–6000 200–700 100–350

Correlation coefficient (R2) 0.9988 0.9987 0.9977 0.9984
Regression equation y = 5.76E−06x + 0.0002 y = 2E−06x + 0.0075 y = 1E-05x + 0.0029 y = 4E−05x + 0.0065

Fig. 2  Linearity densitogram overlay of a rutin and quercetin; b berberine and curcumin

Table 3  Intra–day and inter-day precision results reported as mean 3.2 Development of the optimized mobile phase
area ± SD and %RSD
Intra-day precision Inter-day precision The HPTLC conditions were established by taking different
Mean area ± SD %RSD Mean area ± SD %RSD trials with varying ratios of mobile phase. The mobile phase
of toluene‒ethyl acetate‒methanol‒formic acid (5:3:2:0.5,
Quercetin 0.0136 ± 0.0002 1.4 0.0118 ± 0.0002 1.4 V/V) with a saturation time of 20 min and a development dis-
Rutin 0.0186 ± 0.0002 0.9 0.0177 ± 0.0003 1.7 tance of 8 cm gave good symmetric and resolved peaks. This
Berberine 0.0079 ± 0.0001 1.7 0.0074 ± 0.0001 1.5 mobile phase resulted in RF values of 0.57 ± 0.02 for quercetin,
Curcumin 0.0115 ± 0.0002 1.8 0.0125 ± 0.0002 1.9 0.09 ± 0.02 for rutin, 0.30 ± 0.02 for berberine, and 0.66 ± 0.02
SD standard deviation, %RSD percent relative standard deviation
for curcumin, respectively, and are shown in Fig. 1.

3.3 Linearity
3 Results and discussion
The linearity range, correlation coefficient and regression
3.1 Wavelength selection
equation for the four marker standards are represented in
Table 2. Figure 2 shows the densitogram overlay of each
To acquire the absorption spectrum, all the four marker
standard over their respective concentration ranges.
standards in HPTLC were scanned between 200 and
700 nm. Quercetin, rutin, and berberine displayed maxi-
mum absorbance (max) at 366 nm, while curcumin did 3.4 Precision
so at 425 nm.
The computed intra-day and inter-day precision for each
standard is shown in Table 3. It illustrates that the approach

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68 JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70

Table 4  Robustness results Parameter Markers Conc. (ng/spot) RF mean ± SD %RSD Area mean ± SD %RSD
of the method reported as
mean ± SD and %RSD Mobile phase ratio (toluene‒ethyl acetate‒methanol‒formic acid)
4.8:3.2:2:0.5, V/V Quercetin 1000 0.564 ± 0.006 1.0 0.0139 ± 0.0001 0.9
Rutin 2000 0.096 ± 0.001 0.7 0.0251 ± 0.0003 1.1
Berberine 400 0.307 ± 0.004 1.2 0.0077 ± 0.0001 1.8
Curcumin 150 0.682 ± 0.005 0.7 0.0052 ± 0.0001 1.4
Quercetin 2000 0.591 ± 0.005 0.9 0.0140 ± 0.0002 1.3
5.2:2.8:2:0.5, V/V Rutin 2000 0.094 ± 0.001 1.1 0.0261 ± 0.0004 1.5
Berberine 400 0.318 ± 0.002 0.7 0.0079 ± 0.0001 1.0
Curcumin 150 0.677 ± 0.006 0.9 0.0052 ± 0.0001 1.6
Development distance
75 cm Quercetin 1000 0.564 ± 0.0 0.0 0.0133 ± 0.0002 1.1
Rutin 2000 0.092 ± 0.001 0.7 0.0255 ± 0.0003 1.1
Berberine 400 0.306 ± 0.005 1.5 0.0074 ± 0.0001 1.6
Curcumin 150 0.656 ± 0.012 1.8 0.0052 ± 0.0001 1.0
85 cm Quercetin 1000 0.562 ± 0.008 1.3 0.0132 ± 0.0001 1.0
Rutin 2000 0.097 ± 0.001 0.6 0.0260 ± 0.0003 1.3
Berberine 400 0.326 ± 0.0 0.0 0.0075 ± 0.0001 1.6
Curcumin 150 0.678 ± 0.0 1.2 0.0052 ± 0.0001 1.2
Saturation time
15 min Quercetin 1000 0.577 ± 0.010 1.7 0.0131 ± 0.0001 0.5
Rutin 2000 0.095 ± 0.001 1.2 0.0259 ± 0.0003 1.3
Berberine 400 0.240 ± 0.001 0.5 0.0076 ± 0.0001 1.3
Curcumin 150 0.666 ± 0.009 1.4 0.0052 ± 0.0001 1.1
25 min Quercetin 1000 0.590 ± 0.006 1.0 0.0138 ± 0.0001 1.0
Rutin 2000 0.094 ± 0.001 1.5 0.0261 ± 0.0001 0.3
Berberine 400 0.212 ± 0.002 1.1 0.0075 ± 0.0001 1.3
Curcumin 150 0.676 ± 0.012 1.7 0.0051 ± 0.0001 1.9

RF retention factor, SD standard deviation, %RSD percent relative standard deviation

Table 5  Limit of detection and limit of quantification values for Table 7  Estimation of % content of quercetin, rutin, berberine and
quercetin, rutin, berberine and curcumin curcumin in marketed formulation
Standard LOD (ng/spot) LOQ (ng/spot) Formulation Markers % Content

Quercetin 119.4 361.7 ANTARTH capsule Quercetin 5.26 ± 1.09

Rutin 247.6 750.2 Rutin 4.76 ± 0.83
Berberine 33.0 99.9 Berberine 3.80 ± 0.94
Curcumin 13.9 42.1 Curcumin 7.62 ± 1.58

Table 6  Recovery of quercetin, rutin, berberine and curcumin is repeatable and reproducible because the %RSD values for
quercetin, rutin, berberine, and curcumin did not surpass 2%.
Recovery Standard markers
level (%) % Accuracy
Quercetin Rutin Berberine Curcumin 3.5 Robustness

80 95.61 95.64 94.50 94.51 The results of the robustness study are summarized in
100 97.99 97.43 97.32 98.60 Table 4. The low %RSD values indicate they are less scat-
120 100.80 100.41 99.20 100.73 tered and hence the method can be considered robust.

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JPC – Journal of Planar Chromatography – Modern TLC (2023) 36:63–70
Fig. 3  TLC image of marketed formulation along with four standard markers, i.e., quercetin, rutin, berberine and curcumin on visualization at
254 nm (a). Densitogram overlay of marketed formulation with standard solution at optimized chromatographic condition (b)
3.6 Sensitivity
LOD and LOQ values are summarized in Table 5 for querce-
tin, rutin, berberine and curcumin, respectively, which indi-
cated that the developed method was sensitive.
3.7 Recovery
Recovery studies are useful in evaluating the analytical
method’s accuracy. The results of recovery studies are
shown in Table 6. Recovery was found in the acceptable
range and the values attained were between 95 and 102%.
3.8 Estimation in marketed formulation
Estimation in the polyherbal formulation was performed
by spotting different volumes of the prepared extract of
ANTARTH capsule on TLC plate; the area and RF were
determined. The % content of quercetin, rutin, berberine and
curcumin are summarized in Table 7 and Fig. 3.
4 Conclusion
In the present research work, a novel, precise, accurate and
robust HPTLC method has been developed for the simul-
taneous quantification of quercetin, rutin, berberine, and
69
curcumin. The HPTLC method resulted in the % contents
of 5.26 ± 1.09, 4.76 ± 0.83, 3.80 ± 0.94 and 7.62 ± 1.58 for
quercetin, rutin, berberine and curcumin, respectively, in
ANTARTH formulation. The novel HPTLC method can
be useful for the routine quality control analysis for both
extracts and polyherbal formulations containing the above
phytochemical standards as active ingredients.
Author contributions SL, RD, and SS contributed to the study’s con-
ceptualization and design. AK handled the material preparation, data
collecting, and analysis; she also drafted the text, which was then
modified by all authors. All authors have read and approved the final
manuscript.
Declarations
Conflict of interest The authors have not disclosed any conflicts of
interest that would have an impact on the information in this article.
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