JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577

https://doi.org/10.1007/s00764-020-00075-4

ORIGINAL RESEARCH PAPER

Morphological, cytological and phytochemical studies in naturally

occurring diploid and tetraploid populations of Physochlaina praealta
from high altitudes of Trans‑Himalaya
Younas Rasheed Tantray1 · Mohammad Saleem Wani2 · Saroj Kumar Pradhan1 · Maroof Hamid3 · Ishrat Jan3,4 ·
Vijay Kumar Singhal1 · Raghbir Chand Gupta1 · Talaat H. Habeeb5

Received: 29 July 2020 / Accepted: 7 December 2020 / Published online: 2 February 2021
© Akadémiai Kiadó, Budapest, Hungary 2021

Abstract
Physochlaina praealta samples were studied macromorphologically and cytomorphologically along with their detailed
phytochemical investigation. The concentration of phytoconstituents showed a strong positive correlation with the ploidy
level and altitudinal gradients. The total phenol content was detected maximum in the methanolic extract of leaves and stem
of higher altitudinal plants in both cytotypes (2x, 4x). The maximum content of flavonoids was detected in the methanolic
extract of root and leaves. Root organ from higher elevation possessed the highest DPPH radical scavenging activity, with the
maximum percentage of inhibition being obtained in methanolic extracts. The plants of both cytotypes from higher eleva-
tions accumulate an abundant quantity of secondary metabolites. The two cytotypes differ from each other with respect to
various morphometric characters thereby depicting the drastic affect of polyploidy.

Keywords  Physochlaina praealta · Male meiosis · HPTLC · TPC · TFC · DPPH

Abbreviations KOH Potassium hydroxide

FCR Folin–Ciocalteu reagent QE Quercetin
AlCl3 Aluminum chloride TPC Total phenol content
DPPH 1,1-diphenyl-2-picrylhydrazyl TFC Total flavonoid content
AT Atropine HPTLC High-performance thin-layer chromatography
CA Caffeic acid LOD Limit of detection
CHA Chlorogenic acid LOQ Limit of quantification
SD Standard deviation
PK Panikher
Supplementary Information  The online version of this article
(https​://doi.org/10.1007/s0076​4-020-00075​-4) contains MB Mulbekh
supplementary material, which is available to authorized users. SP Sapi
KH Khardung
* Younas Rasheed Tantray
HU Hundur
younasrasheed53@gmail.com
ICH The International Council for Harmonisation of
Mohammad Saleem Wani
Technical Requirements for Pharmaceuticals for
saleemwani806@gmail.com
Human Use
1
Punjabi University, Patiala 147002, India
2
Govt. Degree College, Surankote, Jammu 185121, India
3
Centre for Biodiversity and Taxonomy, Department
1 Introduction
of Botany, University of Kashmir, Srinagar 190006, India
4 Herbal medicine has been enjoying renaissance among the
Department of Zoology, University of Kashmir,
Srinagar 190006, India purchasers across the globe. But ayurvedic medicines are
5 limited with some hindrances such as scantiness of regular
Biology Department, Faculty of Science at Yanbu,
Taibah University, King Khalid Rd., Al Amoedi, quality control profiles that influence consumer acceptance.
Yanbu El‑Bahr 46423, Saudi Arabia The uses of active components as herbal medicines have

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568 JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577

some safety and efficacy implication. It is difficult to main- and Nelong Valley in Uttarkashi (Uttarakhand) [14]. The
tain quality control profile due to the complex nature and leaves are poisonous and narcotic and hold the belladonna-
intrinsic variability of the chemical constituents of the plant like property of dialting eye pupils and are applied to cure
used as drugs. To address these issues, recently developed boils [15, 16]. They contain up to 1.02% alkaloids, mainly
analytical techniques play a pivotal role in mitigating this hyoscyamine and scopolamine. Its roots and leaves contain
problem [1]. hyoscyamine, which later on gets converted into atropine
High-performance thin-layer chromatography (HPTLC) and hyoscine and thereby serves as an excellent source of
is a widely accepted and frequently employed technique for atropine [17], therefore the importance of the plant is obvi-
the qualitative and quantitative analysis of chemical mark- ous. In the world market, there would be a great demand for
ers in herbal raw materials [2]. HPTLC is having several this plant for being a rich source of alkaloids.
advantages such as ease of application, sensitivity, and preci- As far survey of literature suggests, there is no report on
sion [3]. Moreover, it possesses paramount significance for the phytochemical diversity of P. praealta based on differ-
developing fingerprinting and marker-based standardization ent ploidy levels and also among different populations from
of herbal drugs. This technique is used for the identifica- India. Keeping this in sight, the total phenol and flavonoid
tion, purity, stability, and dissolution of raw materials and content, antioxidant activity and HPTLC analyses of differ-
formulated products [4]. Plant secondary metabolites are ent plant parts, population and cytotypes of P. praealta have
known to play a pivotal role in the adaptation of plants to a been carried out in the present investigation using different
wide range of environmental factors and defense against dis- types of markers.
eases and pathogens [5]. The synthesis and build-up of the
phytochemical component are determined by environmental
induced stresses [6]. External factors such as precipitation, 2 Experimental
light intensity, average temperature, wind speed, tempera-
ture extremes, atmospheric pressure, duration of snow-cover, 2.1 Chemicals
length of the vegetation period and soil attributes influences
the phytochemical profiles [7]. Genetic variation has been The analytical grade solvents were obtained from E. Merck
analyzed as one of the leading factors that affects the con- (Darmstadt, Germany). The HPTLC plates pre-coated with
tents of active constituents. The level of ploidy also influ- silica gel 60 F
­ 254 were purchased from E. Merck. Atropine,
ences both the quantity and diversity of phytochemicals chlorogenic acid, caffeic acid, gallic acid, quercetin, and
within the plants [8, 9]. As far as the effect of altitude on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) were procured from
diversity of secondary metabolites production in plants is Sigma-Aldrich Chemical Company (Steinheim, Germany).
concerned, only meager reports have been documented [10].
Extracts derived from high altitude growing plants showed 2.2 Plant material
higher antioxidant capacity compared to low altitude grow-
ing plants [11]. In the present investigation, the plant samples were collected
The genus Physochlaina G. Don (family Solanaceae) is from high altitudinal cold desert regions of Ladakh-a part
represented by six species throughout the globe, viz., P. cap- of Western Himalayas in the state of Jammu and Kashmir.
itata A.M. Lu, P. infundibularis Kuang, P. macrocalyx Pas- After a proper identification consulting various floras [18]
cher, P. macrophylla Bonati, P. physaloides (L.) G. Don and and finally authenticated from Botanical Survey of India
P. praealta (Decne.) Miers. The plants are perennial herbs (BSI), Dehradun, the voucher specimens were submitted in
and are rich sources of tropane alkaloids, mainly apoatro- the Herbarium, Department of Botany, Punjabi University
pine, apohyoscine, hyoscyamine, scopolamine, 6-hydroxy- Patiala (PUN), a code given by Holmgren and Holmgren
atropine and physochlaine [12]. The species of this genus (1998) [19].
have a long history of use in Chinese traditional medicine
system as remedy with strong anti-inflammatory effects on 2.3 Cytomorphological studies
skin diseases and sexually transmitted diseases, as well as
their positive effects on nervous disorders, both calming and For meiotic studies, Carnoy’s fixative was employed for the
energizing [13]. The herbs of the genusare locally known fixation of anthers for 24 h. The materials were then trans-
as “hun horse” in Tibet and “garag chig tav” in Mongolia ferred to 70% ethanol and kept in a refrigerator at 4 °C for
[13]. The present studied species (P. praealta) grows as a meiotic analysis. Anthers were squashed in acetocarmine
tall perennial herb on high dry stony areas between 2660 (1%) to prepare the slides of meiocytes. Additionally, 1:1
and 4800 m in India, Tibet and China. In India, the species glycerol–acetocarmine mixture was employed for assessing
is found in the cold desert regions including Ladakh (Jammu the viability of pollen grains. For stomatal studies, the abax-
and Kashmir), Lahaul-Spiti and Kinnaur (Himachal Pradesh) ial epidermal peels were obtained from the lower portion of

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JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577 569

mature leaves through KOH treatment. Stomatal index was mixture was then determined at 510 nm versus prepared
calculated by using the formula: SI = S/E + S × 100, where water blank. The results were expressed as quercetin equiva-
SI = stomatal index, S = number of stomata, and E = number lent per gram of dry extract (mg QE/100 g DW, where QE is
of epidermal cells. quercetin equivalent).
The digital imaging system of Leica Qwin has been used
to take photomicrographs from the freshly prepared desir- 2.5.3 DPPH radical scavenging activity
able slides with clear chromosome counts, triads, tetrads,
and pollen grains. The free radical scavenging activity of the extracts and
standard solutions (vit C and atropine) were investigated
2.4 Extraction procedure using DPPH radical scavenging method [22]. A total of 750
µL of the extract or standard was added to 750 µL of DPPH
The root, stem and leaf samples of different populations in in methanol solution (152 µM). After incubation at 37 °C for
two cytotypes of P. praealta (diploid and tetraploid) were 20 min, the absorbance of each solution was determined at
air-dried (temperature of 25 °C ± 2 °C) and grounded to a 517 nm using UV–VIS spectrophotometer. The correspond-
fine powder in mortar and pestle. The dried powdered sam- ing blank readings were also taken and percent inhibition
ples (5 g) were separately placed into a thimble and were was then calculated as follows:
extracted with methanol in a Soxhlet apparatus. Extraction
was carried out for 6 h followed by filtration using Whatman
( )
Ablank − Asample
%Inhibition = × 100
No. 1 filter paper. The filtrates were concentrated to 5 mL in Ablank
a rotary evaporator under vacuum at 50 °C and the resulting
solutions were used as test solutions. where Ablank is the absorbance of the control reaction (con-
taining all reagents except the test compound) and Asample
2.5 Phytochemical analysis is the absorbance of the test compound. The I­ C50 value, the
concentration of sample required for 50% scavenging of
2.5.1 Total phenolic content the DPPH free radical, was determined from the curve of
percent scavenging plotted against the concentration. Each
Folin–Ciocalteu method was used for the estimation of the determination was done in triplicate, and the average I­ C50
total phenolic content. 0.1 mL of sample was mixed with value was calculated.
2 mL of sodium carbonate (2%) freshly prepared, the cock-
tail was robustly mixed on a vortex. After 5 min, 100 mL of 2.5.4 HPTLC instrumentation and conditions
Folin–Ciocalteu reagent (1 N) added to the mixture was left
for 30 min at room temperature and the reading of absorb- Chromatography was performed on HPTLC plates
ance was performed against a blank at 750 nm using an ultra- (10 cm × 10 cm) pre-coated with silica gel 60 ­F254, 0.20 mm
violet–visible (UV–VIS) spectrophotometer V-550 model layer thickness (Merck, Darmstadt, Germany). 2, 4, 6, 8,
(Jasco, Tokyo, Japan). A calibration curve was performed 10, 12 µL of the standard solution (1 mg/mL) along with
in parallel under the same operating conditions using gallic 50 µL of suitably diluted sample solution were applied to
acid as positive control. The results were expressed as mg silica gel plate. Standard solutions and samples were spotted
gallic acid equivalent per gram of dry extract (mg GAE/100 as 6 mm bands, started 15 mm from the left edge, 10 mm
g DW, where GAE is gallic acid equivalent and DW is dry from the bottom edge and 10 mm of track distance, using a
weight) [20]. CAMAG (Muttenz, Switzerland) Linomat 5 sample appli-
cator under a flow of ­N2 gas. The samples were sprayed at
2.5.2 Total flavonoid content a speed of 150 nL/s [23]. The plates were developed in a
CAMAG twin-trough chamber (20 cm × 10 cm) to a dis-
The total flavonoid content was determined according to tance of 80 mm using different mobile phases as methanol‒
the method by Wani et al. [21]. Each sample (500 µL) was acetone‒diethylamine (5:4.8:0.2, V/V, mL) for atropine and
mixed with 2 mL of distilled water and subsequently with toluene‒ethyl acetate‒methanol‒formic acid (75:25:10:6,
150 µL of a sodium nitrite solution (15%). After 6 min, 150 V/V, mL) for caffeic and chlorogenic acid. All the analy-
µL of aluminum chloride solution (10%) was added and ses were carried out in the laboratory at controlled room
allowed to stand for 6 min. Then, an aliquot of 2 mL of temperature (25 ± 3 °C) and 40% relative humidity. After
sodium hydroxide solution (4%) was added to the mixture. development, the plates were dried under warm air and were
Immediately, distilled water was added to bring the final subjected to derivatization with specific reagents. Atropine
volume to 5 mL and the mixture was thoroughly mixed and was identified by derivatization with Dragendorff’s reagent,
allowed to stand for another 15 min. The absorbance of the while derivatization of caffeic acid and chlorogenic acid was

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570 JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577

done in anisaldehyde. After chromatographic development, (3200 m) were diploid cytotypes (n = 21) and the individ-
the peak areas of the bands were measured at 500 nm and uals collected from Sapi (SP) (4000 m), Khardung (KH)
254 nm densitometrically using a CAMAG TLC Scanner IV (4000 m) and Hundur (HU) (3010) were tetraploid cyto-
and the data were analyzed using winCATS software version types (n = 42). Details regarding the localities with altitude,
4.03 (CAMAG). The experiments were done in triplicate meiotic chromosome number, ploidy level and pollen fertil-
and the average areas of markers were taken. The calibra- ity of the worked-out accessions as well as the information
tion curves were prepared by plotting the amount of marker regarding habit and habitat, and comparison of micro- and
compounds spotted (μg/band) as an independent variable (X) macroscopic characters of the two cytotypes are furnished
and the respective peak area as the dependent variable (Y). in Table 2.
The sensitivity of the method was determined with respect to
limit of detection (LOD) and limit of quantification (LOQ). 3.2 Meiotic course
LOD and LOQ were determined from the calibration curve,
based on the standard deviation (SD) of the y-intercepts and Diploid (n = 21) (Fig. 1a).
mean x̄ of the slope of the regression lines according to the The accessions collected from PK (3000 m) and MB
formula, LOD = 3.3 (SD/x̄  ) and LOQ = 10 (SD/x̄  ) (Table 1). (3200 m) revealed the diploid chromosome count of n = 21
which was confirmed from the occurrence of 21 bivalents at
2.6 Statistical analyses metaphase-I (Fig. 1b). Further meiotic course in both acces-
sions was completely normal leading to normal sporad for-
All the statistical analyses were performed in R version mation and 100% pollen fertility (Fig. 1g).
3.6.1 [24]. Analysis of variance (ANOVA) was performed Tetraploid (n = 42) (Fig. 1c).
to evaluate the effects of altitude, ploidy and plant part on The plants collected from HU (3000 m), KH (3900 m)
phytoconstituents specifying ‘altitude’, ‘ploidy’ and ‘plant and SP (4000 m) in Ladakh division (Jammu and Kashmir)
part’ as fixed effects and phytoconstituents as response vari- were found to exist at tetraploid level with a meiotic chromo-
ables. Prior to ANOVA, the data were subjected to Shap- some count of n = 42 as confirmed from the occurrence 42
iro–Wilk’s and Levene’s tests for checking the normality bivalents at metaphase-1 (Fig. 1d). Further meiotic course
and homogeneity of variance, respectively. The data were including sporad formation in these 4x individuals was per-
also log- and square root-transformed to meet the assump- fectly normal leading to high pollen fertility (94–100). The
tions of ANOVA. fertile pollen grains in these accessions were noticed to be
of heterogenous sizes (Fig. 1j).

3 Results 3.3 Morphology

3.1 Cytology 3.3.1 Morphometric analysis and distribution

The present cytological study based on five populations col- The diploid individuals were found to grow at altitudinal
lected from different regions of Ladakh division revealed range of 3000–3200 m, whereas tetraploid populations grow
the existence of diploid (2n = 2x = 42) and tetraploid in the altitudinal range of 3000–4000 m in the cold desert
(2n = 4x = 84) cytotypes based on x = 21. The individuals of Ladakh. The individuals of 2x and 4x cytotypes also look
collected from Panikher (PK) (3000 m) and Mulbekh (MB) alike except that 2x plants were noticed to be more pubescent

Table 1  Method validation Atropine Caffeic acid Chlorogenic acid

parameters of standards by the
proposed TLC–densitometric RF 0.18 0.72 0.77
method
Linearity range 2–12 µg 2–12 µg 2–12 µg
Linear regression equation y = 63.34x + 34.76 y = 301.2x – 13.82 y = 1455x + 1173
Correlation coefficient (r2) 0.9807 0.9993 0.9989
Slope ± SD 1454 ± 12.09 301.2 ± 2.032 63.34 ± 2.224
Intercept ± SD − 28.92 ± 17.32 13.82 ± 15.83 − 1173 ± 94.18
Limit of detection (µg/spot) 0.49 0.17 0.22
Limit of quantification (µg/spot) 1.49 0.53 0.65
Intra-day precision (%RSD) 0.75 1.15 0.92
Inter-day precision (%RSD) 1.04 1.12 1.21

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JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577 571

Table 2  Site of location and Diploid (n = 21) Tetraploid (n = 42)

comparison of macro- and
microscopic characters in the Site of collection Sankoo (3000 m) Hundur (3000 m)
two cytotypes of P. praealta 
Mulbekh (3200 m) Khardung (3900 m)
Sapi (4000 m)
Distribution Less common More common
Habit Perennial herb Perennial herb
Habitat Grows within the rocks Along roadsides and in dry stony areas
Plant height (cm) 35–45 cm 45–60 cm
Leaves
(i) No. of leaves/plant 10–15 25–30
(ii) Size
 Length (cm) 7.4–7.6 cm 8.5–11 cm
 Width (cm) 4.5–4.8 cm 7.4–8.5 cm
Stomata
Size (µm) 30.21–34.42 × 24.55–28.31 33.42–40.53 × 26.31–35.46
(32.13 ± 1.58) × (26.12 ± 2.12) (36.94 ± 2.40) × (31.16 ± 2.84)
Density (1 cm2) 10.12–14.31 (13.20 ± 1.53) 4.21–9.47 (6.31 ± 1.33)
Stomatal index 24.12–35.12 (28.11 ± 5.16) 23.68–35.71 (28.60 ± 3.87)
Trichome
Size (µm) 87.31–198.6 (121.3 ± 32.68) 142.1–201.5 (184.23 ± 26.43)
Density (1 cm2) 5.05–7.31 (6.12 ± 0.59) 2.10–6.31 (4.06 ± 1.60)
Large
46.96–52.68 × 41.34–51.73
(50.21 ± 2.07) × (47.12 ± 3.15)
Pollen grain size(µm) Small
38.14–43.86 × 39.34–43.47
41.31–44.61 × 39.21–42.59 (41.34 ± 1.69) × (41.57 ± 1.52)
(42.15 ± 1.21) × (40.13 ± 1.24)
Pollen fertility (%) 100% 94–96%

than the 4x. Two cytotypes can be differentiated in some revealed that diploid leaves, stem and root samples from MB
micro-characters like stomata, trichome and pollen grains. (3200 m) showed higher phenolic content as compared to
Stomata are significantly large-sized in the 4x cytotype com- samples from PK (3000 m). In the case of tetraploids, TPC
pared to those in the 2x (Figs. 1e, 1 h). As far as the sto- was maximum in leaves, stem, and root samples from KH
matal density is concerned, it is significantly higher in the (4000 m) and SP (4000 m) as compared with HU (3010 m)
2x individuals compared to 4x (Table 2). Detailed analysis (Table 3). Total flavonoid content was the highest in dip-
revealed that trichome frequency per 1 cm2 of leaf surface loid root, leaves and stem from MB (3200 m) population
(Table 2) was significantly higher in 2x compared to 4x. The as compared to roots, leaves and stem from PK (3000 m)
trichomes in 4x plants were noticed to be significantly larger population. Among the populations, the amount of phe-
compared to the diploid (Fig. 1f, i). The fertile pollen grains nols ranged from 1.4 ± 0.3 mg/g DW (diploid, leaves) and
in 4x individuals were noticed to be of two types as large and 43.5 ± 2.7 mg/g DW (tetraploid, roots) and the flavonoid
small as compared to the 2x individuals where pollen grains content ranged from 1.4 ± 0.3 mg/g DW (diploid, leaves)
are of uniform size (Fig. 1g, j). and 56.1 ± 0.4 mg/g DW (tetraploid, roots) (Table 4).

3.4 Total phenol and flavonoid content 3.5 HPTLC analysis

There is a noticeable difference in the phenol and flavonoid The present experiment demonstrates a simple, rapid, precise
content in different extracts with the maximum content in and sensitive HPTLC protocol for the qualitative and quanti-
methanolic extract and minimum content in chloroform tative determination of altitudinal variation, cytotype-based
and petroleum ether extracts. Among the cytotypes high- variation and tissue-specific variation of atropine, caffeic
est content of total phenolics was found in tetraploids in and chlorogenic acid from different populations of P. prae-
comparison to diploids. Tissue-specific chemoprofiling alta. Among all the compounds present, atropine was found

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572 JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577

Fig. 1  a A field photograph

of 2x plant. b A diploid PMC
showing 21 bivalents at meta-
phase-I. c A field photograph of
4x cytotype. d A PMC showing
42 bivalents at metaphase-I.
e Stomata of 2x cytotype.
f Trichomes of 2x cytotype.
g Pollen grains of 2x. h Stomata
of 4x cytotype. i Trichomes
of 4x cytotype. j Pollen grains
of 4x 

Table 3  Total phenol content in different accessions, extracts and plant parts of P. praealta (mg GAE/g DW)
Cytotype Accessions/localities Plant parts Aqueous Methanol Ethanol Chloroform Petroleum ether Hexane

2x Mulbekh (3200 m) Stem 6.1 ± 0.3 13.9 ± 0.3 5.4 ± 0.2 1.6 ± 0.04 1.4 ± 0.09 2.1 ± 0.2

Roots 3.1 ± 0.4 6.9 ± 0.09 3.6 ± 0.2 1.2 ± 0.4 0.9 ± 0.3 1.9 ± 0.3
Leaves 11.6 ± 0.2 20.2 ± 1.1 8.6 ± 1.0 4.2 ± 0.1 6.2 ± 0.14 6.2 ± 0.2
Panikher (3000 m) Stem 5.2 ± 0.2 11.5 ± 0.4 3.1 ± 0.1 2.1 ± 0.6 1.6 ± 0.4 2.9 ± 0.4
Roots 4.7 ± 0.9 4.6 ± 0.1 2.1 ± 0.1 0.5 ± 0.2 0.3 ± 0.04 1.4 ± 0.1
Leaves 10.2 ± 0.3 18.5 ± 1.6 11.1 ± 1.6 2.2 ± 0.1 4.4 ± 0.05 5.1 ± 0.3
4x Sapi (4000 m) Stem 17.2 ± 0.3 38.7 ± 2.6 16.3 ± 0.2 4.3 ± 0.04 5.1 ± 0.2 7.6 ± 0.2
Roots 9.3 ± 1.2 21.3 ± 0.3 16.2 ± 1.3 4.2 ± 0.8 3.2 ± 1.2 5.9 ± 0.4
Leaves 25.3 ± 0.2 42.6 ± 0.9 20.9 ± 1.7 12.8 ± 1.4 14.1 ± 0.5 14.5 ± 0.4
Khardung (3900 m) Stem 19.2 ± 0.2 40.8 ± 2.5 18.2 ± 0.2 6.2 ± 0.2 6.6 ± 0.4 8.6 ± 0.1
Roots 11.4 ± 0.9 21.4 ± 0.1 15.1 ± 1.4 5.5 ± 0.6 4.3 ± 1.2 7.3 ± 0.5
Leaves 23.7 ± 0.2 43.6 ± 0.8 22.5 ± 1.6 8.7 ± 0.15 12.9 ± 0.19 13.6 ± 0.6
Hundur (3010 m) Stem 15.8 ± 0.4 33.5 ± 2.7 14.3 ± 0.3 7.6 ± 0.2 4.7 ± 0.2 8.2 ± 0.2
Roots 13.6 ± 1.3 18.8 ± 0.4 13.1 ± 1.9 3.1 ± 0.08 4.1 ± 0.4 6.2 ± 0.6
Leaves 22.1 ± 0.3 38.5 ± 0.6 23.6 ± 2.2 8.8 ± 0.2 9.8 ± 0.4 11.6 ± 0.9

to be maximum in the tetraploid roots (256.28 ± 0.08 µg/g cytotype from a higher altitude of Khardung (3900 m) and
DW), while caffeic acid was found to be minimum in the Sapi (4000 m) displayed the maximum accumulation of
diploid leaves (0.217 ± 0.09  µg/g DW). The tetraploid atropine in root as compared to lower altitudes of Hundur

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JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577 573

Table 4  Total flavonoid content in different accessions, extracts and plant parts of P. praealta (mg QE/g DW)
Cytotype Accessions/localities Plant parts Aqueous Methanol Ethanol Chloroform Petroleum ether Hexane

2x Mulbekh (3200 m) Stem 4.6 ± 0.2 9.6 ± 0.2 8.2 ± 0.4 3.5 ± 0.3 2.7 ± 0.1 4.8 ± 0.6

Roots 19.8 ± 0.2 26.1 ± 0.4 16.5 ± 0.3 8.7 ± 0.1 3.9 ± 0.2 11.4 ± 0.2
Leaves 7.4 ± 0.7 12.3 ± 0.8 9.1 ± 0.5 1.4 ± 0.3 1.9 ± 0.4 2.2 ± 0.2
Panikher (3000 m) Stem 3.2 ± 0.6 8.2 ± 0.3 7.1 ± 0.4 2.8 ± 0.4 1.8 ± 0.2 4.1 ± 0.5
Roots 18.2 ± 0.6 23.9 ± 0.4 15.1 ± 0.5 7.2 ± 0.03 4.7 ± 0.4 12.9 ± 0.4
Leaves 8.5 ± 0.6 10.2 ± 0.6 10.4 ± 0.7 2.8 ± 0.2 4.3 ± 0.6 3.3 ± 0.1
4x Sapi (4000 m) Stem 10.1 ± 1.6 18.9 ± 0.3 14.9 ± 0.2 6.4 ± 0.2 4.5 ± 0.2 7.3 ± 2.2
Roots 38.8 ± 0.6 54.8 ± 0.5 33.9 ± 3.3 17.4 ± 0.2 7.7 ± 0.09 23.3 ± 0.2
Leaves 18.6 ± 2.2 44.2 ± 8.6 25.1 ± 2.0 7.9 ± 0.7 8.1 ± 3.8 10.1 ± 1.5
Khardung (3900 m) Stem 8.8 ± 1.7 17.4 ± 0.2 13.7 ± 0.3 4.4 ± 0.07 3.5 ± 0.2 6.2 ± 1.8
Roots 40.3 ± 0.1 56.1 ± 0.4 35.3 ± 2.9 19.5 ± 0.2 9.4 ± 0.3 24.6 ± 0.4
Leaves 17.1 ± 1.8 42.8 ± 8.3 23.6 ± 1.8 6.1 ± 0.9 7.2 ± 3.6 8.3 ± 1.6
Hundur (3010 m) Stem 7.3 ± 1.1 15.7 ± 0.3 12.4 ± 0.3 2.6 ± 0.2 2.5 ± 0.3 5.0 ± 1.9
Roots 36.9 ± 0.9 53.1 ± 0.6 32.2 ± 2.9 15.7 ± 0.3 5.8 ± 0.4 20.7 ± 0.3
Leaves 19.80 ± 1.7 40.3 ± 7.6 26.6 ± 2.0 9.0 ± 0.7 9.3 ± 3.8 11.6 ± 1.9

(3010 m). Diploid cytotype from Mulbekh also displayed range, precision, specificity, detection and quantification
the high concentration of atropine in roots followed by Pan- limits (Table 1, Fig. 5). According to the ICH guidelines,
ikher (3000 m) (Fig. 2a, b). Caffeic acid and chlorogenic system suitability tests are considered an integral part of
acid were found the most abundant in the tetraploid leaf chromatographic methods to prove that the system performs
(1.07 ± 0.01, 87.28 ± 0.07 µg/g DW) from higher altitudes adequately [25].
of Sapi (4000 m) as compared to low altitude extracts. The
same trend was observed in the diploid cytotypes (Figs. 3a, 3.6 Antioxidant activity
b, 4a, b). One-way ANOVA revealed a significant effect of
altitude, ploidy and plant part on atropine, caffeic and chlo- The DPPH radical scavenging activity is generally used to
rogenic contents (Tables S1, S2, and S3 in the Supplemen- analyze the ability of antioxidants to scavenge free radicals.
tary Material). The methanolic extract was found to have maximum phe-
Calibration graphs relating the obtained peak areas to the nolic and flavonoid content so DPPH activity was performed
corresponding concentrations of atropine, chlorogenic acid, in this extract only. Two accessions thriving at higher alti-
and caffeic acid were found to be linear over the ranges of tudes one among each cytotype were selected for estimat-
2–12 μg/spot, respectively. The regression equations were ing DPPH activity; the highest scavenging activity for free
computed for each standard, and the proposed method was radicals was observed in the tetraploid accession collected
validated according to the ICH guidelines regarding linearity from Sapi (4000 m) than diploid accession collected from

Fig. 2  a Comparative HPTLC profile of atropine with different plant (leaves, stem, roots); 16–18: Mulbekh (2x) (leaves, stem, roots).
parts and accessions of P. praealta.1–3: standard atropine; 4–6: Sapi b Amount of atropine in methanolic extracts of different plant parts
(4x) (leaves, stem, roots); 7–9: Khardung (4x) (leaves, stem, roots); and accessions of P. praealta (2x, 4x) (µg/g DW)
10–12: Hundur (4x) (leaves, stem, roots); 13–15: Panikher (2x)

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574 JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577

Fig. 3  a Comparative HPTLC profile of caffeic acid with differ- (2x) (leaves, stem, roots); 16–18: Mulbekh (2x) (leaves, stem, roots).
ent plant parts and accessions of P. praealta.1–3: standard atropine; b Amount of caffeic acid in methanolic extracts of different plant
4–6: Sapi (4x) (leaves, stem, roots); 7–9: Khardung (4x) (leaves, stem, parts and accessions of P. praealta (2x, 4x) (µg/g DW)
roots); 10–12: Hundur (4x) (leaves, stem, roots); 13–15: Panikher

Fig. 4  a Comparative HPTLC profile of chlorogenic acid with differ- (2x) (leaves, stem, roots); 16–18: Mulbekh (2x) (leaves, stem, roots).
ent plant parts and accessions of P. praealta.1–3 standard atropine; b Amount of chlorogenic acid in methanolic extracts of different
4–6: Sapi (4x) (leaves, stem, roots); 7–9: Khardung (4x) (leaves, stem, plant parts and accessions of P. praealta (2x, 4x) (µg/g DW)
roots); 10–12: Hundur (4x) (leaves, stem, roots); 13-15: Panikher

Mulbekh (3200 m). In general, the plant extracts of higher praealta (2n = 42, 84) in the present case could be attrib-
altitude showed efficient scavenging ability as compared uted to polyploidy, wherein polyploid individuals depicted
to diploid from lower altitude. Tissue-specific antioxidant increased size in various vegetative and floral parts. Such an
activity revealed significantly higher scavenging potential effect of polyploidy causing gigantism of various vegetative
of root extracts from Sapi (4000 m) followed by leaf and and floral parts has also been reported in a number of wild
stem (Fig. 6). species [30].
The everlasting need of therapeutically essential second-
ary metabolites can be keenly fulfilled if we can record the
4 Discussion variation in their content in medicinal herbs owing to change
in altitudes [31]. Thus the assessment of altitudinal varia-
The intraspecific cytomorphological variations build an tion in secondary metabolites, caused by ecological factors
important factor in the evolution of species. Phenotypic vari- shifting with the altitude of the thriving site, is imperative
ation enable plant species to adopt different growth forms [10]. It is obvious that genetic and environment interaction
while experiencing different environmental stresses [26]. are different in low and high altitude plants and hence there
Morphological variations reflect the phenotypic responses might be a great variation in the composition and the spec-
to environmental gradients and evolutionary history of the trum of phytochemicals [31, 32]. The extents to which plants
species and may indicate the local or regional changes. Gen- accumulate major bioactive phytoconstituents vary at the
erally, morphological variations occur due to differences species/cytotypes level and harbor a good potential for use
in environmental conditions [27], geographical isolation in both modern drug industry and traditional medicinal sys-
[28], and selection and/or genetic drift [29]. Morphometric tems. The present investigation was aimed to explore elite
variations involving qualitative and quantitative traits in P. chemotypes of species with desirable chemical profiles for

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JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577 575

Fig. 5  Calibration curve and

structure of standards atropine
(a, b), caffeic acid (c, d) and
chlorogenic acid (e, f)

pharmaceutical and commercial purposes. Previously, vari- the generation of ROS. Phenols and flavonoid contents vary
ations in the chemical constituents in response to different greatly by the ploidy level and parts of the plant, as is very
altitudes of Himalayas have also been reported for several much clear in the earlier studies [9]. Among tissue level,
plants including Berberis species, Picrorhiza kurroa and the high antioxidant property of the roots and leaves could
Berberis jaeschkeana, etc. [33–35]. We observed a higher be attributed to the presence of atropine and for this reason,
amount of phytoconstituents in stem, root and leaves in the roots and leaves of plants of this genus are commonly used
cytotypes growing at the higher elevation. The increased in medicine [36].
levels of total phenolics and favonoids in the present Mirzamatov et al. (1974) and Evans (1979) have reported
study could be directly correlated with the plant’s defense the identification and quantification of major bioactive com-
mechanism against temperature extremes and this has been pounds in some species of Physochlaina [37, 38]. How-
reported in previous studies [32] and could be a response to ever, the present study revealed variations in the content

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576 JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:567–577

Fig. 6  Antioxidant activity of
diploid and tetraploid leaf, stem
and roots of methanolic extracts

of phytoconstituents with respect to a definite ploidy level, Acknowledgements  Authors wish to thank the University Grant Com-
altitude and different plant parts in P. praealta which is novel mission, New Delhi for providing financial assistance under the DRS,
SAP I, II & III, and ASSIST Programme and also awarding Junior
to the world. A positive correlation between enhancement in Research Fellowship to Younas Rasheed Tantray (Award Letter No.
secondary metabolite content with ploidy level and altitude 2121430298 12/8/2015). Authors are also thankful to Head, Depart-
was noticed and these results were in strong agreement with ment of Botany, Punjabi University, Patiala for providing necessary
previous reports [9, 39–41]. laboratory, herbarium, and library facilities. Thanks are also due to
In-charge IPLS-DBT project (BT/PR 4548/INF/22/146/2012) for labo-
ratory facilities.

Compliance with ethical standards 

5 Conclusion
Conflict of interest  The authors declare that they have no conflict of
We hereby provide the first phytochemical description interests.
of secondary metabolites in Physochlaina praealta from
Ladakh region. Analysis of the TPC, TFC and DPPH radi-
cal scavenging activity of P. praealta extracts showed differ-
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