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Influence of soil variation on diosgenin content profile in Costus speciosus

(Koen. Ex Retz) from Indo‐Gangetic plains

Article in Chemistry & Biodiversity · April 2021

DOI: 10.1002/cbdv.202000977

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DOI: 10.1002/cbdv.202000977 FULL PAPER

Influence of Soil Variation on Diosgenin Content Profile in Costus

speciosus from Indo-Gangetic Plains
Poonam Rawat,a Manish Kumar,a Akanksha Srivastava,a Bhanu Kumar,a Ankita Misra,a
Satyendra Pratap Singh,a and Sharad Srivastava*a
a
Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow
226001, U.P., India, e-mail: [email protected]

Costus speciosus is a rich source of commercially important compound Diosgenin, distributed in different
regions of India. The present investigation was aimed to quantify diosgenin through High Performance Thin
Layer Chromatography in 34 germplasms of Costus speciosus and also to identify the superior sources and to
correlate the macronutrients of rhizospheric soil. The starch content varied in microscopic examination and
correlated inversely (r = 0.266) with diosgenin content. Findings revealed that the extraction process with acid
hydrolysis yielded higher diosgenin content (0.15 –1.88 %) as compared to non-hydrolysis (0.009–0.368 %)
procedure. Germplasms from Uttar Pradesh (NBCS-4), Jharkhand (NBCS-39) and Bihar (NBCS-2) were identified as
elite chemotypes based on hierarchical clustering analysis. The phosphorous content of respective rhizospheric
soil correlated positively (r = 0.742) with diosgenin content. Findings of present study are useful to identify the
new agrotechniques. The elite germplasms can also be used as quality planting material for large scale
cultivation in order to assure a sustained supply to the herbal drug industry.

Keywords: diosgenin, Costus speciosus, acid hydrolysis, chemotype, diversity.

stem in a spiral manner and the flowers are white with

Introduction
shiny reddish bracts. The plant is anchored by under-
Medicinal plants are gaining more attention to ground perennating rhizome which is the storehouse
pharmaceutical industries as they are thought to be a of bioactive metabolite diosgenin. Besides diosgenin,
sink of industry-oriented bioactive and therapeutic the rhizome of C. speciosus is also a good source of
components.[1] There is a huge demand of such curcumin and curcuminoids.[4] Ethnobotanically, the
bioactive compounds worldwide, for which plants are rhizomes are used to cure various ailments like
the only sources. Costus speciosus (Koen. Ex Retz) Sm. jaundice, bronchial asthma, dropsy, diabetes, leprosy
(syn. Cheilo Costus speciosus) (Costaceae) is emerging and, severe headache.[5] The cardiotonic, hydrochlo-
as a new source of steroidal sapogenin-diosgenin and retic and CNS depressant activities of rhizome have
the content is at par with Dioscorea spp.[2] The plant is also been reported.[4,6] Kannikars, the hill tribe of South
native to South-East Asia, widely distributed in the India, uses the rhizome of this plant as food and
regions of Asia, Africa, and America.[3] In India, it is medicine.[3] Inflammation, hyperlipidemia and acute
naturally growing almost in all the phytogeographical tonsillitis have been cured by the juice of rhizome.
zones having moist soil including Western and Eastern Other ethno-medico-botanical properties include
Himalayas, Gangetic plains, Eastern and Western ghats, treatment of acute pharyngitis, anti-adipogenesis, anti-
etc. C. spciosus starts growing in rainy season till angiogenic, antioxidant, antipyretic.[7]
October. Lanceolate leaves are arranged on the erect Diosgenin is a therapeutically and commercially
valuable molecule as it serves as a precursor for the
synthesis of corticosteroids, i. e., cortisone, progester-
Supporting information for this article is available on the one, and pregnenolone.[8] Moreover, investigations
WWW under https://doi.org/10.1002/cbdv.202000977 have reported the use of diosgenin as an anti-

Chem. Biodiversity 2021, 18, e2000977 © 2021 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2021, 18, e2000977

tumorigenic agent against various types of cancer Results and Discussion

through the activation of p53, induction of apoptosis,
Quality Parameters for Commercial Adequacy
cell cycle arrest at G1 phase and suppression of
metastasis.[9,10] Diosgenin also exhibits anti-allergic A total of 34 germplasms were collected at the
activity by the inhibition of IgE production, mast cell flowering stage from different locations and variable
infiltration and degranulation.[11] altitudes (26 to 2314 feet) of Indo-Gangetic plains
In C. speciosus diosgenin is majorly present in its (Table 1). The reason behind collection at a similar
glycosidic form as dioscin in different parts of the developing stage was to eliminate the other abiotic
plant, where aglycone molecule is attached with sugar and biotic factors that affect the plant metabolism.
moiety through a glycosidic bond.[12] The metabolic Furthermore, a database regarding the mapping of C.
variation of diosgenin in C. speciosus has been speciosus was very important to identify the elite
reported through various analytical methods such as germplasms and beneficial to rectify the issues
HPLC,[13] and HPTLC[14] however, such studies were subjected to site-specific commercial cultivation by
carried out in a localized population only. The effect of identification of natural conditions. Moreover, macro-
various abiotic factors on the production and accumu- scopically, the observations of C. speciosus germplasms
lation of secondary metabolites is well established[15] have evidenced that the tubers were irregularly
and thus, it is quintessential to elaborate the metabolic cylindrical, soft with a smooth surface, and pale brown,
variation of diosgenin existing in the natural popula- measuring from 3 –15 cm. Apart from that, non-
tion of C. speciosus. Indeed, it is well established that significant variations were found in morphological
the diosgenin content varies after acid hydrolysis (pre- features (i. e., shape and size of leaves, rhizomes, and
and post-acid hydrolyzed conditions) in the rhizome of flowers) in all the collected samples (Figure 1). These
C. speciosus.[16] samples were in healthy condition with no significant
In the wake of the therapeutic and industrial structural variation (Figure 1).
relevance of diosgenin, the chemotaxonomic studies Briefly, the transverse section of rhizome showed
on C. speciosus are indispensable for the sustainable an outermost uniseriate layer of epidermis consisting
availability of good quality raw material to the of compactly arranged barrel-shaped cells. Some of
industry. Hence, the present study was aimed to the epidermal cells possessed multi/unicellular hair-
analyze the intra-specific variation of diosgenin in the like structures. Under epidermis, variable layers of
natural population of C. speciosus (hydrolyzed extract) periderm, made of stratified cork cells were observed.
collected from different locations of Indo-Gangetic The periderm was 10– 15 cells thick in NBCS-03, NBCS-
plains for the identification of site-specific elite chemo- 42 and NBCS-141 whereas the periderm consist of 5–
types. The rhizospheric soil (from natural location of 10 cell thickness in NBCS-02, NBCS-47, NBCS-130 and
sampling) characteristics were also evaluated and NBCS-132 (Figure S1). Vascular bundles were localized
correlated with diosgenin content of the collected in cortex, endodermis, and central Steller region
germplasms. This study will provide baseline data (ground tissue). Xylem vessels were found surrounded
which will aid in site-specific commercial cultivation of by phloem cells (amphicribal type of vascular bundle).
elite germplasms of C.specious for sustainable avail- The cells of cortex and central Steller region were
ability of good quality raw material to the industry. polygonal, thin-walled parenchymatous with intercel-
Further, the edaphic factors affecting the production lular spaces. Starch granules densely filled in cells were
of diosgenin will be identified for their possible distributed particularly at both the sides of endoder-
implications in developing suitable agro-technologies mal layer surrounding the vascular bundles in the
for commercial cultivation of elite germplasms. This upper cortical region (Figure 2). Distribution of starch
study will be helpful in the promotion of conventional granules was comparatively lesser in peripheral region
methods for cultivation of the species, propagating of the rhizome (Figure 2, S1-1). NBCS-20, NBCS-23 and
good quality planting material for sustainable avail- NBCS-50 exhibited high starch density per unit area
ability of good quality raw material to meet out the while NBCS-17, NBCS-18, NBCS-28, NBCS-31, NBCS-32,
commercial demand. and NBCS-33 possessed low starch density per unit
area. Starch granules were elongated and oval; their
density was higher near to the endodermal region and
size was also smaller (ranging from area = 79.39 μm2 �
1.23 to 256.97 μm2 � 2.45) whereas they were bigger
(ranging from area = 356.42 μm2 � 2.05 to

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 Table 1. Passport data sheet of Costus speciosus (Koen. Ex. Retz.) samples collected from Indo-Gangetic plains.
S. No. Collection No. Voucher number Date of Collection Location/Dist./State Alt. (ft) Latitude Longitude
(N) (E)
1 NBCS-01 308001 3. 09. 2016 Ranchi, Jharkhand 2314 23°21’56.07’’ 85°49’51.94’’
2 NBCS-02 308002 8. 09. 2016 Pusa Campus, Samastipur, Bihar 172 25°58’55.59’’ 85°38’55.05’’
3 NBCS-03 308003 10. 09. 2016 Baghi devi temple, Sitamarhi, Bihar 221 26°35’42.89’’ 85°28’51.02’’

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4 NBCS-04 308004 10. 09. 2016 Bagha, Lakhimpur, U.P. 519 28°00’11.69’’ 80°25’47.53’’
5 NBCS-36 308036 12. 08. 2017 Birsa Agriculture University Ranchi, Jharkhand 2081 23°25’00.02’’ 85°19’00.31’’
6 NBCS-37 308037 13. 08. 2017 Rajrappa, Ramgarh, Jharkhand 930 23°37’54.07’’ 85°42’38.03’’
7 NBCS-38 308038 13. 08. 2017 Paterwar, Bokaro, Jharkhand 778 23°36’39.62’’ 85°51’07.12’’
8 NBCS-39 308039 13. 08. 2017 Rajrappa, Ramgarh, Jharkhand 930 23°37’54.07’’ 85°42’38.03’’
9 NBCS-40 308040 15. 08. 2017 Bokaro, Jharkhand 702 23°40’09.46’’ 86°09’04.00’’
10 NBCS-41 308041 16. 08. 2017 Topchanchi, Dhanbad, Jharkhand 981 23°54’02.58’’ 86°11’25.56’’
11 NBCS-42 308042 17. 08. 2017 Dhanbad, Jharkhand 802 23°47’44.35’’ 86°25’49.39’’
12 NBCS-43 308043 18. 08. 2017 Ranka Forest Garhwa, Jharkhand 1143 23°59’29.49’’ 83°47’10.66’’
13 NBCS-46 308046 29. 08. 2017 Ahmad Nagar, Lakhimpur, U.P. 521 28°05’21.96’’ 80°25’07.47’’
14 NBCS-47 308047 29. 08. 2017 Ahmad Nagar, Lakhimpur, U.P. 521 28°05’21.96’’ 80°25’07.47’’
15 NBCS-50 308050 10. 09. 2017 Bhagalpur, Bihar 128 25°20’52.08’’ 86°58’56.74’’
16 NBCS-57 308057 28. 10. 2017 Chahaniya, Chandauli, U.P 263 25°25’08.05’’ 83°12’42.41’’
17 NBCS-58 308058 29. 10. 2017 Jaugarh, Sakteshgarh, Mirzapur, U.P 636 24°57’44.38’’ 82°48’56.11’’
18 NBCS-59 308059 29. 10. 2017 BHU, Varanasi, U.P. 281 25°16’03.79’’ 82°59’28.53’’

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19 NBCS-64 308064 10. 12. 2017 Kakdwip, West Bengal 26 21°52’33.70’’ 88°11’06.99’’
20 NBCS-65 308065 11. 12. 2017 Kharagpur, West Bengal 174 22°20’45.64’’ 87°13’55.11’’
21 NBCS-66 308066 12. 12. 2017 Bankura, West Bengal 294 23°13’56.69’’ 87°04’42.98’’
22 NBCS-67 308067 15. 12. 2017 Murshidabad, West Bengal 70 24°10’33.25’’ 88°16’48.64’’
23 NBCS-68 308068 16. 12. 2017 Pakur, West Bengal 278 24°33’13.25’’ 87°39’29.21’’
24 NBCS-69 308069 17. 12. 2017 Maldatown, West Bengal 103 25°00’39.03’’ 88°08’27.95’’
25 NBCS-70 308070 18. 12. 2017 Katihar, West Bengal 108 25°32’55.83’’ 87°37’07.10’’
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26 NBCS-93 308093 28. 07. 2018 Kasta, Lakhimpur, U.P. 497 27°50’51.82’ 80°31’47.83’
27 NBCS-132 308094 1 .09. 2018 Dalma, Jamshedpur, Jharkhand 714 22° 53’47.02’’ 86°12’23.46’’
28 NBCS-133 308095 5. 09. 2018 Saraikela, Jharkhand 167 22°43’12.50’’ 85°55’44.38’’
29 NBCS-135 308096 7. 09. 2018 Dumaria forest, Jharkhand 366 22°24’10.12’’ 86°31’38.57’’
30 NBCS-136 308097 21. 09. 2018 Duarsini forest, West bengal 141 22°45’49.78’’ 86°27’04.19’’
31 NBCS-138 308098 15. 09. 2018 Gamharia, Jamshedpur, Jharkhand 176 22°49’10.56’’° 86°06’11.27’’
32 NBCS-139 308099 10. 09. 2018 Chandil forest, Jamshedpur, Jharkhand 236 22°57’29.48’’ 86°10’23.33’’
33 NBCS-140 308100 13. 09. 2018 Banka forest, Jharkhand 277 24°33’13.09’’ 86°34’30.43’’
34 NBCS-141 308101 29. 09. 2018 Gorumahisani, Odisha 294 22°18’59.82’’ 86°13’07.81’’

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 Chem. Biodiversity 2021, 18, e2000977

Figure 1. Morphology of Costus speciosus. a- Vegetative plant, b- Rhizome.

Figure 2. Prototype of anatomical features of Costus speciosus (Koen.) (NBCS-40). Plate (a)- Showing trichome (bar size- 100 μm),
Plate (b)- vascular bundle, surrounded by starch granules (bar size- 100 μm), Plate (c)- overview of cortical vascular bundle,
endodermis, and ground tissue (bar size- 100 μm), Plate (d)- showing multi-storied cork cells (bar size- 500 μm). Abbreviation: ep –
Epidermis, pd – periderm, tr – trichome, ct – cortex, cvb – cortical vascular bundle, st – starch grains, ed – endodermis, evb –
endodermal vascular bundle, xy – xylem, ph – pholem.

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 Chem. Biodiversity 2021, 18, e2000977

458.15 μm2 � 2.68) in size in regions where density almost similar environmental conditions. It indicates
was less such as in ground tissue region. The present that there might be some microclimatic factors or
investigation is well corroborated with recent findings endophytic microflora playing role in the synthesis
of Kumar et al.[17] that also reported the presence of and accumulation of starch in the rhizomes. The
starch granules in cortical and pith regions during the results of pharmacognostic standards were found to
microscopic observations of C. speciosus germplasms. be within the limits of Ayurvedic Pharmacopoeia of
Whereas another research on the microscopical study India which signifies the identity, purity, and strength
of C. speciosus rhizome[18] reported the absence of of the collected samples.
starch granules. In a few samples like NBCS-1, NBCS-
16, NBCS-17, NBCS-25 and NBCS-29, oleoresin was
Variability in Diosgenin Content
found at certain places in cortical region.
Pharmacognostic standards are a measure of Densitometric scanning of C. speciosus samples re-
quality and the authenticity of raw material. Any vealed that there is a considerable variation in
spurious or adulterated raw material can be identified diosgenin content of methanolic extract (ME) and acid
with the help of these parameters. In the present hydrolyzed methanolic extract (AME) of each sample
study, Loss on drying (LOD) of collected samples (Figure 3). On analyzing ME (10 mg ml 1) of collected
ranged from 5.14 % to 11.4 % (Figure S2). Extractive samples, diosgenin content ranged from 0.009 % �
values viz, water soluble, alcohol soluble and hexane 0.02 to 0.368 % � 0.03. Minimum content was recorded
soluble ranged from 2.1 % to 15.9 %, 1.1 % to 6.9 % and in NBCS-50 from Bhagalpur (Bihar) and the maximum
1.1 % to 7.1 %, respectively (Figure S3). The evaluated was in NBCS-37 from Rajrappa (Ramgarh, Jharkhand).
values for LOD and extractive values were found quite Further, densitometric scanning of AME (1 mg ml 1)
higher than reported previously[14] which further revealed that diosgenin (active) content ranged from
validated the superior quality of collected germplasms. 0.151 % � 0.01 to 1.877 % � 0.03. The highest and low-
Total starch content in the collected samples ranged est content was reported in NBCS-4 from Bagha
from 0.6 %–4.8 %. It was found that in the rhizomes (Lakhimpur, U.P.) and NBCS-133 (Saraikela, Jharkhand).
collected from different locations of Indo-Gangetic The maximum diosgenin content reported earlier was
plains contain variable amount of starch in spite of found to be 4.78 % in C. speciosus collected from

Figure 3. HPTLC chromatogram and overlay spectra of 34 germplasms of Costus speciosus. Plate (a)- pre-acid hydrolysis, plate (b)-
post-acid hydrolysis. Plates are scanned at λmax 440 nm and visualized after derivatizing with anisaldehyde sulfuric acid reagent.

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 Chem. Biodiversity 2021, 18, e2000977

Maharashtra region of India.[19] Results of increased It was also observed that multiple overlapping
diosgenin yield in the present study are in agreement bands were found in samples prior to hydrolysis
with previous reports.[20,21] whereas, the resolution was clearer with lesser number
Literature suggests that diosgenin is synthesized in of bands after hydrolysis (Figure 3). Noticeably, the
its glycosidic form through phenylpropanoid pathway sample having highest amount of diosgenin before
in the leaves and then, translocated to other plant acid treatment was not necessarily having highest
parts, highest concentration being in the rhizomes.[22] amount after treatment (Figure 5). Further, the results
The glycosidic form of diosgenin is pharmacologically of this study also indicate that starch and diosgenin
less active and thus, for the production of active content are inversely related (r = 0.266) to each other
molecule the glycosidic bond needs to be hydrolyzed in all the collected germplasms. Diosgenin content in
through acids,[16] enzymes[23] and, microorganisms.[22] NBCS-50 was reported to be least among all the
It is well-known that glycosides have potential samples while starch content was highest (Figure 6,
therapeutic values because of the aglycone component, Figure 7). Although, the role of starch in the biosyn-
whereas sugar moiety is responsible for the absorption thesis of diosgenin is not reported yet. These are some
and transportation of aglycone molecule to the site of primary observations about the interrelationship of
action.[24,25] To quantify the diosgenin, acid hydrolysis is starch and active diosgenin, however, an extensive
one of the preferred methods. In this context, it was study is needed for some concrete output.
found that the extractive yield of each sample in its un- The findings based on the diosgenin content were
hydrolyzed state was relatively higher than its hydrolyzed further evaluated by hierarchical cluster analysis. A
state (Figure 4). The possible reason for this may be the dendrogram bifurcated the population into two
loss of associated metabolites like fiber, sugar, and starch branches separating NBCS-04, NBCS-39 and NBCS-2 in
content due to repeated washing of the hydrolyzed one cluster and rest of the samples in another cluster
sample. The compounds soluble in water may get eluted (Figure 8). Clustering of NBCS-04 (Lakhimpur, U.P.), NBCS-
out during the complete process of acid hydrolysis. The 39 (Ramgarh, Jharkhand) and NBCS-2 (Samastipur, Bihar)
similar observation was also reported earlier by Azmi together implies the high yielding samples and are
et al.[26]

Figure 4. Comparative extractive yield (mg) of Costus speciosus, collected from Indo-Gangetic plain. Values are presented as mean
� standard deviation (n = 3). Significant (p > 0.05) difference was found in the extractive yield before and after acid hydrolysis.

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 Chem. Biodiversity 2021, 18, e2000977

Figure 5. Comparative diosgenin content (%) of Costus speciosus, collected from Indo-Gangetic plain. Values are presented as mean
� standard deviation (n = 3). Significant (p > 0.05) difference was found in the diosgenin content in each sample before and after
acid hydrolysis.

Figure 6. Variation of starch content in Costus speciosus germplasms. Sections were stained with Lugol. Plate (a)- Showing low
density of starch grains (NBCS-04) in endodermal region (10X, bar size- 100 μm), Plate (b)- Showing high density of starch grains
(NBCS-50) in endodermal region (10X, bar size-100 μm)

identified as superior sources of diosgenin in Indo- ing absorption spectra of samples which confirms the
Gangetic plains. specificity of diosgenin in plant samples and standard.
Peak purity of diosgenin was tested by comparing
spectra at the start, middle and end points. To
Standardization of HPTLC Conditions
determine the sensitivity and selectiveness of method,
Superimposable spectrum was obtained by comparing limit of detection and limit of quantitation were
the absorption spectrum of standard with correspond- calculated with a formula of standard deviation and

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 Chem. Biodiversity 2021, 18, e2000977

Figure 7. Inverse relation of starch and diosgenin content in rhizomes of Costus speciosus. Values are presented as mean � standard
deviation (n = 3). Starch and diosgenin content are showing negative correlation (r = 0.266).

Figure 8. UPGMA dendrogram of Costus speciosus population for identifying elite chemotype(s), based on diosgenin content.

reported to be 8.532 ng spot 1 and 25.856 ng spot 1, and were expressed as per cent relative standard
respectively (Table S1). A calibration curve for the deviation (RSD).
marker compound was developed and was found to For Intra-day precision, the absorption spectrum of
be linear with respect to the peak area ranging from individual sample was analyzed thrice in a day. Like-
0.2 to 1 μg spot 1. Regression coefficient (R2) was wise, inter-day repeatability was analyzed over three
found to be 0.997, indicating a linear relationship consecutive days, three times a day at a single level.
between standard area vs. concentration. Inter-day The precision (% RSD) of developed method was
and Intra-day studies were carried out to check the found within the limit of standards, i. e., less than 5 %
precision of method at a single level (0.2 μg spot 1) (Table 2). The accuracy of method was tested by

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 Chem. Biodiversity 2021, 18, e2000977

Table 2. Inter-day and intra-day precision studies for validation of HPTLC method.
Population code Statistical value* Intra-day Inter-day
Day 1 Day 2 Day 3
NBCS-04 S.D. 0.0008 0.0008 0.0002 0.0004
Average 0.0287 0.0287 0.0273 0.0272
RSD (%) 2.901 2.901 0.993 1.807
NBCS-02 S.D. 0.0005 0.0005 0.0005 0.0003
Average 0.0379 0.0379 0.0369 0.0361
RSD (%) 1.427 1.427 1.582 0.888
NBCS-39 S.D. 0.0003 0.0003 0.0002 0.0008
Average 0.03264 0.0326 0.032 0.0312
RSD (%) 0.947 0.947 0.921 2.601
* SD = Standard Deviation, RSD = Relative Standard Deviation, values are mean � SD (n = 3).

recovery of standard diosgenin (0.425 ng spot 1) in consequently induce the synthesis of diosgenin in C.
sample and was ranging from 97.678 to 99.29 % speciosus.
(Table 3). The method was also tested for robustness The pH and electrical conductivity (EC) of rhizo-
by slight deliberate changes in the composition of spheric soil were ranging from 6.8 to 7.7 and 73 to
mobile phase and observed to be robust. Hence, the 358 μs, respectively. The pH ranging from 5.7 to 7.5 is
method was found linear, accurate, precise, and robust reported as optimum for the commercial cultivation of
for the quantification of diosgenin in C. speciosus and C. speciosus.[30] This might be the possible reason
other herbal formulations containing diosgenin. behind the non-significant correlation of diosgenin
with soil pH. Moreover, difference in the pattern of
diosgenin content and EC of rhizospheric soil was
Abiotic Factors and their Correlation with Diosgenin
possibly attributed to the wide range of environmental
Content
parameters.[31]
The macronutrient profiling of rhizospheric soil of the Previous reports showed that variability in metabol-
respective plant samples demonstrated a variable ic content in plants within phytogeography is directly
range of macronutrients, ranging from 69 mg kg 1 to associated with the mineral elements present in it.[32]
994.6 mg kg 1 Potassium (K) (henceforth, elements are The crucial role of soil macro-micro nutrients in the
represented with symbols in bracket), 39.46 kg ha 1 to growth, development and production of secondary
98.47 kg ha 1 (P), 0.024 % to 0.077 % (N) and 2.026 % to metabolites has been reported.[33] There are several
14.844 % (C) (Table 4). Out of these, the (P) content of edaphic factors such as macro- and micro-nutrients
rhizospheric soil was significantly (r = 0.742) correlated along with soil physical parameters significantly affect
with the diosgenin content of samples (Table 5). The the plant metabolite content because these factors
findings of current study corroborated with previous perform a decisive role in not only plant fitness but
reports that revealed the importance of (P) in the also as a building block for primary and secondary
preparation of intermediate products (as a structural metabolites.[34] This can be inferred from the results
component) in mevalonate (cytosolic) and meth- obtained in the study that the nutrient level may also
ylerythritol phosphate (plastidial) pathways[27,28,29] and

Table 3. Recovery studies of diosgenin through proposed HPTLC method.

Sample Amount of Amount of Theoretical Observed Recovery Average SD* Mean
standard standard value value (%) recovery RSD*
(μg) added (μg) (μg) (%) (%)
(%)
NBCS-04 0.425 80 2.925 2.857 97.678
0.425 100 3.625 3.599 99.296 98.630 0.84 0.856
0.425 120 4.225 4.179 98.915
* SD = Standard Deviation, RSD = Relative Standard Deviation, values are mean � SD (n = 3).

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 Chem. Biodiversity 2021, 18, e2000977

Table 4. Analysis of rhizospheric soil from respective locations of germplasms collected from Indo-Gangetic plains.
Samples Soil type K (mg/kg) P N OC pH E.C (μs)
(kg/Ha) (%) (%)
NBCS-01 Loamy & clayey 102 80.21 0.054 2.432 6.8 191
NBCS-02 Alluvial 130.2 96.21 0.047 3.447 7 137
NBCS-03 Loamy & clayey 150.2 64.31 0.050 2.133 6.9 102
NBCS-04 Alluvial 120.5 98.47 0.057 4.041 6.8 136
NBCS-36 Loamy & clayey 115.1 55.16 0.050 6.305 7.2 224
NBCS-37 Loamy & clayey 104.3 82.14 0.043 2.026 7.1 198
NBCS-38 Loamy & clayey 85.5 39.46 0.057 2.602 7.1 110
NBCS-39 Alluvial 301.4 95.45 0.077 2.787 6.9 358
NBCS-40 Loamy & clayey 348 64.45 0.050 2.121 7.1 174
NBCS-41 Loamy & clayey 305.6 91.94 0.050 2.713 7.1 195
NBCS-42 Loamy & clayey 310.8 50.69 0.043 4.589 6.7 125
NBCS-43 Loamy & clayey 302 44.34 0.045 6.044 6.9 116
NBCS-46 Loamy & clayey 78.5 89.4 0.043 2.396 7.2 189
NBCS-47 Loamy & clayey 74.6 86.4 0.053 3.019 7.3 200
NBCS-50 Alluvial 70 56.14 0.050 2.714 6.9 252
NBCS-57 Alluvial 118.4 67.42 0.048 11.15 6.8 145
NBCS-58 Gravel Sandy 113.5 67.12 0.046 3.814 7.2 242
NBCS-59 Red Sandy 53.9 68.41 0.058 4.119 7.3 115
NBCS-64 Sandy 205.1 69.67 0.049 2.356 7.4 104
NBCS-65 Red Sandy stony 140.2 92.41 0.049 3.295 7.7 208
NBCS-66 Red Sandy stony 160.7 78.45 0.045 6.057 7.5 96
NBCS-67 Red Sandy stony 147.1 84.12 0.045 2.2147 7.4 110
NBCS-68 Red Sandy stony 177.1 64.12 0.068 6.729 7.1 104
NBCS-69 Red Sandy stony 152 92.14 0.064 3.651 6.8 108
NBCS-70 Red Sandy stony 141 88.91 0.065 7.924 7.1 165
NBCS-93 Red Sandy stony 69 69.45 0.058 5.921 6.8 73
NBCS-132 Loamy & clayey 120.9 67.45 0.035 3.145 7.1 156
NBCS-133 Red Sandy stony 159 65.45 0.064 5.921 7 139
NBCS-135 Loamy & clayey 101 84.45 0.024 2.950 6.9 98
NBCS-136 Loamy & clayey 99.5 87.45 0.024 2.680 6.8 115
NBCS-138 Red Sandy 994.6 63.75 0.067 3.8147 7.4 124
NBCS-139 Loamy & clayey 115.3 89.25 0.025 4.119 7.25 113
NBCS-140 Loamy & clayey 127 64 0.065 14.844 7.1 112
NBCS-141 Loamy & clayey 127.4 68.52 0.365 5.153 6.9 111

Table 5. Co-relation matrix between diosgenin content of 34 germplasms of Costus speciosus and abiotic factors of soil from
respective locations.
K P N OC Diosgenin (%) Starch pH EC
(mg/kg) (kg/Ha) (%) (%) (μs)
K (mg/kg) 1
P (kg/Ha) 0.124 1
N (%) 0.021 0.101 1
OC (%) 6E-17 2E-15 2E-17 1
Diosgenin (%) 0.006 0.742 0.111 6E-18 1
Starch 0.155 0.186 0.004 8E-17 0.26605 1
pH 0.217 0.036 0.148 6E-15 0.0674 0.163 1
EC (μs) 0.016 0.195 0.076 3E-16 0.228984 0.303 0.03 1

play a crucial role in the diosgenin content in C.

speciosus.

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 Chem. Biodiversity 2021, 18, e2000977

An Approach Towards Agro-Techniques for the speciosus. Acid hydrolysis was found to be a suitable
Cultivation of C. speciosus method for obtaining a higher yield of diosgenin in
free form and this method can be a better option for
Diosgenin is serving as a precursor for about 60 % of industrial-scale extraction of diosgenin. A significant
all the steroidal drugs and currently, the world positive correlation between soil phosphorus and
demand for diosgenin is 55,000 –75,000 kg year 1. In diosgenin content was found. This indicates the
India, Pharmaceutical industries are completely de- development of site-specific agro-techniques at differ-
pendent on diosgenin for the commercial production ent phytogeographical zones for developing quality
of steroidal drugs.[35] planting material leading to quality raw material. The
Being a stockpile of industrially valuable metabo- study also expresses the need for the initiation of
lite, C. speciosus has been exploited so much that it is cultivation practices of C. speciosus to conserve the
now counted in a near-threatened category.[36] Efforts plant and to ensure their sustainable availability to the
for in situ conservation of this medicinal plant is being phytopharmaceutical industries.
undertaken to conserve it. With an eye towards mass
propagation, biotechnological approaches like micro-
propagation, somatic embryogenesis and cryopreser- Experimental Section
vation etc. have been initiated for sustainable produc-
Chemicals and Reagents
tivity in terms of its bioactive metabolites.[1] However,
it is found that cultivation of C. speciosus in the natural The marker compound diosgenin was procured from
conditions has been neglected due to its poor ChromaDex Inc., USA and HPTLC pre-coated silica gel
regeneration capacity and very slow propagation.[3] A 60 GF254 (20 × 20) plates were purchased from Merck,
previous report suggests that since 1980,[37] there is no India. All the chemicals and reagents used were of
concerted effort for the commercial or large scale analytical grade and procured from Thermo Fischer,
cultivation of this plant. Mumbai (India).
In the present investigation, it has been tried to
find out the suitable environmental conditions, favor-
Collection of Plant Samples
able edaphic characteristics, and specific sites for
conventional cultivation of C. speciosus for high yield Samples of C. speciosus (rhizome) were collected from
of diosgenin. The findings of present investigation July to December from Indo-Gangetic plain in the year
suggested that (P) is very crucial for the synthesis of 2016 –2018, covering the four states of India namely
diosgenin, as the three high diosgenin containing Uttar Pradesh, West Bengal, Bihar, and Jharkhand.
samples identified through cluster analysis possessed Guidelines of good agricultural and field collection
high amount of (P) in their respective rhizospheric soil practices of World Health Organisation were followed
namely, NBCS-04 (98.47 kg Ha 1) followed by NBCS-02 for the collection of germplasms.[38] Identification and
(96.21 kg Ha 1) and NBCS-39 (95.45 kg Ha 1). Whereas authentication of plant samples were done by Dr.
(K) and (N) content in soil were not found to so any Sharad Srivastava (Senior Principal Scientist, Pharma-
positive correlation with diosgenin content. All the cognosy Division, CSIR-NBRI, Lucknow). A passport
three high diosgenin yielding germplasms were found data sheet was prepared for each sample. The
to be growing under optimum pH conditions as earlier herbarium specimen of each sample was deposited in
reported.[30] The EC of the soil does not affect the the Herbarium of CSIR-National Botanical Research
diosgenin content in samples. Therefore, (P) rich soil Institute, Lucknow, India (LWG), with individual field
with optimum pH can be recommended as an agro- specimen number (Table 1). The collected rhizomes
technology to fortify the soil for increasing the yield of were washed, chopped, and dried in a hot air oven at
diosgenin. 40 °C and then, powdered (mesh No. 40) with an
electric grinder.

Conclusions Pharmacognostic Evaluation of C. speciosus Rhizome

The present investigation established that C. speciosus The anatomy was done as per the standard protocol[39]
is a rich source of diosgenin. The variations in micro- with slight modifications. The fresh samples were
climatic conditions even within specific phytogeogra- washed and preserved in ethanol (70 %) for anatomical
phy significantly affect the synthesis of diosgenin in C. studies. Rhizomes were hand sectioned to get trans-

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 Chem. Biodiversity 2021, 18, e2000977

verse sections. The thin sections were dehydrated by solution in methanol. The working solutions of
passing through a gradient of alcohol. Staining was standard and plant extracts were filtered through a
done with ethanolic safranin (50 %) for 30 seconds Millipore membrane filter (0.45 mm), (Pall, USA).
followed by ethanolic fast green (90 %). The sections
were then treated with a series of xylene (10– 100 %)
HPTLC Instrumentation and Chromatographic Condi-
and finally mounted with Canada balsam. Photomicro-
tions
graphs were taken with Eclipse 80i, Nikon Instruments
Inc., America. The pharmacognostic parameters, i. e., TLC fingerprinting for quantification of diosgenin was
moisture content, water soluble extractive, alcohol performed through CAMAG visionCATS software on
soluble extractive and hexane soluble extractive were HPTLC (CAMAG, Switzerland), consisting of Linomat V
determined in each sample as per standard protocols sample applicator, CAMAG ADC-2 development cham-
of Ayurvedic Pharmacopoeia of India.[40] The total ber (20 × 10 cm) and TLC Scanner III. The silica gel 60
starch content in each sample was estimated through GF254 (20 × 10 cm) plates were washed thrice with
Mont Gomery method.[41] methanol and activated (120 °C for 20 min) before
sample application. 6 mm bands of sample (10 μL)
were applied, positioned 10 mm from the bottom and
High-Performance Thin Layer Chromatography (HPTLC) 15 mm from side of the plate. Linear ascending
development was carried out with a binary mobile
Extract Preparation
phase, consisted of hexane and ethyl acetate (7 : 2, v/v)
Powdered rhizome (5 g) was cold macerated with in CAMAG automated developing chamber, pre-satu-
methanol, kept on a rotatory shaker for 6 h and then, rated with the vapors of mobile phase for 20 min at
allowed to stand at room temperature for 18 h. The room temperature (27 °C � 2) for better resolution. The
process was repeated thrice and the solution was scanning of plate was performed at λmax 440 nm using
filtered, pooled filtrates were dried under reduced TLC Scanner III with a slit width of 5 × 0.45 mm,
pressure in a rotatory evaporator (Büchi, Switzerland) scanning speed 20 mm/s. Quantification of the marker
at 40 °C. The concentrated methanolic extract (ME) was done on the basis of standard area unit versus
was freeze-dried (Labconco, USA) and stored at 4 °C concentration and expressed on per cent dry weight
for HPTLC analysis. The yield of methanol extract was basis of raw drug.
calculated and expressed as a % value on dry weight
basis.
Validation of HPTLC Method
The method for High-Performance Thin Layer Chroma-
Acid Hydrolysis of C. speciosus Rhizome
tography was validated according to the ICH guide-
The powdered sample of rhizome was acid hydrolyzed lines under which various parameters such as specific-
as per the method of Jahfar et al.[16] with slight ity, linearity, sensitivity, accuracy, recovery, precision,
modifications. Powdered rhizome (5 g) of C. speciosus and robustness were evaluated.[42]
was refluxed with HCl (50 mL, 2.5 N) for 4 h. The
mixture was cooled, filtered, and washed with hot
Analysis of Abiotic Factors in Soil
water until the residue was deprotonated up to pH 7.
The residue was allowed to dry and further extracted The content of macroelements in rhizospheric soil of
with methanol up to complete extraction. The yield of each sample was analyzed as per standard protocols.
acid hydrolyzed methanolic extract (AME) of each Nitrogen content (N) was estimated through auto-
sample was calculated and expressed as a % value. analyzer,[43] phosphorus content (P) was analyzed
through spectrophotometer[44] and, potassium content
(K) was evaluated by a flame photometer.[45] Various
Preparation of Stock and Working Solutions
physical properties of rhizospheric soil, viz, organic
The stock solution of standard diosgenin was prepared carbon content (OC), pH and electrical conductivity
by dissolving it (1 mg) in analytical grade methanol (EC) were also analyzed as per the method of
(1 ml, 99.5 %) and was diluted further to prepare Faulkneret al.[46]
working solution (0.1 mg ml 1) on the day of analysis.
For HPTLC analysis, sufficient amount of ME and AME
of each sample was weighed to prepare 10 mg ml 1

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 Chem. Biodiversity 2021, 18, e2000977

[6] M. N. Prabhu, P. G. Pai, S. V. Kumar, A. Gaurav, C. S. D’Silva,

Statistical Analysis S. D. Ullal, K. A. Shenoy, D. S. Sushma, ‘Diuretic activity of
aqueous ethanolic extract of leaves of Costus speciosus in
Analysis of each sample was done in triplicate. The
normal Wistar albino rats’, Res. J. Pharm. Biol. Chem. Sci.
results were recorded as mean � standard deviation 2014, 5, 127 –132.
(SD) and analysis of variance (ANOVA) was used to [7] P. Muthukumaran, G. Thiyagarajan, R. A. Babu, B. S.
calculate the critical f value. The statistical significance Lakshmi, ‘Raffinose from Costus speciosus attenuates lipid
for analyzed diosgenin content was calculated using synthesis through modulation of PPARs/SREBP1c and
GraphPad Prism (GraphPad Software Inc., San Diego, improves insulin sensitivity through PI3 K/AKT’, Chem.-Biol.
Int. 2018, 284, 80 –89.
CA, USA), the difference was considered significant at [8] Y. A. Jasem, M. Khan, A. Taha, T. Thiemann, ‘Preparation of
P < 0.05. steroidal hormones with an emphasis on transformations
of phytosterols and cholesterol – a review’, Mediterr. J.
Chem. 2014, 3, 796–830.
[9] D. S. Kim, B. K. Jeon, Y. E. Lee, W. H. Woo, Y. J. Mun,
Acknowledgements ‘Diosgenin induces apoptosis in HepG2 cells through
generation of reactive oxygen species and mitochondrial
The authors are thankful to the Director CSIR-NBRI for pathway’, Evid.-Based Complement. Altern. Med. 2012,
providing facilities and inspiration all through the 981675.
work (CSIR-NBRI_MS/2020/07/03). Authors are also [10] S. Selim, S. A. Jaouni, ‘Anticancer and apoptotic effects on
thankful to NASF, Indian Council of Agricultural cell proliferation of diosgenin isolated from Costus specio-
Research, New Delhi, India, for financial support sus (Koen.) Sm’, BMC Complement. Altern. Med. 2015, 15,
301.
through project No. NASF/ABA-6009-2016-17/350.
[11] C. H. Huang, C. Y. Ku, T. R. Jan, ‘Diosgenin attenuates
allergen-induced intestinal inflammation and IgE produc-
tion in a murine model of food allergy’, Planta Med. 2009,
75, 1300 –1305.
Author Contribution Statement [12] J. Raju, C. V. Rao, ‘Diosgenin, a steroid saponin constituent
Sharad Srivastava designed and conceptualized the of yams and fenugreek: emerging evidence for applica-
tions in medicine’, Bioact. Compd. Phytomed. 2012, 125,
study, collected, and authenticated the plant sample,
143.
finalized manuscript draft. Poonam Rawat performed [13] J. Karthikeyan, V. Reka, R. V. Giftson, ‘Characterization of
the acid hydrolysis, prepared plant extracts, performed bioactive compounds in Costus speciosus (Koen.) by reverse
and validated the HPTLC, performed microscopy of the phase HPLC’, Int. J. Pharm. Sci. Res. 2012, 3, 1461– 1465.
plant samples, wrote original draft. Manish Kumar [14] N. Verma, R. L. Khosa, ‘Development of standardization
collected the plant and soil samples, performed soil parameters of Costus speciosus rhizomes with special
reference to its pharmacognostical and HPTLC studies’,
analysis. Akanksha Srivastava performed soil analysis. Asian Pac. J. Trop. Med. 2012, 2, S276-S283.
Bhanu Kumar collected the plant and soil samples, [15] B. Ncube, J. F. Finnie, V. J. Staden, ‘Quality from the field:
corrected the manuscript draft. Ankita Misra analyzed The impact of environmental factors as quality determi-
data, helped in planning and interpretation of experi- nants in medicinal plants’, S. Afr. J. Bot. 2012, 82, 11– 20.
ments, corrected draft manuscript. Satyendra Pratap [16] M. Jahfar, A. K. A. Rahim, A. Jincy, K. P. Unnikrishnan, ‘Assay
Singh analyzed data, corrected draft manuscript. of Steroidal Sapogenin in Costus speciosus rhizomes’, Asian
J. Chem. 2008, 20, 1382 –1388.
[17] M. Kumar, A. Misra, A. Srivastava, P. K. Shukla, L. M. Tewari,
S. Srivastava, ‘Comparative Pharmacognostical and Phar-
References macological Evaluation of Costus speciosus (Koen) JE Sm.
Germplasm Collected from Eastern Ghats of India’, Pharma-
[1] P. Maji, D. G. Dhar, P. Misra, P. Dhar, ‘Costus speciosus (Koen cogenomics J. 2020, 12, 1.
ex. Retz.) Sm.: Current status and future industrial [18] C. Kala, S. S. Ali, S. Chaudhary, ‘Comparative pharmacog-
prospects’, Ind. Crops Prod. 2020, 152. nostical evaluation of Costus speciosus (Wild Ginger) and
[2] B. Dasgupta, V. B. Pandey, ‘A new Indian source of Zingiber officinale (Ginger) rhizome’, Int. J. Curr. Pharm.
diosgenin (Costus speciosus)’, Experientia 1970, 26, 475 – Res. 2016, 8, 19 –23.
476. [19] J. Y. Nehete, M. S. Bhatia, ‘Quantitative analysis of diosge-
[3] V. A. Pawar, P. R. Pawar, ‘Costus speciosus: An Important nin in Costus speciosus extracts by HPTLC with ultraviolet
Medicinal Plant’, Int. J. Sci. Res. 2014, 3, 28 –33. absorption densitometry’, Anal. Chem.: Indien J. 2008, 7,
[4] S. Srivastava, P. Singh, G. Mishra, K. K. Jha, R. L. Khosa, 879 –883.
‘Costus speciosus (Keukand): A review’, Pharm. Sin. 2011, 2, [20] Y. Peng, Y. Wang, Z. Yang, J. Bao, H. Peng, ‘Content
118 –128. Increase of Spirostanol Saponins during Enzymatic Hydrol-
[5] R. K. Gupta, ‘Medicinal and Aromatic Plants’, CBS Publishers ysis of Dioscorea zingiberensis C. H. Wright.’, Ind. Eng. Chem.
and Distributors 2010, 234, 499. Res. 2010, 49, 8279 –8281.

www.cb.wiley.com (13 of 14) e2000977 © 2021 Wiley-VHCA AG, Zurich, Switzerland

 Chem. Biodiversity 2021, 18, e2000977

[21] Y. L. Zhu, W. Huang, J. R. Ni, W. Liu, H. Li, ‘Production of fiber: A review’, Renewable Sustainable Energy Rev. 2015,
Diosgenin from Dioscorea zingiberensis tubers through 43, 1006 –1015.
enzymatic saccharification and microbial transformation’, [34] A. Hassan, ‘Effect of mineral nutrients on physiological and
Appl. Microbiol. Biotechnol. 2009, 85, 1409–1416. biochemical processes related to secondary metabolites
[22] B. Cai, Y. Zhang, Z. Wang, Z. D. Xu, Y. Jia, Y. Guan, A. Liao, production in medicinal herbs’, Med. Aromat. Plant Sci.
G. Liu, C. Chun, J. Li, ‘Therapeutic Potential of Diosgenin Biotechnol. 2012, 6, 105–110.
and Its Major Derivatives against Neurological Diseases: [35] H. A. Deshpande, S. R. Bhalsing, ‘Plant derived novel
Recent Advances’, Oxid. Med. Cell Longevity 2020, 3153082. biomedicinal: diosgenin’, Int. J. Pharmacogn. Phytochem.
[23] M. Wei, Y. Bai, M. Ao, W. Jin, P. Yu, M. Zhu, L. Yu, ‘Novel Res. 2014, 6, 780–784.
method utilizing microbial treatment for cleaner produc- [36] U. Schippmann, D. J. Leaman, A. B. Cunningham, ‘Impact of
tion of Diosgenin from Dioscorea zingiberensis C. H. Wright cultivation and gathering of medicinal plants on biodiver-
(DZW)’, Bioresour. Technol. 2013, 146, 549– 555. sity: global trends and issues’, Biodiversity and the
[24] A. Akhila, M. M. Gupta, ‘Biosynthesis and Translocation of ecosystem approach in agriculture, forestry and fisheries.
diosgenin in Costus speciosus’, J. Plant Physiol. 1987, 130, 2002.
285 –190. [37] S. Singh, M. Gupta, R. Lal, R. Thakur, ‘Costus speciosus seeds
[25] V. Kren, ‘Glycoside vs. Aglycon: The Role of Glycosidic as an Additional Source of Diosgenin’, Planta Med. 1980,
Residue in Biological Activity. In: Fraser-Reid B. O., Tatsuta 38, 185 –186.
K., Thiem J. (eds) Glycoscience. Springer, Berlin, Heidelberg, [38] World Health Organization, ‘WHO guidelines on good
2008, 2589, https://doi. org/10.1007/978-3-540-30429- agricultural and collection practices [GACP] for medicinal
6_67. plants’, 2003.
[26] A. S. Azmi, M. I. A. Malek, N. I. M. Puad, ‘A review on acid [39] C. K. Kokate, A. P. Purohit, S. B. Gokhale, ‘Analytical Pharma-
and enzymatic hydrolysis of sago starch’, Int. Food Res. J. cognosy’, 2005. Thirtieth ed. Pune, India.
2017, 24, S265 –S273. [40] Anonymous, ‘The Ayurvedic pharmacopoeia of India’,
[27] E. Vranová, D. Coman, W. Gruissem, ‘Structure and 2008, 2. Government of India: Ministry of Health and
Dynamics of the Isoprenoid Pathway Network’, Mol. Plant Family Welfare, New Delhi.
2012, 5, 318 –333. [41] R. Montgomery, ‘Determination of glycogen’, Arch. Bio-
[28] L. Zhao, W. Chang, Y. Xiao, H. Liu, P. Liu, ‘Methylerythritol chem. Biophys. 1957, 67, 378– 386.
Phosphate Pathway of Isoprenoid Biosynthesis’, Ann. Rev. [42] ICH, ‘ICH Topic Q2 (R1) Validation of Analytical Procedures:
Biochem. 2013, 82, 497–530. Text and Methodology’, Int. Conf. Harmon. 2005, 17.
[29] A. Banerjee, T. D. Sharkey, ‘Methyl Erythritol 4-phosphate [43] B. V. Subbiah, G. L. Asija, ‘A rapid method for the
(MEP) Pathway Metabolic Regulation’, Nat. Prod. Rep. 2014, estimation of nitrogen in soil’, Curr. Sci. 1956, 26, 259 –260.
31, 1034 –1055. [44] R. A. Olson, M. B. Rhodes, A. F. Dreier, ‘Available
[30] A. Pandey, S. Gupta, K. R. Yadav, ‘AgroTechniques of Costus Phosphorus Status of Nebraska Soils in Relation to Series
speciosus: An important Endangered Medicinal Plant’, Na- Classification, Time of Sampling and Method of Measure-
tional Conference on Forest Biodiversity: Earth’s Living ment’, Agron. J. 1954, 46, 175–180.
Treasure. 2011. [45] S. J. Toth, ‘Quantitative determination of eight elements in
[31] A. N. Iwanycki, R. N. Havskov, B. Markussen, H. H. C. Bruun, plant tissue: A systematic procedure involving the use of a
F. F. Eiriksson, M. Thorsteinsdóttir, N. Rønsted, C. J. Barnes, flame photometer’, Soil Sci. 1948, 66, 459–466.
‘Untargeted metabolic profiling reveals geography as the [46] H. Faulkner, R. Alexander, B. R. Wilson, ‘Changes to the
strongest predictor of metabolic phenotypes of a cosmo- dispersive characteristics of soils along an evolutionary
politan weed’, Ecol. Evol. 2018, 8, 6812 –6826. slope sequence in the Vera badlands, southeast Spain:
[32] J. Chen, S. C. Saunders, T. R. Crow, R. J. Naiman, K. D. implications for site stabilisation’, Catena 2003, 50, 243 –
Brosofske, G. D. Mroz, B. L. Brookshire, J. F. Franklin, ‘Micro- 254.
climate in Forest Ecosystem and Landscape Ecology’,
BioScience 1999, 49, 288– 297.
[33] H. P. S. A. Khalil, M. S. Hossain, E. Rosamah, N. A. Azli, N.
Saddon, Y. Davoudpoura, M. N. Islam, R. Dungani, ‘The role Received November 30, 2020
of soil properties and its interaction towards quality plant Accepted April 9, 2021

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