Use of spinach (Spinacia oleracea) containing phytoecdysteroid (β-ecdysone) as a crop saver in sericulture industry

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The Pharma Innovation Journal 2023; 12(2): 2301-2305
ISSN (E): 2277-7695
ISSN (P): 2349-8242
NAAS Rating: 5.23
TPI 2023; 12(2): 2301-2305
© 2023 TPI
www.thepharmajournal.com
Received: 08-12-2022
Accepted: 11-01-2023
Sameena Maqbool
College of Temperate Sericulture.
Sher-E-Kashmir University of
Agricultural Sciences and
Technology of Kashmir, Jammu
& Kashmir, India
Zia H Rufaie
College of Temperate Sericulture.
Sher-E-Kashmir University of
Agricultural Sciences and
Technology of Kashmir, Jammu
& Kashmir, India
Baqual MF
College of Temperate Sericulture.
Sher-E-Kashmir University of
Agricultural Sciences and
Technology of Kashmir, Jammu
& Kashmir, India
Sanna Parvez
College of Temperate Sericulture.
Sher-E-Kashmir University of
Agricultural Sciences and
Technology of Kashmir, Jammu
& Kashmir, India
Corresponding Author:
Sameena Maqbool
College of Temperate Sericulture.
Sher-E-Kashmir University of
Agricultural Sciences and
Technology of Kashmir, Jammu
& Kashmir, India
Use of spinach (Spinacia oleracea) containing
phytoecdysteroid (β-ecdysone) as a crop saver in
sericulture industry
Sameena Maqbool, Zia H Rufaie, Baqual MF and Sanna Parvez
Abstract
Spinach (Spinacia oleracea) belonging to family Chenopodiaceae was reported to be having insect
moulting hormone β-ecdysone or 20-hydroxyecdysone. The Leaves and soft shoots of this plant were
collected, washed, shade dried and later on powdered for obtaining plant extract by using methanol. (Hot
extraction by Soxhlet apparatus). The presence of moulting hormone was confirmed under high
performance liquid chromatography (HPLC) by running the marker 20-hydroxyecdysone along with this
plant extract with a mobile phase of methanol and water in the ratio of 55:45 respectively. Results
confirmed high concentration of 20-hydroxyecdysone (β-ecdysone) in Spinach (Spinacia oleracea) The
effect of this plant extract containing phytoecdysteroid (β-ecdysone) on the spinning behaviour of
silkworm double hybrid (CSR6×CSR26) × (CSR2×CSR27) was investigated in the present study. Fully
grown 5th instar larvae of silkworm Bombyx mori L. were fed with mulberry leaves sprayed with
different concentrations of this plant extract. The application of plant extract containing ecdysteroid
reduced 3-4 feeds and finished mounting earlier than other treatments. However, a crop was harvested
earlier than normal harvesting time. The reduction in the period is almost one day in both the trails. This
means a quantum reduction in leaf consumption i.e. 3-4 feeds were saved by application of plant based
ecdysteroid at these times of application. The reduction in the larval duration and consumption of
mulberry leaves manifests in adverse effect on the quantitative traits like yield etc Usually no farmer
would go for such a drastic reduction of yield as well as in cocoon characters unless he is compelled to
do so under adverse conditions, like safety of entire crop from diseases and unforeseen shortage of
mulberry leaf. However, the farmer would naturally prefer a reduced yield to a much bigger crop loss.
Due to this reason, phytoecdysteroid emerges as a crop saver. The present study recommends the
application of Spinach (Spinacia oleracea) containing 20- hydroxyecdysone at the onset of spinning,
however under stress and adverse conditions the application can be done on and after 5th day of Vth
instar for saving cocoon crop to some extent.
Keywords: β-ecdysone, hastening, maturation, Bombyx mori L.
Introduction
It is known that analogues of ecdysteroids also occur in certain proportions in plants. These
plants synthesize these ecdysteroids as a defence mechanism and these occur in them in large
quantities (Schmelz, et al., 2000) [8]. The ecdysteroid derived from plant source is popularly
known as Phytoecdysteroid. These phytoecdysteroids have been seen to be 20 times more
active than Zoo-ecdysteroids (Nair et al., 2002) [4]. As a defence mechanism in plants it is also
believed that they provide some degree of protection to the plant against non-adopted
phytophagous insects (Bergamasco and Horn, 1983; Kubo and Hanke, 1986) [1, 3]. Ecdysteroid
(ES) in the context of sericulture can be any phyto-sterol structurally closer to the original
insect ecdysteroid i.e. 20- hydroxyecdysone, which can induce a response in silkworm equal to
that of the natural ecdysteroid and could be used for synchronizing the maturation activity
among the silkworms in order to spin cocoon within short span of time (Rufaie et al., 2012) [7].
Ecdysterone or 20-hydroxyecdysone is the most widely occurring phytoecdysteroid and many
locally available plants like Taxus wallichiana Zucc. (Himalayan Yew), and Cupressus
tularosa Linn (Cupreous) have been identified to have shown reasonable concentration of 20-
hydroxyecdysone (Rufaie et al., 2011) [6]. This Phytoecdysteroid can also advance the
maturation activity and hasten cocoon spinning process in silkworms especially when a partial
or complete crop loss is feared by the rearers either due to mulberry leaf shortage or disease
attack.

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Materials and Methods
Spinach (Spinacia oleracea) was taken for detection of 20-
hydroxyecdysone i.e. Insect moulting hormone (β-ecdysone)
which can be used later on during silkworm rearing. These
plant species are as under.
The plant materials (leaf and tender branches) of Spinach
(Spinacia oleracea) was taken and washed with distilled
water first and later on shade dried till brisk. The dried plant
material was powdered in a grinder in order to make a fine
powder for getting plant extract. The hot extract of this plant
material was prepared by boiling powdered plant material
along with methanol and water in the ratio of 3:2 in a Soxhlet
Apparatus continuously for 6 hours. The extract was
separated from the residue by filtration and centrifugation.
This extract was concentrated at a temperature of 60 °C in a
rotary evaporator and again dissolved in methanol and water
solvent. The crude extract was defatted in a solvent system by
a partition between n-Hexane and 80% aqueous MeOH
(Hoffman and Hetru, 1983) [1].
The presence of 20-hydroxyecdysone- like substance was
identified in the extracts by thin layer chromatography,
keeping ecdysone (20-hydroxyecdysone) procured from M/S
Sigma, St. Louis, M.O., USA as standard. The sample and
standard (20E) was run on the Silica gel G coated plate for
which mobile phase (thin-layer chromatography solvent) used
was chloroform: methanol in the ratio of 4:1. After
developing this plate was initially placed in iodine chamber
and later on sprayed with anisaldehyde reagent for steroid
visualization. This plate were also viewed in a UV hood at
UV 254 nm, where identical spots between the standard (20E)
and plant sample was identified. In this study High
performance liquid chromatography (HPLC) technique was
used to identify a particular compound from a mixture.
Therefore this technique was used for confirmation of
presence of phytoecdysteroid (20-hydroxyecdysone) from
plant extract. Those plant extract of Spinach (Spinacia
oleracea) which showed identical spots coinciding with
standard under TLC thereby giving a preliminary clue
regarding the presence of 20-hydroxyecdysone was taken for
final identification and confirmation under High Performance
Liquid Chromatography. This was done at Indian Institute of
Integrative Medicine Srinagar. Sample of plant extract and
standard (20-E) was run for identification and quantification
of 20-hydroxyecdysone in a High performance liquid
chromatography (HPLC). About 400 µl of this sample was
taken in HPLC viol for identification and quantification. The
mobile phase used for this purpose was MeOH: H2O
(Methanol: Water) in the ratio of 55:45 and flow rate of
HPLC was maintained at 0.4ml/min. About 3-5 µl of each
sample were run for identification of 20-hydroxyecdysone.
After running the plant extracts along with standard 20-
hydroxyecdysone, this plant extract was identified for
possessing 20-hydroxyecdysone as its retention time
coincided with that of the standard which was run along with
this plant extract. As such it was confirmed by HPLC studies
that the plant extract of Spinach (Spinacia oleracea) contain
phytoecdysteroid in the form of 20-hydroxyecdysone.
In the present study Silkworm double hybrid of (CSR6 ×
CSR26) × (CSR2 × CSR27) was reared on mulberry leaves
under recommended environmental conditions
(Krishnaswami, 1986) [9] up to the onset of spinning activity.
The larvae were grouped into various batches each containing
250 larvae per replication. After it was confirmed under high
performance liquid chromatography (HPLC) that plant extract
of Spinach (Spinacia oleracea) is possessing 20
hydroxyecdysone like substance it was taken for further
studies. This plant extract was re-dissolved in methanol to
prepare 10% stock solution (10g in 100 ml) which was used
to prepare dilution of 1:100 (1%) and 1:50 (2%) (w/v)
concentrations in distilled water (Jayapaul et al., 2003) [2] for
further study during silkworm rearing. The plant extract was
sprayed on mulberry leaves in uniform quantity (@ 60 ml ml /
200 g of leaf for 100 larvae (Tantray, et al., 2011) [10] was
sprayed with an atomizer for per os administration to the
larvae in each replication. The phytoecdysteroid was mostly
used for synchronization of maturation at the start of
seriposition. However in this study the phytoecdysteroid was
used as a crop saver under certain adverse conditions which
hastened the maturation process in silkworm double hybrid
when applied on different timings i.e 72 hrs (3rd day) and 120
hrs (5th day) during Vth age of silkworm larva. In addition to
this plant extract a commercial formulation of plant extract
(branded hormone SAMPOORNA) was also used as Control-
I as per its recommended concentration. Another Control i.e
without any treatment was also maintained side by side for
evaluating the performance of plant extract containing 20-
hydroxyecdysone like substance. The time and number of
worms mounted was recorded at regular intervals till the
completion of mounting process in both experimental and
control batches.
Results and Discussion
The most common and appropriate use of phytoecdysteroid is
to hasten the maturation and to induce simultaneous or
synchronized cocoon spinning. However phytoecdysteroid
can be used as a crop saver under certain adverse conditions
when applied at a particular time during Vth age of silkworm
larva. The effect of the administration from 72 hours (3rd Day)
onwards was most remarkable as far as crop saving treatment
is concerned. The treatments done at 72, and 120 hours (3rd&
5th Day) had a clear impact on the larval duration and
obviously on other related characters such as larval weight
and cocoon traits. In case of treatment at 120 hours (5th Day),
it was observed that shortest larval duration of 642.57hrs was
recorded in this time of application. In addition to this shortest
Vth age larval duration of 162.52 h was also recorded in this
time of application. This reduction in 5th age larval duration
was observed due to per OS administration of plant extract
containing 20-hydroxyecdysone like substance to the larvae in
each replication. A difference of nearly 17 hours was
observed in Vth age larval duration between control-II
(179.53 hrs) and plant extract fed batches (162.52 hrs). This
162.52 hrs. Was the lowest Vth age larval duration. There was
also significant difference in 5th age larval duration between
Control-I i.e 163.97hrs and the lowest recorded after
application of plant extract i.e 162.52 hrs. However the
difference was only 1-2 hours. This decrease in 5th age larval
duration was also measured in percentage as against the value
of control-II. The highest decrease of 9.43 per cent was
observed after applying plant extract of Spinacia oleraceaat a
concentration of 1:100. In 120 hrs treatment, where
maturation of worms for mounting started at 128 hrs (i.e 8
hours after the treatment) and cumulative maturation in this
treatment was achieved at 155-160 hours in Vth age. It took
nearly 36-40 hours (from treatment to end) for culmination of
mounting i.e. on the 7th day. During this treatment lowest

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number of feeds i.e 26.5 were taken by treated batches as
against 29.25 by the control-II (which was not treated). The
application of plant extract containing ecdysteroid reduced 3-
4 feeds and finished mounting earlier than other treatments.
Similarly in case of 72 hrs treatment it was observed that
maturation was also hastened as compared to control.
However it progressed slowly as compared to 120 hrs
treatment. The maturation process started 32 hrs after
treatment (at 104 hours) and progressed slowly and completed
mounting of silkworms for seriposition within 64 hrs (at 168
hours) as compared to 120 h treatment. The possible reason
for this was that treatment at 120 hrs i.e. On 5th day of 5th
instar was more effective due to the reason that more number
of feeds (more than 20) were taken by this batch as compared
to 72 hrs treatment. However the quantity harvested was less
as there was a difference of nearly 7 grams between the
lowest recorded weight of 10 mature larvae and that of
Control-II which resulted in reduced cocoon weight and other
cocoon characters. However a crop was harvested earlier than
normal harvesting time. A moderate pupation was observed in
these batches. The highest pupation was recorded in control
batches where worms were fed fully as compared to treated
batches where plant extract was applied, due to which less
feeds were taken by the larvae as they matured earlier. Nair et
al., 2005 [5] has also reported that when larvae were treated at
72 hrs, the economic traits were adversely affected but the
larval duration was shortened considerably. While as the
present study indicated that economic traits get adversely
affected in 72 hrs as well as in 120 hrs treatments, however in
case of 120 hrs treatment the larval duration gets shortened by
nearly 17 hours and in 72 hrs treatment by nearly 12-13 hrs. It
is evident from results that this technology can’t be utilized on
seed crops as there should be a pupation of more than 90 per
cent which is not achievable under this technology. Although
the larval duration and the survival are inversely related, the
survival as such was good in almost all the treatments. This
also gives an impression that treatments did not influence the
survival adversely.
The most important effect of these treatments at 120 hrs and
72hrs during Vth instar was reduction in larval duration and
formation of a cocoon crop. In this treatment the longest total
larval period of 659.53hrs was recorded in control-II (without
treatment), and as such there was a difference of about 18 h
between the shortest larval period (642.57 hrs) and the
control-II (659.53 hours). This has happened due to the
application of plant extract containing 20-hydroxyecdysone
like substance in this crop saving use. The reduction in the
period is almost one day in both the trials. This means a
quantum reduction in the leaf consumption i.e 3-4 feeds were
saved by application of plant based ecdysteroid at these
application timings. The reduction in the larval duration and
consumption of mulberry leaves manifests a rather strong
adverse effect on the quantitative traits like yield etc. Usually
no farmer would go for such a drastic reduction of yield as
well as in cocoon characters unless he is compelled to do so
under adverse conditions, like safety of entire crop from
diseases and unforeseen shortage of mulberry leaf. However
the farmer would naturally prefer a reduced yield to a much
bigger crop loss. Due to this reason, phytoecdysteroid
emerges as a crop saver. In case of untimely leaf shortage and
sudden disease outbreak, phytoecdysteroid can be applied at
any time after 3rd day of Vth instar (after 72 hours) which is
obligatory feding period to reduce larval duration. The present
study recommends the application of extracts of Taxus
wallichiana Zucc. Cupressus tularosa Linn and Datura
stramonium Linn containing 20-hydroxyecdysone like
substance at the onset of spinning, however under stress
conditions the application can be done after 5th day of Vth
instar i,e after 120 h (Table-1, Fig. 1).
Phytoecdysteroid as a crop saver under stress conditions
The phytoecdysteroid can be used as a crop saver under stress
conditions when applied at a particular time during Vth age.
The effect of administration from 72 h onwards was most
remarkable as far as crop saving treatment is concerned. The
treatment done at 72, and 120 h had a clear impact on larval
duration and also on other related characters such as larval
weight and cocoon traits. In case of 120 hours it was observed
that shortest larval duration of 642.57 h was recorded in this
time of application. In addition to this shortest Vth age larval
duration of 162.52 h was also recorded in this time of
application and this decrease in Vth age larval duration was
observed due to plant extract application containing ecdysone.
A difference of nearly 18 h was observed between control-II
179.53 h and plant extract 162.52 h which was the lowest
larval duration. The decrease in 5th age larval duration was
also measured in percentage as against the value of control-II.
The highest decrease of 9.47 per cent was observed after
applying plant extract of Spinacia oleracea at a concentration
of 1:100 In 120 h treatment maturation started at 128 h (8 hrs
after the treatment) and cumulative maturation in this
treatment was achieved at 162-163 h. It took nearly 32-36 h
(from treatment to end) for culmination of mounting i.e. on
the 7th day. During this treatment lowest number of feeds i.e
26.500 were taken by treated batches as against 29.250 by
control –II (which was not treated). The application of plant
extract containing ecdysteroid reduced 3-4 feeds and finished
mounting earlier than other treatments. Similarly in case of 72
h treatment it was observed that maturation also got hastened
as compared to Control-II. However it progressed slowly as
compared to 5th day (120 h) treatment. The maturation process
started 32 h after treatment (at 104 h) and progressed slowly
and mounting was completed within 64 h (at 168 h) as
compared to 5th day (120 h) treatment. Here also difference of
nearly 12-13 h was observed between control-II and the
lowest value recorded in this treatment. The possible reason
for this may be that treatment at 120 h i.e on 5th day of 5th
instar was more effective due to the reason that more number
of feeds were taken by this batch as compared to 3rd day (72
h) treatment. In 3rd day (72 h) treatment also 2-3 feeds were
saved and a crop was harvested. However the quantity
harvested was less as there was a difference of nearly 5-7
grams in larval weight of these treatments and other normal
treatments, which resulted in deterioration of certain
characters. However a crop was harvested earlier than normal
harvesting time. A moderate pupation was also observed in
these batches. The highest pupation was recorded in control
batches where worms were fed fully as compared to treated
batches where plant extract was applied, due to which less
feeds were taken by the larvae as they matured earlier. (Nair
et al., 2005) [5] has reported that when larvae were treated on
3rd day (72 h), the economic traits were adversely affected and
the larval duration was shortened considerably. While as the
present study indicated that economic traits get adversely
affected on 3rd day (72 h) treatment as well as on 5th day (120
h) treatment, however in case of 5th day (120 h) treatment the

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larval duration gets shortened by nearly 18 h and in 72 h
treatment by nearly 12-13 h. It is evident from result that this
technology can’t be utilized on seed crop as pupation in seed
crop should be 90-95 per cent which is not achievable under
this technology. Although the larval duration and the survival
are inversely related, the survival as such was good in almost
all the treatments. This also gives an impression that treatment
did not influence the survival adversely, and mortality due to
feeding of plant extract containing ecdysone as well as
branded hormone did not took place.
The most important effect of these treatment on 5th day (120
h) and 3rd day (72 h) during Vth instar was reduction in larval
duration and formation of a cocoon crop. In these treatments
the longest larval period of 659.53 h was recorded in control-
II (without treatment), and as such there was a difference of
about 17 h between the shortest larval duration i.e 642.57 h
and the 659.53 recorded in control–II. This has happened due
to the application of plant extract containing 20-
hydroxyecdysone like substance in this crop saving use. The
reduction in the period is almost one day in both the trails.
This resulted in sizable reduction in mulberry leaf
consumption. As a result of this 2-3 feeds were saved by
application of this plant based ecdysteroid at this time of
application. This reduction in larval duration and consumption
of mulberry leaves is beneficial for the rearers however it has
resulted in the form of adverse effect on various quantitative
traits of cocoon like cocoon weight, shell weight, like yield
etc. Usually no farmer would go for such a drastic reduction
of yield as well as in cocoon characters unless he is compelled
to do so under stress/ adverse conditions, like safety of entire
crop from disease and unforeseen shortage of mulberry leaf.
However the farmer would naturally prefer a reduced yield to
a much bigger crop loss. Due to this reason, phytoecdysteroid
emerges as a crop saver under such stress conditions. In case
of untimely leaf shortage and sudden disease outbreak,
phytoecdysteroid can be applied at any time after 3rd day of
Vth instar (after 72 h) which is obligatory period to reduce
larval duration. The present study recommends the application
of Spinacia oleracea (spinach) (containing 20-
hydroxyecdysone) at the onset of spinning, however under
stress conditions the application can be done after 5th day of
Vth instar (Table 1) which can save the crop to some extant
under stress conditions and farmers will get some crop even
though that will be inferior as far as its qualitative characters
are concerned.
Table 1: Effect of Phytoecdysteroid on maturation of silkworm Bombyx mori L. (Crop Saving Use)
S. No
Duration in 5th Instar
(Hours)
Maturation Percentage
(72 hours)
Maturation Percentage
(120 hours)
Pos. Control (Hormone)
Maturation (% age)
Abs. Control
(Maturation %age)
1
3rd Day (72h)
Application of P. Extract
2
80
3
88
5
4th Day (96h)
6
104
9
7
112
17
8
5th Day (120h)
28
Application of P. Extract
Application of P. Extract
9
128
47
10
13
10
136
58
21
28
11
6th Day (144h)
75
39
51
12
152
91
67
74
13
160
95
100
100
14
14
7th Day (168h)
100
36
15
176
77
16
184
95
17
8th Day (192h)
100
18
200
19
208
20
9th Day (216h)
Fig 1: Effect of Phytoecdysteroid on maturation of silkworm Bombyx mori L.

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