The Effect of Fortificated Mulberry Leaves with Echinacea Purpurea on Silkworm ( Bombyx mori

protein, peroxidase activity, and phenoloxidase). while larvae fed at 1% concentration recorded high ascorbic acid values.


INTRODUCTION
Silkworm Bombyx mori L. has been domesticated in Egypt for more than 100 years.Due to its sensitivity, it is subjected to some bacterial and viral infections that cause loss of cocoon production, which affects an expected income for farmers or even youth who want to have a project generate an income return.Having information about silkworm's nutrition is essential for determining the health and growth of silkworm larvae and their cocoon parameters.Feeding with nutritionally enriched leaves has a positive impact on the quality and quantity of silk produced.Conversely, any nutritional deficits in the silkworm's diet can affect their health and subsequently affect the quality of silk produced.One of the most important supplements that confuse any deficient in the silkworm diet is the botanical extract due to its secondary metabolites that affect silkworm physiology Etebari and Matindoost (2004) All over the world, there are no silkworm types resistant to bacterial infection or pests.Therefore, during the rearing period, we added some additives that have an immune power and also, increase the production.Recently, mulberry leaves have been fortified with plant extracts to boost production (Sujatha and Purushotham, 2003;Kuntamalla andAkkinapally, 2004 andTakhlique, 2012).
Also, mulberry leaves play a vital role in the silk production.To improve the economic parameters of the silkworm, it must feed on nutritional leaves (Khezrian et al.,2021).The new technique in silkworm rearing to increase their performance and economic parameters is feeding them on enhanced mulberry leaves with supplementary nutrients (Etebari and Matindoost, 2005).Vitamins, amino acids, and proteins that added to mulberry leaves to increase their nutritional efficiency and improve the silkworm's economic traits ( Singh, et al., 2005;Rani and Singh, 2011).Echinacea purpurea is one of the most important medicinal plants.Studies revealed Echinacea extracts have alkamides that have immunomodulatory properties in vitro and in vivo.(Goel et al., 2002;Gertsch et al., 2004).Also, it contains polysaccharides that inhibit cell apoptosis and oxidant stress (Vimalanathan et al., 2017;Hou et al., 2020).Echinacea extract might be able to boost the function of monocytes and natural killer cells, which are essential components of the body's immune system and help fight off infections (Declerck et al., 2021).
Proteins are the major component of the hemolymph plasma.Protein-rich mulberry leaves are responsible for silk production.As well, as a protease enzyme that is responsible for the conversion of mulberry protein to silk protein (Kumar and Kalpana, 2009).Phenol oxidase plays a vital role in the defence mechanism of invertebrates (Liu et al., 2007).
The main role of the peroxidase enzyme is to protect the organism from oxidation stress.Damaged cell caused by free radicals is prevented by the presence of peroxidase activity that has immune function (Sun et al., 2009).
Ascorbic acid, also known as vitamin C, is a water-soluble vitamin with the chemical formula C6H6.Despite not having a fully known function yet, it plays a role in tyrosine metabolism, steroid synthesis, and carotenoid synthesis, as well as acting as a feeding stimulant immunity and detoxifications.It possesses antioxidant power and is a potential phage stimulant.Additionally, possess a growth promoter and a booster of silk production in silkworms (Javed and Gondal 2002;Hussain and Javed 2002;Prassad 2004;Etebari et al., 2004;Etebari and Matindoost, 2005).Our investigation studies the effect of Echinacea purpurea supplemented with mulberry leaves on silkworm production and immunity.

MATERIALS AND METHODS
The investigation was carried out at the Sericulture Research Department in Plant Protection Research Institute, Agricultural Research Center.

Plant Extract Preparation:
Echinacea purpurea shrubs were purchased from IMTENAN shop for medical and healthy products.Dried and ground using an electric grinder: 500 g of plant powder was added into the flask of a Soxhlet apparatus along with 400 ml of 70% ethanol.The apparatus was run for 12 h and thereafter the flask containing active plant hydroalcoholic extracts was discharged in a desiccator to dry off in vacuum condition.Then, the extract was preserved at −20 °C (Harikrishnan et al.,2010).Experimental Design.
Young larvae (1st~3rd instars) were reared at 27~28 °C and 85%~90% relative humidity, and the late-age larvae (4 th and 5 th instars) were maintained at 24~26 °C with a relative humidity of 70%~80%.Rearing was carried out under hygienic conditions according to (Marwa 2020).At the beginning of the 4 th instar, larvae were divided into three groups, each one containing 300 larvae that were divided into three replicates (each one containing 100 larvae).Each group was treated with one concentration of Echinacea extract (0.5, 1 and 2%) till spinning.

A-Biological Index:
Larval weight at the end of the 4 th and 5 th instar, pupation (%), and cocooning percentage (%) were registered.B-Cocoon Traits.

C-Biochemical Analysis:
Hemolymph was collected from treated larvae on the 6 th day of the 5 th larval instar by removal of the thoracic leg in 1.5 ml Eppendorf tubes with a small amount of phenyl thio-urea crystal(PTU) as an anti-coagulant substance (Mahmoud 1988).The tubes were kept at -20°c, and the blood samples were centrifuged at 10000 rpm for 10 minutes at 5°c. the supernatant was assayed to determine.a-Phenol Oxidase Activity.was determined according to a modification of (Ishaaya1971).b-Peroxidase Activity.
was determined according to the procedure given by (Hammerschmidt et al., 1982).c-Total Proteins were determined by the method of (Bradford 1976).

A-Biological Index:
Table (1) showed that the application of Echinacea purpurea to the 4 th larval instar recorded significantly, the highest weight for control (0.498g.) significantly, while all treatments recorded less weight on par with each other (0.421, 0.417 and 0.408g.)for (2%, 1%, and 0.5%) respectively.Otherwise, the 5 th instar weight was maximum for 2% treatment (2.49g.) with a significant variance among other treatments and control, minimum results were (2.05 and 1.96g.)for (0.5%, and Control) respectively, with no significant variance between each other.
The cocooning percentage showed no significant variance between 2% and 1% treatments (100.00,98.45%).Also, pupation ratio, data were recorded (100.00 and 100.00) for (2% and 1%) with no significance, respectively.However, all treatments differed from the control significantly, which recorded the lowest value in the previous criteria (78.00 and 81.90 %), respectively.

B-Cocoon Traits:
As shown in Table (2) compared with the control group.Feeding on Echinacea purpurea increases all cocoon parameters compared with the control group especially the cocoon shell ratio which is an important indicator for evaluating the success of the investigation.It was obvious that feeding on 2% concentration is the most effective that recorded (1.28g., 0.272g., 1.01g., and 21.12%) significantly in females for (C.W., C.S.W., P.W., and C.S.R%) respectively.But, for the male, 2% treatment recorded maximum results in cocoon weight, pupa weight, and cocoon shell ratio compared to other concentrations (0.5% and 1%) whereas, the results registered were (0.993, 0.950 and 0.956g.)for male cocoon weight, (0.748, 0.723 and 0.730) for pupa weight and (23.96%, 23.14 and 23.02%) for cocoon shell ratio for (2%, 1%, and 0.5%) respectively.Cocoon shell weight 2% treatment recorded the highest data significantly (0.238 g.).
Table 2: Effect of fortified mulberry leaves with Echinacea purpurea on cocoon traits.C-Biochemical Analysis.
Among the different concentrations 2% treatment recorded significantly high results in total protein, peroxidase, and phenol oxidase that were (79.53 mg\ml, 17.66 ∆ O.D. /min/ ml, and 13.63 O.D.units/min/ ml ) respectively.However, the lowest value was (64.533 mg\ml) in negatively treated control for total protein while, peroxidase and phenol oxidase enzymes recorded the minimum results (7.63 ∆ O.D. /min/ ml and 8.30 O.D.units/min/ ml ) for 1% treatment respectively, with no significant variance between it and control.
On the other hand, values of larvae fed on mulberry leaves fortified with 1% of Echinacea purpurea showed the highest result significantly, (139.66 ug A.A./ ml) while data was registered for larvae fed on 2% concentration (107.33 ug A.A./ ml) without significance compared to other treatments.

DISCUSSION
Nutritional requirements, conversion efficiency and strength immunity of silkworms play a significant role in determining the cost-benefit ratio of rearing them.These factors are crucial for identifying superior breeds of silkworms.

A-Biological and Cocoon Characters:
This present study revealed that feeding silkworm larvae on mulberry leaves enriched with Echinacea purpurea only during the 4 th larval instar did not affect larval weight as shown previously, these results are in line with (Saad et al., 2014) who referred to the mulberry leaves enriched with food supplements affect significantly the weight of full-grown larvae, silk gland, and cocooning percentage.However, the results took another direction during the 5 th instar, the larval weight increased, cocooning and pupation ratio also, increased, significantly.
These findings are in agreement with (Shinde et al., 2014) who proved that the important factors affecting on silk industry are mulberry variety and nutritive additives.Also, (Etebari et al., 2004) referred to nutritional supplements, such as proteins, carbohydrates, vitamins, minerals, hormones, and antibiotics, which can enhance larval weight, pupal rate, and cocoon weight in silkworm larvae.Nutrient quality of mulberry leaves has a significant impact on biomass gain and economic traits.E. purpurea extract has a positive impact on all economic parameters of silkworms.(Khezrian et al.,2021) in conformity with our results, many botanical extracts (Echinacea purpurea) have been impacted by silkworm economic characteristics.(Saravanan et al.,2011) , Changes in silkworm larval weight when different feeding supplements were used might have been due to the increase in protein production.Also, (Guiqin et al., 2020) who studied the effect of flavonoids as secondary metabolites on silkworm performance, found that, a positive impact on the activity and silk production.Cucurbita moschata extract boosts silkworm growth and increases larval weight, cocoon, shell weight also cocoon shell ratio at higher concentrations (Jamela et al., 2023).10% of C. longa extract has a positive impact on all cocoon traits and economic characteristics of mulberry silkworms (Rajitha and Savithri 2015).Some medicinal plant extracts (A.vasica, B. spectabilis, P. niruri, T. arjuna and Pongamia glabra) enhance all biological and cocoon traits of silkworm larvae after feeding during 4 th and 5 th larval instars (Latha et al.,2011).

B-Biochemical Analysis:
The increase in hemolymph total protein indicating to an increase in digestive activities that lead to protein accumulation and transport by heamolymph to other tissues.These findings go in line with (Nagata and Kobayashi 1990) refer to the elevations of hemolymph protein during the 5 th instar due to the active secretion of proteins by other tissues.(Suramya et al., 2020) who proved that Echinacea is a potent anti-oxidant, phage stimulant, growth promoter, and silk production booster.Also, (Marwa 2020) goes in our line feeding silkworms on 2.5% of Aleo vera increase total protein.Echinacea contains caffeic and ferulic acid derivatives, caftaric acid, chicoric acid, and chlorogenic acid as major phenolic compounds, along with polysaccharides and glycoproteins that, explain the elevation of peroxidase, and phenol oxidase activities in larvae fed on mulberry leaves fortified with 2% concentration extract as shown in previous results.Echinacea enhances immune function in both healthy and immuno-compromised animals (Mariangela et al., 2018).Polysaccharides protect the body by preventing the absorption of free radical species and safeguarding them from oxidation before they can be absorbed.the addition of 1 g. of polysaccharides boosted peroxidase activity (Chang and Chen 2003).Also, (Fu et al.,2007) revealed that the ingestion of polysaccharides obtained from Utricularia rigida, a type of seaweed, can play a crucial role in preventing the deterioration of antioxidant defence.(Manjula et al.,2020) phenoloxidase enzyme increased after feeding S. litura on P.B of Manihot esculenta leaf extract containing phenolic compounds.2.5% of Aleo vera increase peroxidase activity in silkworm heamolymph as reported by (Marwa 2020).
On the other hand, larvae fed on mulberry leaves supplemented with 1% of Echinacea purpurea extract only recorded high ascorbic acid levels, which declined gradually in treatments fed on 2% and 0.5% without significance.This result may be due to the increasing of Echinacea purpurea dose that contains polysaccharides and antioxidants, which are in conformity with (Lamikanra and Watson 2001) who referred to the inverse relation between ascorbic acid and peroxidase activity.However, the total protein level in the same group (1%) was low compared with the 2% treatment group.The result of increasing ascorbic acid leads to an increase in blood glucose which decreases stimulation of the hunger center decreases feed intake and affects silk production.These results are supported by Etebari et al. (2004) findings, The act of providing silkworms with excessive quantities of ascorbic acid has been found to result in hypervitaminosis, leading to a decrease in their overall weight that leads to silk reduction.

CONCLUSION:
The target of this study is finding an extract that has double power, and effect depending on its concentration: activate antioxidant enzymes to reduce infection and enhance silk production.Also, despite the positivity of a 1% concentration (which contains a high amount of ascorbic acid), on immunity and that prevents cell apoptosis, a high (2%) concentration of Echinacea purpurea also has flavonoids and phenolic compounds that raise antioxidant enzymes.Therefore, may be the most effective concentration (2%) increases total protein, phenol oxidase, and peroxidase enzymes.,The other two concentrations (0.5%, 1%) are responsible for increasing immunity which reduces pathogenic invasion, which is affecting silk production.
d-Vitamin C or ascorbic acid method (A.O.A.C 1975) is based on the measurement of the extent to which a 2,6-dichlorophenol-indophenol dye solution is decolorized by the presence of ascorbic acid.Data Analysis: Obtained data was analyzed as one-way ANOVA, using Proc ANOVA in SAS (Anonymous 2003) and means were compared by LSD (P= 0.05 level) in the same program.

Table 1 :
Effect of fortified mulberry leaves with Echinacea purpurea on some biological parameters.

Table 3 :
Effect of fortified mulberry leaves with Echinacea Purpurea on some biochemical analysis.