Antioxidant and Phenolic Activities in EMS Induced Mutants of Mulberry Genotype RFS 135 and Effec

International Journal of Biological Research and Development (IJBRD) ISSN (P): 2250–0022; ISSN (E): Applied Vol. 10, Issue 1, Jun 2020, 5–12 © TJPRC Pvt. Ltd.

ANTIOXIDANT AND PHENOLIC ACTIVITIES IN EMS INDUCED MUTANTS OF MULBERRY GENOTYPE RFS135 AND EFFECTS OF MUTAGEN ON MORPHOECONOMIC CHARACTERS ANIL KUMAR H. V1* & MURALIDHAR. S. TALKAD2 1

Professor, Laboratory of Applied Biological Sciences, DVS College of Arts and Science, Shimoga, Karnataka, India

2

Professor, Herbal Research & Project Consultant –R&D, Natural Remedies Pvt Ltd. Hosur Road, Bangalore, India

ABSTRACT Mulberry (Morus alba L.,) is perennial and arboreal form of family Moraceae. The leaves have certain potent phytochemical components with antioxidant abilities. In the present investigation, the chemical mutagen EMS, concentrations of 0.1% and 0.3% was used to induce mutations in mulberry variety RFS 135. Ethyl Methane Sulphonate (EMS) is a mutagen, through random mutations it induces nucleotide substitution. The 180 days old M1V1 mutant clones maintained in field were evaluated for morpho-economic traits. The bio-phytochemical assay for protein, total reducing sugars, total chlorophyll and phenolic and antioxidant activities were assessed in tender leaves of mutants.

economic traits against control. Total chlorophyll 3.708 ± 0.030 when compared to control 2.791± 0.048, protein content in treated 0.3% was 43.33 ± 0.71 against control 29.66 ±1.73, reducing sugar content was 12.0 ±0.8670 against control 2.16 ± 0.2647. Plant height (cms) in EMS treated 0.3% were 937.53 ± 10.57 against control 662.5 ± 28.87, phenolic content, in clones of 0.3% treated were 38.0 ± 4.0 against control 26.4 ± 2.5 (GAE mg/g DW) and Antioxidant activity (RSA % inhibition), 70.26 ± 0.20 in treated against control 52.40 ± 0.52 by DPPH method. The leaf area was 811.51 cm2 ± 31.39 at 0.3%, against control 282.64 cm2 ± 33.65, an indicator of biomass increase.

Original Article

Mutants recovered at 0.3% treatment showed higher levels of bio-phytochemical response and morpho-

The variations in gross morpho-economic traits in the M1V1 mutant clones probably could be beneficial for therapeutics and high yield in biomass.

KEYWORDS: EMS, Mutation, Mulberry Genotype RFS135, Phenolic & Antioxidant Activities, DPPH, BioPhytochemical Constituents, Morpho-Economic, High Yield & M1V1 Clones

Received: Mar 13, 2020; Accepted: Apr 03, 2020; Published: Apr 29, 2020; Paper Id.: IJBRDJUN20202

INTRODUCTION Mulberry of family Moraceae is grouped under order Urticales. It is widely distributed in the eco-climatic regions of tropical, sub-tropical, temperate and sub-arctics. (Jolly et al., 1986, Sanjappa, 1989). Perhaps, it might have first originated in the foothills of Himalayas and distributed later to the continents like Asia, Europe, Africa, and America (Sanchez, 2000a, b). Mulberry thrives well in regions between 50oN Lat. and 10oS Lat. (Yokoyama 1962); and in the altitudes of 4000 MSL (Machii et al., 1999; Tutin et al., 1996). 68 species of genus Morus have been reported and classified. Most of them seen distributed in Asia and extensively cultivated for sericulture industry aimed at producing commercial silk.

Mulberry is basically deciduous species and primarily cultivated for biomass (leaf) to rear silk worm in sericulture industry. The mulberry leaf protein is basis for bio synthesis of silk by silkworm Bombyx mori L., so the

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mulberry genotypes of high protein content in leaves is key factor for economic profitability in silk production. In recent past, many researchers have reported that, apart from its use as forage for silk worm, mulberry has several functional diversified medicinal and nutritional properties, which has been effectively utilized in food and pharmaceutical industry.

The various parts of mulberry have a base in an ayurvedic preparation. Root has a drug constituent “Glycosidase” which is useful in treatment of hypertension, its juice agglutinates the blood and acts as deworming agent and its extract is Hypoglycemic. Root bark is an anthelminthic. The stem bark is used as purgative and vermifuge. Apart from its importance in silk production, the mulberry leaves possess aplenty of medicinal values.

Gamma-aminobutylic acid rich in leaves is proved effective against high blood pressure (Nae-Cherng Yang et al., 2012). It is established that blood-sugar level related to diabetes is cured by deoxynojirimycin that is abundantly found in mulberry leaf. (Akira Asai et al., 2011). The elevation of blood sugar in postprandial has been suppressed by DNJ-rich mulberry leaf extract due to possession of an inhibitor of α-glucosidase (Yoshihiro Kojima et al., 2010). The leaves have a diaphoretic and emollient effect. Thus the decoction can be used as a health drink.

Leaves of Mulberry possess certain phenolic compounds with antioxidant properties toward Reactive Oxygen Species (ROS) which results in oxidative damage of cellular system (Van Acker, et al 1996 & Naowaratwattana, W et al., 2010). Phenolic derivatives such as rutin, quercetin and isoquercetin, including some flavonoids, have been reported in mulberry leaves (Jia, Z et al., 1999). Some studies reveal significant effect on the contents of anthocyanin, other phenolic compounds and antioxidants activities in mulberry cultivated under varied conditions and harvest seasons (Wang, Z. J, et al 2006 & Li, Y et al., 2008). Studies have been carried out to confirm the consistency of bio efficacy of phenolics with the cultivars and seasons (Jia, Z et al 1999, Matsuoka, T et al., 1994 & Onogi, A et al., 1993). Mulberry leaves are not only the forage for insect and animals, but also acts as an excellent antioxidative compound coupled with diuretic, hypoglycemic and hypotensive activities. (Li, Y et al 2008).

The present study is focused on the Antioxidant and Phenolic Activity, biochemical parameters and morphological characters on EMS induced mutation in Mulberry genotype RFS135

MATERIALS AND METHODS The healthy cuttings of Mulberry genotype RFS135 procured from Central Sericulture Germplasm Research Station, Hosur, Tamil Nadu, India, were planted and established at Germplasm bank at Bangalore University, as per the recommended package of practices (Krishnaswami, 1971). Further, the cuttings were prepared following the standard procedure (Sujathamma and Dandin, 1998) and were planted in earthen pots of propagation mixture and maintained under standard nursery conditions (Hartmann and Kester, 1986).

The sprouting buds were treated with 0.1% and 0.3% concentrations of Ethyl methane sulphonate (EMS) solution following cotton swab method intermittently for 12 hours. After 90 days the treated population was transplanted to the field and maintained under recommended package of practices for 180 days. The beneficial variants clones of M1V1 generation recovered from 0.3% EMS treated population were screened for the experimentation.

MULBERRY LEAF EXTRACTION Healthy tender leaves were collected, dried and powdered, further followed by extraction using 70% ethanol, the dried extract was stored at −20 °C (Yuxiao Zou, et al., 2012).

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Antioxidant and Phenolic Activities in EMS Induced Mutants of Mulberry Genotype RFS135 and Effects of Mutagen on Morpho-Economic Characters

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TOTAL PHENOLS ESTIMATION Mulberry leaf extract were subjected to Total phenols (TP) estimation and the results expressed as equivalents (GAE) mg/g of gallic acid (Chen, C. Y et al., 2008) FRAP ASSAY The leaf extract was incubated with the FRAP reagent, the redox-linked reduction of Fe3+ -2,4,6-tripyridyl-S-triazine was measured for absorbance at 593 nm, using a Shimadzu UV1700 spectrophotometer, the FRAP value was expressed as (TE)/100g of Trolox equivalents (Chen, C. Y et al., 2008).

DPPH SCAVENGING ACTIVITY ASSAY The free radical scavenging capacity, the DPPH assay was carried out with slight modifications (Bae SH, et al 2007). The assay was performed in a 96-well micro plate, the absorbance of samples were measured at 517 nm. The scavenging activity was expressed by the DPPH free radical inhibition rate which was calculated using the formula: DPPH scavenging activity (%) = [1 – (As - Ac /Ao) X 100] “As” = absorbance of the sample with DPPH, “AC”= absorbance of the sample with ethanol, and “Ao” = the absorbance of DPPH with ethanol, analysis were performed in triplicate.

STATISTICAL ANALYSIS Statistical analysis was performed using Analysis Of Variance (ANOVA) and t-tests were implemented to evaluate the correlation between mean values of control against treated samples.

RESULTS Table 1: Total Phenolic, FRAP and RSA % Inhibition in the leaves of Mulberry Genotype RFS135 Maturity of Leaves - Tender TP FRAP RSA (% inhibition) Groups (GAE mg/g DW) (mmol TE /100 g DW) DPPH method 26.4 ± 2.5 10.4 ± 1.0 52.40 ± 0.52 CC: Control 1C: 0.1% EMS 32.6 ± 3.2 12.2 ± 1.2 64.50 ± 0.34 Treated 3C: 0.3% EMS 38.0 ± 4.0 16.5 ± 2.0 70.26 ± 0.20 Treated

Table 2: Protein and total Chlorophyll Content in the leaves of Mulberry genotype RFS 135 Maturity of Leaves - Tender Total Chlorophyll Groups Protein % Reducing sugar % (mg/gm/wt) 2.791 ± 0.048 29.66 ± 1.73 2.16 ± 0.2647 CC: Control 1C: 0.1% EMS 2.909 ± 0.012 37.33 ± 0.62 3.16 ± 0.1058 Treated 3C: 0.3% EMS 3.708 ± 0.030 43.33 ± 0.71 12.00 ± 0.8670 Treated

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Table 3: Plant Morpho- economic Parameters of the M1V1 Clones of Mulberry Genotype RFS 135 Maturity of Leaves – Tender, Medium Coarse, Coarse Mean Branches per Mean Leaves per Height of the plant Leaf Area Groups plant Plant (cm) (cm2) 660.933 ± 17.30 21.26 ± 0.54 662.5 ± 28.87 282.64 ± 33.65 CC: Control 1C: 0.1% EMS Treated 3C: 0.3% EMS Treated

28. 06 ± 1.41

872.55 ± 29.17

778.06 ± 7.25

38.4 ± 0.73

989.66 ± 29.74

937.53 ± 10.57

716.914 ± 24.34 811.518 ± 31.39

DISCUSSIONS 0.3% EMS concentration affected the gross morphological characters in M1V1 clones of mulberry RFS135 variety, which showed great degree of genetic variations and more unique alleles against the control. Leaf extract of mulberry is used as nutritional intake due to its functional characteristics in controlling blood glucose level. The major constituents like phenolics and flavonoids probably might have antioxidant activities (Mudra M, et al 2007). The RAPD studies using random primers ((OPW- 04/05), has revealed that 0.3% EMS had altered the molecular characters of total genomic DNA with polymorphs of significantly variable size of different base pairs and probably influenced variations in the phytochemical constituents like phenolics and flavonoids (Kumar H. V. A, et al., 2013).

The mean Total Phenol (TP) content of the six cultivars ranged from 30.4 to 44.7 equivalents (GAE) mg/g, harvested in the month of May showed highest Total Phenol content (Yuxiao Zou, et al., 2012). Similarly, our study reveals that M1V1 mutant clones recovered from 0.3% EMS treatment under same conditions after 180 days of harvest recorded the total phenolic content of 38.0 ± 4.0 against control 26.4 ± 2.5 (GAE mg/)( table.1). Mutant clones of 0.3% EMS treated showed higher levels of biochemical response for total chlorophyll 3.708 ± 0.030 when compared to control 2.791 ± 0.048, similarly protein contents was 43.33 ± 0.71 against control 29.66 ± 1.73. The reducing sugar in the clones of 0.3% was 12.00 ± 0.8670 compared to control 2.16 ± 0.2647 that showed significant increase (table.2).

The morpho-economic traits such as plant height (cm) in treated 0.3% were 937.53 ± 10.57 against control 662.5 ± 28.87. Mutants clones evolved from 0.3%. EMS treatment exhibited increased plant height. Similarly, the clones showed higher number of branches 38.40 ± 0.73 compared to control 21.26 ± 0.54. Significant increase in number of branches and plant height were confirmed from the present studies and was proved statistically (p = 0.0006). The variations in plant height and number of branches has been well established through bud mutations induced by radio sensitivity of gamma radiations in mulberry varieties M5 and Mysore local (Hazama et al.,1968, Rao et al., 1984). Strong correlation has been proved between the plant height and leaf yield ( Sarkar et al.,1987, Susheelamma et al., 1989 and Bari et al., 1989).

Further, the increased shoot height is due to cell elongation, they attributed the differential response of root and shoot to their anatomical and physiological differences and difference between their growth mechanisms (Sinha and Godward, 1972).

The more number of branches and shoots per plant are the indicators of high yield obviously associated with biomass (leaves).

Bigger leaf size is attributed to increase in biomass. The mean leaf area increased to 811.51 cm2 ± 31.39 in the clones of 0.3% EMS treated population, against the control 282.64 cm2 ± 33.65. The increase in leaf area is attributed to

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Antioxidant and Phenolic Activities in EMS Induced Mutants of Mulberry Genotype RFS135 and Effects of Mutagen on Morpho-Economic Characters

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enlargement in tissues of palisade and spongy. It is proved from the findings of variants obtained from induction of mutations by colchicines. The increase in size and thickness of leaf observed probably must have increased the biomass.

(Dwivedi et al., 1989, 1985) In the present studies, the EMS might have influenced through mutations to enlarge the palisade and spongy tissues, likely to have increased the size of leaf.

Antioxidant activity (RSA % inhibition), 70.26 ± 0.20 in treated when compared control 52.40 ± 0.52 by DPPH method. Many researchers claimed on the hypoglycemic and hypolipidemic effects of Morus indica in patients with type II diabetes (Andallu, B et al., 2001) and hypoglycemic and antioxidant activity in streptozotocin-diabetic rats (Chen, F. et al 1995, Andallu, B et al., 2003). In Morus alba L., such antioxidant activity of various solvent extracts has been established (Yen, G et al 1996), similarly the free radical scavenging activity of flavanoids has been proved (Zhishen, J et al., 1999).

Many other such phytoconstituents has been well established in the leaves of Morus alba L., (Kim, S. Y. et al., 1999)( Sharma, R et al., 2001).

CONCLUSIONS The present study establishes a significant increase in biochemical-Phytochemical constituents in M1V1 mutant clones recovered from 0.3% EMS induced Mulberry genotype RFS135. The phenolic content and antioxidant activity also shows significant increase, which would lead to a strong therapeutic response, may be useful in near future for preventing ROS oxidative induced damaging effects. The significant enhancement in the morpho-economic traits indicates evolving promising high yielding variety of mulberry benefiting sericulture industry. Probably EMS might have gross effects on variations in biochemical – phyto constituents, morpho-economic traits and antioxidant phenolic activities. Further, molecular profiling would help in better understanding of variations observed among the M 1V1 mutant clones.

ACKNOWLEDGEMENTS The corresponding author Dr. H. V. Anil Kumar profusely express deep sense of gratitude to Dr. Munirajappa, Rtd Director, Center for Applied Genetics, Bangalore University for his research guidance. I thank Dr. H. T. Krishna murthy, the Principal DVS College of Arts and Science, Shimoga, for his constant encouragement extended to publish this research article. My sincere thanks to Shri K. Basappa Gowda, the President, Shri. K. N. Rudrappa Kolale, VP and Shri. S. Rajashekar, Secretary of DVS management committee for their valuable support to all my research endeavours and progress at this esteemed institute.

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