Effect of oligomeric sodium alginate and chitosan on ...

Journal of Functional and Environmental Botany Volume 7, Number 2, November, 2017, 101-111

DOI: 10.5958/2231-1750.2017.00012.9

Research Article

Effect of oligomeric sodium alginate and chitosan on Growth Attributes, Physiology and Essential Oil Composition in Mentha arvensis L. in Northern Himalayas M. Afaan Fazili1,2*, Abdul Hamid Wani1 and Zahoor A. Bhat1 1

Downloaded From IP - 103.55.108.2 on dated 30-May-2018

www.IndianJournals.com

Members Copy, Not for Commercial Sale

Section of Mycology and Plant Pathology, Department of Botany, University of Kashmir, Srinagar-190006, J&K, India 2 Genetics, Cytogenetics and Plant Breeding Laboratory, Department of Botany, Aligarh Muslim University, Aligarh-202002, UP, India *Corresponding author email id: [email protected]

ABSTRACT Mentha is a genus of plants in the family Lamiaceae. It is estimated that 13 to 18 species exist. The oil derived from Mentha is of great economical use.Natural occurring marine polysaccharides such as sodium alginate, chitosan, carrageenan after undergoing depolymerisation by different techniques have shown positive influence on growth, morphological and yield attributes of plants. In this study, the gamma-degraded polysaccharides like sodium alginate and chitosan have been applied to check the effect on growth, physiological and essential oil composition. A pot experiment was conducted to evaluate the influence of different doses of irradiated sodium alginate (ISA) and chitosan on the growth, physiological and biochemical attributes as well as essential oil composition in Mentha arvensis L. A single dose of ISA was used (80 mg L -1) in combination with six doses of irradiated chitosan (20, 40, 60, 80, 100, 120 mg L -1). It was found from the results that the treatment T4 (ISA 80 + IC80) proved to be optimum and enhanced most of the growth parameters. The plant height was recorded higher by 16.88%, fresh weight increase by 41.62%, and dry weight by 31.74% over the control, at the treatment T4 (ISA 80 + IC80) respectively. Similar trend was noticed while analysing the physiological and biochemical parameters where the total chlorophyll content and carotenoid content was increased by 40.25% and 37.30%. The carbonic anhydrase (CA) activity increased by 16.26 %.Total nitrogen (N) content showed the maximum increase by 27.60% P content by 14.34% and K content by almost 5.01% over the control plants. The combined treatment (ISA 80 + IC80) enhanced the oil yield (as revealed by the Gas Chromatography GC/GC– MS (Mass Spectroscopy) analysis) by 2.89% in menthol content, 39.74% in menthol content, L menthone content by 80.05%, L -menthone yield by 319.28%, isomenthone content by 31.73%, menthylacetate content by 36.32%, menthylacetate yield by 200% over the control in M. arvensis L. Keywords: Mentha arvensis L., GC, GC-MS analysis, Irradiated sodium alginate, Essential oil content, Essential oil yield

INTRODUCTION Mentha arvensis, the corn mint, field mint, or wild mint, is a species of flowering plant in the mint family Lamiaceae. It has a circumboreal distribution, being native to the temperate regions of Europe and western and central Asia, east to the Himalaya and eastern Siberia, and North America. Alginates have occupied

Journal of Functional and Environmental Botany

a prominent position among natural polysaccharides and are important structural parts of brown algae like Sargassum (Anthony et al., 2007) in appreciable amounts. Chitosan is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of fungi. Such oligomers are applied to plants in the form of foliar sprays, regulate 101




M. Afaan Fazili, Abdul Hamid Wani and Zahoor A. Bhat

and elicit various kinds of biological as well as physiological activities, including enhancement of plant growth, seed proliferation and germination, root and shoot elongation, flower production, alleviation of heavy metal stress and others. However, the application of these oligomers can shorten the harvesting period of various crops and may help in reducing the use of insecticides and chemical fertilizers (Hien et al., 2000; Nagasawa et al., 2000; Ahni et al., 2001; Cabalfin, 2002; Hafeez et al., 2003; Luan et al., 2003; Lee et al., 2003; Sabharwal, 2004). Essential oils comprise metabolites which are mainly in the form of monoterpenes that are the product of plants secondary metabolism (Weiss, 2002; Sangwan et al., 2001). The radioactive degraded natural polymers (using gamma rays) are also reported to have been used as plant growth promoters in the form of foliar sprays (Kume et al., 2006; Mollah et al., 2009; Khan et al., 2011; Idree set al., 2011, 2012; Sarfaraz et al., 2011; Aftab et al., 2011, 2013). The biosynthesis from mevalonate and methyl-erythitol phosphate, the production of essential oil not only depends upon genetic variations but also on the environmental conditions at different developmental stages of plants which in turn alter and modify the quantity and quality of essential oils. The endogenous and exogenous application of plant growth hormones could enhance the essential oil production in plants involving the various plant signalling mechanisms. Utilisingcrosses between Mentha species, it has been shown that single Mendelian gene(s) control the presence or absence of major compounds such as carvone, menthone, menthol and piperitone. Several mint species are industrial crops, a source of essential oils enriched in certain monoterpenes that are widely used in food, flavour, cosmetic and pharmaceutical industries. Different species of mint are used across the globe for their medicinal and culinary properties. Mint is usually taken after a meal for its ability to reduce indigestion and colonic spasms by reducing the gastrocolic reflex. Since they are often perennial and produce suckers, Mentha species reproduce both by reproductive and vegetative means. In Mentha crops, the purity of cultivars is maintained by vegetative means of propagule generation. Mint species are widely distributed and are prone to attack by a variety of diseases and pests. Mentha is a genus of aromatic perennial herbs belonging to the family Lamiaceae, distributed mostly

102

in temperate and sub-temperate regions of the world. Mentha arvensis L. also known as Japanese mint, corn mint or menthol mint is cultivated commercially on large scales and is an important essential oil-bearing plant. Mentha has a large number of species that differ widely in their characteristics and polyploidy level. It is known to comprise about 40 recognisable species. The mint plants have been tremendously used in the drug, medicinal and other uses due to their essential oil content which are usually monoterpene and sesqueterpenes. It is believed that M. arvensis L. (Japanese mint) is a hybrid between M. arvensis L. and Mentha aquatica L. The monogenicbasis for conversion of menthone to menthol showed that gene R, either homozygous (RR) or heterozygous (Rr), is responsible for the reduction of menthone tomenthol or carvone to carveol. Enzymes involved inmonoterpene biosynthesis are described. Pests andpathogens of Mentha that cause substantial damageto the crop and considerable loss in oil yield are alsodescribed. A number of microbes, namely Macrophomia phaseolina, Puccinia menthae, termites, cutworms, whitefly and semi-loopers damage the Mentha plant. MATERIAL AND METHODS Plant Material and Growth Conditions The pot experiment was conducted in the net house of department of botany, University of Kashmir, Srinagar, JK, India. Before transplanting, each pot was filled with 5-kg homogenous mixture of soil and organic (cow-dung) manure (4:1) along with the plant material of M. arvensis L. (Var. Himalayan). Physicochemical characteristics of the experimental soil mixture (4 parts soil:1 part cow-dung manure) were: texture – sandy loam, pH (1:3), E.C. (1:2) 0.48 dS m-1, available N,P and K 102.4, 7.8 and 145.9 mg kg-1 of soil, respectively. A uniform recommended basal dose of N, P and K (25:11:21 mg kg -1 soil, respectively) was applied in the form of urea, single superphosphate and muriate of potash at the time of planting. However, the experiment was conducted in the randomised block design using earthen pots (25 cm diameter × 25 cm height). Each treatment was replicated five times. The plants were watered as and when required.

Volume 7, Number 2, November, 2017

Effect of oligomeric sodium alginate and chitosan on Growth Attributes, Physiology and Essential Oil Composition

Irradiation of Sodium alginate and Chitosan Solid material of sodium alginate and chitosan (SigmaAldrich, USA) was sealed in a glass tube with atmospheric air at different time intervals. The samples of sodium alginate and chitosan were irradiated in a Gamma Chamber (Cobalt-60, GC-5000) at Bhabha Atomic Research Centre (BARC), Srinagar, India. The samples were irradiated to 520 kGy gamma radiation dose at a dose rate of 2.4 kGy h -1. Polyvinyl alcohol polymers of known molecular weight were used as standards. Aqueous concentration of ISA and IC was finally prepared using double distilled water for spray treatments.




Analyses of Growth Parameters The plants of M. arvensis L. from each pot were uprooted carefully after 120 days of planting and then growth parameters (plant height, fresh weight, dry weight) were recorded. Plants were washed with fresh water to remove the additional foreign particles. Plant height and fresh weight was recorded. The plant samples were kept in a hot air oven at 80°C for 46 to 48 h. Later, the total dry weight of the plants was recorded. Biochemical Parameters Estimation of Total Chlorophyll and Carotenoid Contents Total contents of chlorophyll and carotenoids in fresh leaves were estimated using the method of Lichtenthaler and Buschmann (2001). Fresh tissues extracted from the interveinal leaf area were grinded using mortar-pestle using 70–80% acetone. At 662 and 645 nm, optical densities were recorded and carotenoid content of samples was recorded at 470 nm using a spectrophotometer (Shimadzu UV-1700, Tokyo, Japan). The photosynthetic pigments were expressed as mg g -1 FW. Estimation of Carbonic Anhydrase Activity Carbonic anhydrase (CA) activity was quantified in fresh leaves using the method as described by Dwivedi and Randhawa (1974). 200 mg of fresh leaf pieces were incubated in Petri dishes, containing 10 mL of 0.2 M cysteine hydrochloride solution for 20 min at 4°C. 4 mL of 0.2 Msodium bicarbonate solution


and 0.2 mL of 0.022% bromothymol blue were added. The reaction mixture was titrated against 0.05 N HCl using methyl red as an indicator. The enzyme was expressed as µM CO 2 kg-1 leaf FW s-1. Estimation of N, P and K Content From each treatment, the leaf samples were taken and digested for the estimation of leaf -N, -P and -K contents. The leaves were dried in a hot air oven at 75–80°C for 24 h. Using the mortar and pestle, the dried leaves were ground, and the leaf-powder was passed through a 72 mesh. For the estimation of N, P and K contents, the sieved leaf-powder was used. A quantity of 100-mg ovendried leaf powder was carefully poured into a digestion tube, and to which the 2 mL of analytical reagent grade concentrated sulphuric acid was added subsequently. This solution was heated on a temperature controlled assembly at 100°C for about 2 h. The content was cooled for about 15 min at room temperature. To the cooled content, 0.5 mL of 30% hydrogen peroxide (H 2O2) was added frequently drop by drop. The addition of H 2O2 was followed by gentle heating of the content, and then the content was cooled at room temperature. This step was repeated until the content of the tube turned colourless. The estimation of the per cent N, P and K contents in the leaves was estimated through aliquot (peroxidedigested material). Determination of N content Leaf nitrogen (N) content was estimated according to method of Lindner (1944). The protocol followed by Novozamsky et al. (1983) was taken into consideration for modification. By using temperaturecontrolled Kjeldahl assembly, the dried leaf-powder samples were digested with H 2SO4 in the digestion tubes. Into a 50 mL volumetric flask, a 10-mL aliquot (peroxide-digested material) was poured. To neutralise the excess acid and prevent turbidity, the 2 mL of 2.5 N sodium hydroxide and 1 mL of 10% sodium silicate solutions were added. A 5 mL aliquot of the peroxide-digested plant material was poured into a 10 mL graduated test tube followed by addition of 0.5 mL of Nessler’s reagent. The OD (optical density) of the solution was recorded at 525 nm using the spectrophotometer.

103


oil was dried over anhydrous sodium sulphate and preserved in sealed glass vials at 4°C for the gas liquid chromatography (GLC) analysis of the oil. The active constituent (menthol content) of the essential oil was determined using GLC (Nucon 5700, New Delhi, India) equipped with the AT-1000 stainless steel column, flame ionisation detector and integrator. Nitrogen was used as the carrier gas. The flow rates of nitrogen, hydrogen and oxygen were maintained at 0.5, 0.5 and 5 mL s -1, respectively. GLC temperature schedule was as follows: detector temperature, 250°C; oven temperature, 160°C and injector temperature, 250°C. The sample size was 2 L in variably. The identification of active constituent (menthol content) was based on retention time. It was quantified as the percent content comparing their peaks with the peaks obtained from the reference standard reported in the literature.

Determination of P content The method of Fiske and Row (1925), with slight modification by Rorison et al. (1993), was used to estimate the leaf P content in the peroxide-digested material. A 5-mL aliquot was poured into a 10-mL graduated test tube. To it, 1 mL of molybdic acid (2.5%) was added, followed by addition of 0.4 mL of 1-amino-2-naphthol-4-sulfonic acid. When the colour of the content turned blue, the volume of the test tube was made up to 10 mL, using double distilled water. The OD of the solution was recorded at 620 nm using the spectrophotometer.




Determination of K content Leaf K content was determined in the peroxide digested plant material by a flame-photometer (Hald, 1947) (Model, C150, AIMIL, India) with the help of emission spectra using specific filter. In the flamephotometer, the solution (peroxide-digested material) was discharged through an atomiser in the form of a fine mist into a chamber, where it was drawn into a flame. Combustion of the elements produced the light of a particular wavelength [max for K = 767 nm (violet)]. The light, thus produced, was passed through an appropriate filter to impinge upon a photoelectric cell that subsequently activated a galvanometer to get the reading.

Observed volume of oil (mL) Oil content (% v/w) =

x 100 Weight of sample (g)

Statistical Data Analysis The data were analysed statistically according to randomised block design using SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA). Mean values of the results were statistically compared using Duncan’s Multiple Range Test at p< 0.05.

Determination of Yield and Quality Attributes The yield and quality attributes were determined estimating the essential oil content and essential oil yield and herbage yield per plant of M. arvensis L.

RESULTS It was observed from Table 1 that irradiated chitosan (IC) and sodium alginate brought about improvement in the growth, physiological, biochemical as well as yield and active constituents of M. arvensis L.

Isolation and compositional analysis of essential oil The essential oil was extracted and then quantified gravimetrically according to Guenther (1972). The fresh leaves were chopped into small pieces. Essential oil content in the leaves was extracted by distillation for 3 h, using Clevenger’s apparatus. The extracted

Growth Attributes As revealed from Table 1, growth attributes like plant height, fresh weight as well as dry weight were

Table 1: Effect of degraded sodium alginate (ISA 80) along with different concentrations of irradiated chitosan (IC 20, IC40, IC60, IC80, IC100, IC120) on growth parameters of Mentha arvensis L. Growth Parameters

T1 (ISA 8+IC 20)

T2 (ISA 8+IC 40)

T3 (ISA 80+IC 60 )

T4 (ISA 80+IC 80 )

Plant height (cm)

94.61±1.29 cd

95.81±2.44 bcd

98.01±1.30 bc

105.34±2.29 a 101.33±1.45 ab

92.44±1.92 cd

90.12±2.07 d

Fresh weight (g)

42.31±1.13 d

46.46±1.25 c

51.10±1.32 b

55.22±0.67 a

50.54±1.02 b

40.44±0.89 d

38.99±1.12 d

d

cd

bc

a

ab

d

12.98± 0.83 d

Dry weight (g)

104

13.21±0.54

14.28±0.35

15.16± 0.55

17.10±0.29

T5 T6 (ISA 80 +IC 100 ) (ISA 80 +IC 120 )

16.01±0.59

13.10±0.44

T7 (Control)



profoundly enhanced by doses of ISA and chitosan as compared with the control plants sprayed with DDW in M. arvensis L. Among various doses applied, ISA80 + IC80 proved best in the respective experiments. Soil applied with ISA along with different doses of chitosan significantly influenced all the growth parameters. The effect of ISA 80 + IC80 was significant on plant height, fresh weight as well as dry weight. In comparison with the controlled plants, the ISA 80 + IC80 improved plant height by over 16.88%, the fresh weight by over 31.74% and dry weight by over 41.62%, respectively (Table 1).

Photosynthetic Pigments

It was observed from Table 2 that depolymerised form of sodium alginate and chitosan significantly enhanced the total chlorophyll content in the plants under treatment. Of all the ISA and IC concentrations applied in combination, the dose ISA 80 + IC100 [ISA (80 mg L-1) and IC (80 mg L -1)] proved best in enhancing the photosynthetic content. Depolymerised form of sodium alginate and chitosan significantly enhanced the total chlorophyll content in the plants under treatment. However, the combined treatment of ISA 80 + IC80 was much significant as compared with other treatments of ISA and IC. The total carotenoid content was increased by almost 37.30% at the T4 (ISA 80 + IC80) over the control plants.

Chlorophyll content

Carbonic anhydrase activity

It was observed from Table 2 that depolymerised form of sodium alginate and chitosan significantly enhanced the total chlorophyll content in the plants under treatment. Of all the IC and ISA concentrations applied in combination, the dose ISA 80 + IC80 [ISA (80 mg L-1) and IC (80 mg L -1)] proved best in enhancing the photosynthetic content. Depolymerised form of sodium alginate and chitosan significantly enhanced the total chlorophyll content in the plants under treatment. However, the combined treatment of ISA 80 + IC 80 was much significant as compared to other treatments of ISA and IC. The total chlorophyll content was increased by 40.25% at the T4 (ISA 80 + IC80) over the control plants (Table 2).

ISA and IC at different concentration increased the CA activity. However, when applied at the dose rate of 80 mg L -1 increased the CA activity maximally. The maximum enhancement shown by T4 (ISA 80 + IC80) was about 16.26% over the control plants (Table 2).



Physiological and Biochemical Characteristics


Carotenoid content

Leaf-N, -P and -K content Nutrient utilisation, absorption and transport of photosynthesis in plants are facilitated by the increased membrane permeability (Crozier and Turnbull, 1984). Leaf -N, -P and -K contents were also significantly enhanced by the ISA 80 + IC80 application, proving to be the best dose over all doses in contrast to the control plants. The approximate increase in the leaf-

Table 2: Effect of foliar application of irradiated sodium alginate (ISA 80) along with different concentrations of irradiated chitosan (20, 40, 60, 80, 100 and 120 mg L -1) on physiological and biochemical attributes of Mentha arvensis L. Means within a column followed by the same letter(s) are not significantly different (p < 0.05). The data shown are means of five replicates±SE Physiological and BioT1 chemical Parameters (ISA 80+IC 20 )

T2 (ISA 80+IC 40 )

T3 (ISA 80+IC 60 )

T4 T5 T6 (ISA 80+IC 80 ) (ISA 80 +IC 100 ) (ISA 80 +IC 120 )

0.95±0.004 f

0.97±0.003 e

1.12±0.007 c

1.32±0.008 a

Total chlorophyll content (mg g -1)

1.14±0.008 b

T7 (Control)

1.02±0.006 d 0.87± 0.0012 g

Total carotenoid content (mg g -1)

0.532±0.003 e 0.560±0.005 e 0.630±0.005 d 0.703±0.002 a 0.590±0.006 c 0.674± 0.007 b 0.512±0.004 f

CA activity (µM CO2 kg-1 FW s-1)

251.21±1.57 d 263.77±1.46 c 271.28±1.24 b 280.45±2.05 a 265.44±1.22 c 255.03±1.74 d 241.21± 1.11 e

Leaf-N content (%) Leaf-P content (%) Leaf-K content (%)

4.09±0.56 e

4.26±0.72c

4.31±0.65 b

4.48±0.54 a

4.18 ±0.41 d

3.87±0.33 f

3.51±0.81 g

0.242±0.001 bc 0.248±0.002 bc 0.251±0.001 b 0.263±0.002 a 0.253±0.001 b 0.237 ±0.002 c 0.230 ±0.002 c 2.63±0.77 a

2.65±0.84 a


2.68±0.99 a

2.72± 0.83 a

2.71±0.79 a

2.61± 0.61 a

2.59±0.45 a

105





Figure 1: GC chromatogram showing peaks of different constituents of essential oil of Mentha arvensis L. at T4 [(ISA 80 + IC80) 80 mg L -1] Table 3: Volatile oil composition of M. arvensis L. (Var. Himalaya) as revealed by GC–MS from Kashmir division of J&K S.No. Retention Time

Constituents

S.No.

Retention Time

Constituents

1

3.881

dl-Limonene

16

17.977

Piperitone oxide

2

4.046

Eucalyptol

17

18.216

Butyloctadecanoate

3

4.665

§-3-Carene

18

24.584

Caryophyllene oxide

4

5.174

£-Phellandrene

19

27.778

2,5-Dimethyl-3-hexyne-2,5-diol

5

8.372

3-Octanol

20

29.988

Thymol

6

10.186

L -Menthone

21

31.594

a-Aminoisobutanoic acid

7

10.363

cis-Sabinene hydrate

22

39.075

18,18-Bi-l,4,7,10,13,16-hexaoxacyclononadecane

8

10.962

L -Menthone

23

40.227

18,18-Bi-l,4,7,10,13,16-hexaoxacyclononadecane

9

12.918

L-Linalool

24

40.333

(2S,2S)-2,2-Bis [1,4,7,10,13-pentaoxacyclopentadecane]

10

13.869

ß-Caryophyllene

25

40.550

(2S,2S)-2,2-Bis [1,4,7,10,13-pentaoxacyclopentadecane]

11

14.211

Isoneomenthone

26

40.675

2-Hydroxyhexadecyl-2,3-isopropylidene glycerol

12

15.533

Menthol

27

40.793

Dodecyltriglycol

13

16.301

Neo-menthol acetate

28

40.842

Dodecyltriglycol

14

17.139

trans-Anethole

29

40.922

Dodecyltriglycol

15

17.389

Piperitone oxide

30

41.00

(2S,2S)-2,2-Bis[1,4,7,10,13-pentaoxacyclopentadecane]

In the above Table 3 the volatile oil composition of M. arvensis L. (Var. Himalaya) was revealed by GC–MS from Kashmir division of J&K. The GC-MS analysis was done in SKAUST-K. The compounds identified from the GC-MS are shown in the Table 3 above. Nearly, thirty compounds were isolated from this technique on the basis of retention time (RT).

106



N, -P and -K content was 27.60%, 14.34 % and 5.01%, respectively, over the control plants. In relation to these results, Naeem et al. (2011) reported profound increase in the uptake of these elements (N and P) by IC concentration of 80 mg L -1 in M. arvensis L. However, such results can be concorded to the ISA which also showed optimal increase at the same concentration that is 80 mg L -1. Such an impact of ISA and IC application could be ascribed to the ISA and IC-mediated increase in overall growth of plants which accordingly demanded for higher up take of these nutrients from the soil, leading to their significant accumulation in the leaves (Table 2).




Yield Attributes The combined treatment of the plant growth regulators at T4 (ISA80 + IC80) enhanced the oil composition (as revealed by the GC/GC–MS analysis) by 2.89% in menthol content, 39.74% in menthol yield per plant, L -menthone content by 80.05%, L -menthone yield by 319.28%, isomenthone content by 31.73%, menthyl acetate content by 36.32%, menthyl acetate yield by 200% over the control in M. arvensis L. (Table 5).

DISCUSSION Only few records are known which report the effect of IC on agricultural crops. However, this is not true in the case of ISA. This survey has been concorded to check the influence of degraded polysaccharides like chitosan and sodium alginate on growth, physiological and biochemical as well as yield and active constituents of M. arvensis L. Going through the earlier studies, ISA 80 (80 mg L-1) has proven to be the best dose for enhancing the growth, physiological as well as yield attributes in M. arvensis L. (Figure 2). So here in this study, the unique optimal concentration of ISA that is ISA80 was quantified along with the different concentrations of IC (IC 20, IC 40, IC 60 , IC 80 , IC 100 , IC 120mg L -1 ).Among the various doses, ISA 80 + IC 80 proved to be the optimal for almost all parameters studied. Growth Attributes The effect of ISA 80 + IC80 was significant on plant height, fresh weight as well as dry weight. In accordance with the earlier studies, Idrees et al.

Table 4: Effect of hydro-distillation process on essential oil content of Mentha arvensis L. at various stages of growth Effect of Extraction Time of Oil Obtained Through Clavenger’s Apparatus from Mint Leaves at Different Agrochemical/Environmental Conditions

Shade Dried Mint Leaves

Sun Dried Mint Leaves (Ave. Temp. 15–25°C)

Mint Plants Grown in Poor Moisture Content Soils (Dry Soil Conditions)

Mint Plants Grown in Average Moisture Content Soils

Mint Plants Grown in High Moisture Content Soils

Time (min)

Oil content (mL)

10

2.39

20

2.62

1.04

0.40

1.13

1.04

1.11

0.44

1.44

1.14

30 40

2.88

1.42

0.53

1.52

1.27

2.97

1.56

0.59

1.71

1.56

50

3.23

1.83

0.62

1.82

2.28

60

3.38

1.94

0.63

1.94

2.36

70

3.55

2.12

0.68

2.52

2.38

80

3.68

2.16

0.74

2.88

2.41

90

4.63

2.56

0.79

3.42

2.46

100

5.44

3.19

0.92

3.88

2.58

110

5.89

3.48

0.97

3.91

2.62

120

6.77

3.53

0.98

3.94

2.77

The effect of hydro-distillation process on essential oil content of Mentha arvensis L. was studied at various stages of growth (Table 4). Taking into the consideration, the various agrochemical, environmental conditions, the total effect on oil content of Mentha arvensis L. was observed. The oil was measured in ml. Among the various agrochemical, environmental conditions studied it was concluded that the shade dried mint leaves yielded more oil as compared to the other conditions observed.


107


Table 5: Effect of foliar application of irradiated sodium alginate along with different concentrations of irradiated chitosan (20, 40, 60, 80, 100 and 120 mg L-1) on yield attributes of Mentha arvensis L. Means within a column followed by the same letter(s) are not significantly different (p < 0.05). The data shown are means of five replicates ± SE. Content and yield of active constituents

T1 (ISA80+IC20)

T2 (ISA80+IC40)

T3 (ISA80+IC60)

T4 (ISA80+IC80)

Menthol content (%)

91.21± 0.01c

91.34±0.01c

92.94±0.01b

93.83±0.02a

Menthol yield per plant (mL)

0.502±0.002f

0.523±0.002e

0.558±0.003c

0.668±0.003a 0.541± 0.002d 0.602±0.004b 0.478±0.003g

4.54±0.26f

4.63±0.31e

5.45 ±0.28c

6.77±0.023a

4.93±0.34d

5.90±0.031b

3.76±0.41g

0.21±0.003f

0.29±0.002e

0.47±0.003c

0.58±0.003a

0.31±0.002d

0.51±0.002b

0.14±0.003g

Iso-menthone content(%) 3.10±0.010f

3.31±0.010e

3.55±0.021c

4.11±0.010a

3.42±0.020d

3.78±0.020b

3.12±0.021g

2.22±0.020f

2.34±0.010e

2.65±0.021c

2.89±0.020a

2.51±0.020d

2.77±0.010b

2.12±0.021g

0.009±0.002f

0.011±0.001e

0.14±0.002c

0.021±0.002a 0.016± 0.001d 0.018±0.002b 0.007±0.001g

L

-Menthone content (%)

-Menthone yield perplant (mL) L

Menthyl acetate content (%)

91.55±0.01c

93.34±0.02a

T7 (Control) 91.19±0.02c




Menthyl acetate yield per plant (mL)

T5 T6 (ISA80+IC100) (ISA80+IC120)

Figure 2: GPC of un-irradiated and irradiated sodium alginate (ISA). The molecular weight distribution of un-irradiated and irradiated sodium alginate. The average molecular weights of the un-irradiated sodium alginate samples were estimated to be about 0.695 MDa. The distribution curve in the GPC profile shows shifting of whole graph to higher retention time indicating radiation degradation of sodium alginate on irradiation and forming lower molecular weight oligomers. This average molecular weight of 0.695 MDa was observed in the control and 0.595 MDa for the irradiated samples.

108



(2011, 2012) and Khan et al. (2011) reported significant improvement in plant growth parameters with the application of radiation degraded oligosaccharides of chitosan and sodium alginate. A key role is played by the degraded sodium alginate in enhancement of the biological activities of the plants. Thus, our findings about the enhancement of growth parameters like plant height, fresh weight and dry weight are in relation with those of the findings of Idrees et al. (2011, 2012) and Khan et al. (2011).




Physiological and Biochemical Attributes Of all the IC and ISA concentrations applied in combination, the dose ISA 80 + IC80 [ISA (80 mg L -1) and IC (80 mg L -1)] proved best in enhancing the photosynthetic content. Depolymerised form of sodium alginate and chitosan significantly enhanced the total chlorophyll content in the plants under treatment. It has been reported that ISA induce cell signalling, leading to the stimulation process of different processes in various plants, including ISA and IC-mediated development in the content of photosynthetic pigments and net photosynthetic rate (Farmer et al., 1991). This study is related to earlier findings of Aftab et al. (2011), Khan et al. (2011) and Sarfaraz et al. (2011). Being one of the most abundant zinc-containing proteins in plants, CA has played a crucial role in the process of photosynthesis, which is obvious by its dominance in photosynthetic tissues. It catalyses the reversible hydration of CO 2 to carbonic acid, thereby enhancing the availability of CO2 to RuBisCO in photosynthesis (Badger and Price, 1994). The depolymerised natural polysaccharides have been reported to increase the stomatal conductance significantly for such plant responses (Naeem et al., 2011), which may be responsible for the diffusion of additional amounts of CO 2 through the stomata to be inducted upon by CA, resulting in the CA activity. As reported by Patier et al. (1995) and Akimoto et al. (1999), their occurs biosynthesis of various enzymes in the tissue culture as a result of application of certain irradiated natural polysaccharides. However, a probable reason for the enhancement of CA activity could be the ISA and ICmediated de novo synthesis of CA, that might engulf transcription/translation of the genes linked, as it has been verified for other degraded natural polysaccharides (Knowles and Ries, 1981) (Table 2).


Yield Attributes The combined treatment of the plant growth regulators at T4 (ISA80 + IC80) enhanced the oil composition (as revealed by the GC/GC–MS analysis) (Figure 1). The exogenous application of IC and ISA has been found positively effective in improving growth, yield and essential oil content of M. arvensis L. (Naeem et al., 2011). Increment in the yield of essential oil and that in the contents of its active constituents might be due to the degraded polysaccharide (ISA and IC)stimulated vegetative growth, population of leaf oil glands, nutrient accumulation (N and P) and also due to the beneficial effect of IC and ISA on plant metabolism and enzymes activities responsible for mono or sesquiterpenes biosynthesis, which could have been subsequently used to enhance the formation of metabolites with regard to oil formation. This conclusion is in accordance with the findings of Naeem et al. (2011) on M. arvensis L. CONCLUSION Conclusively, the foliar application of ISA and IC resulted in the enhancement of growth characteristics, physiological and biochemical parameters, essential oil yield and contents of its main constituents in M. arvensis L. with 80 mg L -1 proving the best optimal concentration. These findings are in relation with the earlier findings carried out regarding the effect of other degraded natural polysaccharides on growth, yield and/or quality and life span of other medicinal crops. The combination of two different degraded polysaccharides ISA and IC have been tested for the first time in this study to enhance the crop production in M. arvensis L. Further, this research may also help to find out the optimum concentration of IC and ISA and/or other irradiated natural polysaccharides for different medicinal and aromatic plants to enhance the productivity, quality and production of essential oil and other active constituents. ACKNOWLEDGEMENTS The financial support provided to the first author from the University Grants Commission New Delhi, for carrying out this study is gratefully acknowledged. The author is highly thankful to BRNS/BARC Mumbai/ Srinagar for successfully carrying out the degradation of the samples at various dose rates.

109


REFERENCES Aftab T, Khan MMA, Naeem M, Idrees M and Varshney L, 2011. Enhancing the growth, photosynthetic capacity and artemisinin concentration in Artemisia annua L. by irradiated sodium alginate-A plant growth promoter. Radiation Physics and Chemistry, Vol. 80, pp. 833–836. Aftab T, Naeem M, Direst M, Khan MMA, Moinuddin and Varshney LS, 2013. Cumulative role of irradiated sodium alginate and nitrogen fertilizer on growth, biochemical processes and artemisinin production in Artemisia annua. Industrial Crops and Products, Vol. 50, pp. 874–881. Ahni KJ, Park L and Byun MW, 2001. The production of chitosan and alginate monomer and oligomer by irradiation. In: Session 59A, Carbohydrate, 2001. IFT Annual Meeting. New Orleans, LA, USA.




Akimoto C, Aoyagi H and Tanaka H, 1999. Endogenous elicitorlike effect of alginate on physiological activities of plant cells. Applied Microbiology and Biotechnology, Vol. 52, pp. 429– 436. Anthony J, Gabapathy A, Coothan KV, Streenivasan PP, Arjuna R and Palaninathan V. 2007. Beneficial effects of sulphated polysaccharides from Saragassum wightii against mitochondrial alterations induced by cyclosporine A in rat kidney. Molecular Nutrition and Food Research, Vol. 51, pp. 1413–1422. Badger MR and Price GD, 1994. The role of carbonic anhydrase in photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology, Vol. 45, pp. 369–392. Cabalfin EG, 2002. Status of radiation treatment of liquid samples in the Philippines. In The FNCA Workshop on Application of Electron Accelerator, December 16–20, 2002. Takasaki, Japan.

Hien NQ, Nagasawa N, Tham LX, Yoshii F, Dang HV, Mitomo H, Makuuchi K and Kume T, 2000. Growth promotion of plants with depolymerised alginates by irradiation. Radiation Physics and Chemistry, Vol. 59, pp. 97–101. Idrees M, Naeem M, Alam M, Aftab T, Hashmi N, Khan MMA, Moinuddin and Varshney L, 2011. Utilizing the gamma irradiated sodium alginate as a plant growth promoter for enhancing the growth, physiological activities and alkaloids production in Catharanthus roseus L. Agricultural Sciences in China, Vol. 10, pp. 101–105. Idrees M, Nasir S, Naeem M, Aftab T, Khan MMA, Moinuddin and Varshney L, 2012. Gamma irradiated sodium alginate induced modulation of phosphoenolpyruvate carboxylase and production of essential oil and citral content of lemongrass. Industrial Crops and Products, Vol. 40, pp. 62–68. Khan ZH, Khan MMA, Aftab T, Idrees M and Naeem M, 2011. Influence of alginate oligosaccharides on the growth, yield and alkaloids production of opium poppy (Papaver somniferum L.). Frontiers of Agriculture in China, Vol. 5, pp. 122–127. Knowles NR and Ries SK, 1981. Rapid growth and apparent total nitrogen increases in rice and corn plants following applications of triacontanol. Plant Physiology, Vol. 68, pp. 1279–1284. Kume T, Nagasawa N and Yoshii F, 2002. Utilization of carbohydrates by radiation processing. Radiation Physics and Chemistry, Vol. 63, pp. 625–627. Lee DW, Choi WS, Byun MW, Park HJ, Yu YM and Lee CM, 2003. Effect of irradiation on degradation of alginate. Journal of Agricultural and Food Chemistry, Vol. 51, pp. 4819–4823.

Crozier A and Turnbull CGN, 1984. Gibberellins: Biochemistry and Action in Extension Growth. What’s New in Plant Physiology, Vol. 15, pp. 9–12.

Lichtenthaler HK and Buschmann C, 2001. Chlorophylls and carotenoids: Measurement and characterization by UV–vis spectroscopy. In: Wrolstad RE, ed. Current Protocols in Food Analytical Chemistry. pp. F4.3.1–F4.3.8, John Wiley and Sons, New York.

Dwivedi RS and Randhawa NS, 1974. Evaluation of rapid test for the hidden hunger of zinc in plants. Plant and Soil, Vol. 40, pp. 445–451.

Lindner RC, 1944. Rapid analytical methods for some of the common inorganic constitutes of plant tissue. Plant Physiology, Vol. 19, pp. 76–89.

Farmer EE, Thomas DM, Michael JS and Clarence AR, 1991. Oligosaccharide signaling in plants. Journal of Biological Chemistry, Vol. 266, pp. 3140–3145.

Luan LQ, Hien NQ, Nagasawa N, Kume T, Yoshii F and Nakanishi TM, 2003. Biological effect of radiation-degraded alginate on flower plants in tissue culture. Applied Biochemistry and Biotechnology, Vol. 38, pp. 283–288.

Fiske CH and Subba Row Y, 1925. The colorimetric determination of phosphorus. The Journal of Biological Chemistry, Vol. 66, pp. 375–400. Guenther E, 1972. The essential oils: History-origin in plants production-analysis, vol. 1. Huntington: Robert E. Krieger Publishing Company.

Mollah MZI, Khan MA and Khan RA, 2009. Effect of gamma irradiated sodium alginate on red amaranth (Amaranthus cruentus L.) as growth promoter. Radiation Physics and Chemistry, Vol. 78, pp. 61–64.

Hafeez M, Muhamad O, Muhammad SY and Ahmad MS, 2003. Irradiated chitosan as plant promoter. In Prosiding Simposium Biologic Gunaan Ke-7, Mines Beach Resort & Spa, 3–4 June, 2003, Seri Kumbangan, Selangor.

Naeem M, Idrees M, Aftab T, Khan MMA, Moinuddin and Varshney L, 2011. Irradiated sodium alginate improves plant growth, physiological activities and active constituents in Mentha arvensis L. Journal of Applied Pharmaceutical Science, Vol. 2, No. 5, pp. 28–35.

Hald PM, 1947. The flame photometer for the measurement of sodium and potassium in biological materials. Journal of Biological Chemistry, Vol. 167, pp. 499–510.

Nagasawa N, Mitomo H, Yoshii F and Kume T, 2000. Radiation induced degradation of sodium alginate. Polymer Degradation and Stability, Vol. 69, pp. 279–285.

110



Novozamsky I, Houba VJG, van Eck R and van Vark W, 1983. A noval digestion technique for multi-element plant analysis. Communications in Soil Science and Plant Analysis, Vol. 14, pp. 239–248. Patier P, Potin P, Rochas C, Kloareg B, Yvin JC and Liénart Y, 1995. Free and silica-bound oligokappa-carrageenans elicit laminarase activity in Rubus cells and protoplasts. Plant Science, Vol. 110, pp. 27–35. Rorison IH, Spencer RE and Gupta PL, 1993. Chemical analysis. In: Hendry GAE, Grime JP, eds. Methods in Comparative Plant Ecology. pp. 156–161, Chapman and Hall, New York, NY, USA.

Sarfaraz A, Naeem M, Nasir S, Idrees M, Aftab T, Hashmi N, Khan MMA, Moinuddin and Varshney L, 2011. An evaluation of the effects of irradiated sodium alginate on the growth, physiological activities and essential oil production of fennel (Foeniculum vulgare Mill.). Journal of Medicinal Plants Research, Vol. 5, pp. 15–21. Weiss EA, 2002. Essential oil crops. In Myrtaceae. Eucalyptus citriodora. Wallingford, Oxon, Ox 1 8 DE, UK: CAB International. Received 12.07.2017 Accepted 18.12.2017




Sabharwal S, 2004. Radiation processing in India: Current status and future programme. In Radiation Processing of Polysaccharides. Vienna, Austria: International Atomic Energy Agency.

Sangwan NS, Farooqi AHA, Shabih F and Sangwan RS, 2001. Regulation of essential oil production in plants. Plant Growth Regulation, Vol. 34, pp. 3–21.


111