Evaluation of Allelopathic Effect of Andrographis paniculata Nees on Biochemical

and Growth Parameters of Chenopodium murale L.

*K. M. Abdul Raoof, M. B. Siddiqui and Shazia Department of Botany, Aligarh Muslim University, Aligarh, UP (India), 202 002 *Correspondent author e- mail: abdulraoofkm@gmail.com

This experiment was conducted to determine the effects of leaves of Andrographis paniculata Nees in the growth performance of Chenopodium murale. Aqueous leachate of Andrographis paniculata at 0.5, 1, 2, and 4% concentrations were applied to determine their effect on seed germination, seedling growth and biochemical activity of test plants. Germination percentage and seedling growth decreased with increasing concentration of the treatment solution as compared to control. Protein and chlorophyll content were decreased with the aqueous leachates and an organic fraction compared to the control setup. Acid soluble carbohydrate content and water soluble carbohydrate were increased by both treatments Keywords: Aqueous leachates, Andrographis paniculata, Chenopodium murale, chlorophyll, carbohydrate, protein

1. INTRODUCTION

Weeds are the major enemy of the crop plants and create harmful effects on agricultural crops due to several factors such as competition for space, light and nutrients. Organic chemicals released as leaf leachates, root exudates affect the crop plants. Weed species are considered as a rich source of secondary metabolites (Allelochemicals) and these chemicals improve a certain kind of environmental system on other plants growing in their vicinity and the phenomenon known as allelopathy [1]. Allelopathy is a potential field of research all over the world. Few researchers consider only the deleterious interactions as allelopathy, while, the latest thinking includes allelopathy to both harmful and beneficial interactions between the plants [2].

Andrographis paniculata Nees is described a valuable medicinal plant in many popular systems of medicine including Ayurveda, Siddha and Unani. It cures dysentery, antivenom, fever, cholera, diabetes influenza and swelling [3,4,,5,6,7,8] The herb is used in preparation of household medicines. A decoction of this plant is used to cure jaundice. 'Alui' is a medicine prepared by using leaves and tender shoots. Extract of this plant exhibit antifertility, antifungal and antinematicidical activities. The primary medicinal component of being andrographolide, which is a “diterpene lactone”, water soluble substance and is distributed all over the plant body in different proportions. The leaves contain the highest amount of andrographolide (2.5%) and in stem (less than 2%), the most medicinally active phytochemicals in the plant, while the seeds contain the lowest. The other medicinal chemicals are also bitter principles: diterpenoids viz. deoxyandrographolide, 19 -D glucoside, and neo-andrographolide, all of which have been isolated from the leaves. The present study was to evaluate the allelopathic effect of aqueous leachates of leaves of A. paniculata on seed germination, seedling growth and biochemical activities of Chenopodium murale.

2. MATERIAL AND METHODS 2.1. Collection of plant materials

Leaves of Andrographis paniculata were collected from the premises of the Aligarh Muslim University campus, Aligarh. Healthy and freshly collected leaves were surface cleaned, dried and powdered. The seeds are also collected from the agricultural fields of the Aligarh.

2.2. Extraction of leachable allelo-chemicals

Based on the methods devised by Kumari et al. [9] healthy and freshly collected leaves of Andrographis paniculata were cut roughly into pieces after clearing their surface and their dry weight per unit fresh weight were determined by desiccating the tissue in the oven.

The weighed amount of fresh leaf pieces of the plant was soaked in the requisite amount of pure water (resistivity more 18.5 mega ohms cm and conductivity less than 0.05µScm at 25°C) for a period of 20hrs at room temperature. It was filtered completely through a triple layer of muslin cloth and the requisite concentration was made with water. One half

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Biosciences International, 2(2): 14-17

ResearchcPaper

Received March, 2013

Accepted June, 2013

Raoof et al. 2013:Evaluation of Allelopathic Effect of Andrographis paniculata Nees on Biochemical and Growth Parameters of Chenopodium murale L.

Biosciences International Volume 2, Issue 2, Page(s) 14-18 Juner, 2013

of this filtrate referred to as the aqueous leachates was used as such, while the other part was chilled and subjected to acid hydrolysis using pre-chilled, 3N HCl. The precipitate so formed were recovered through centrifugation (2000rpm). These were washed 5-6 times with pure water. Every time the recovery was made through mild centrifugation. For experimental purposes, the requisite amount of the precipitate was dissolved in a few drops of ethyl alcohol and the final volume was made with pure water. A drop of tween 20 was added to it, to serve as a surfactant. This is referred to as aglycone or aglyconic or organic component of aqueous leachates.

2.3. Extraction of organic fraction

Freshly collected, surface cleaned and healthy leaves of the requisite plant were dried under shade and powdered. The powder was immersed in Petroleum ether (60°-80°C) for 20hrs. The liquid was separated from the residue (Marc), through mild centrifugation (500rpm for 2 min). From the liquid portion the solvent (Petroleum ether) was recovered on a hot water bath. The requisite amount of the residue so obtain was weighed and a few drops of xylene, apart from a drop of tween- 20 (to act as a surfactant) were added to it. Final volume was made with pure water. This was termed Petroleum ether fraction.

The marc (residue from petroleum ether suspension) was suspended in methanol for 20hrs and filtered, from one half of the filtrate, methanol was covered with a hot water bath. The residue, so obtained was dissolved in a drop of methanol and the final volume was made with pure water. It has been called Methanol fraction. From another half of methanol filtrate the solvent was removed and the residue was partitioned between chloroform and water (1:1 V/V). The two layers so formed were separated in a separating funnel. The chloroform was recovered over a hot water bath. For the requisite amount of residue a few drops of methanol were added and the final volume was made with pure water. This has been termed as the chloroform fraction. The water from the aqueous layer after separating chloroform fraction was dried under low pressure on a rotary flash evaporator. The solution made with water has been termed as the Water fraction.

2.4. Growth performance in laboratory condition

Different concentration of aqueous leachates were prepared (4, 2, 1, 0.5%). The effects of aqueous leachate on germination, root length and shoot length were tested by placing 10 seeds of test plants in Petri dishes of 9 cm diameter containing two layers of filter paper saturated with the aqueous leachate. A separate control series was set up using distilled water. Both treated and control Petri dishes were kept continuously moist by applying extracts of respective concentration and distilled water whenever needed. The dishes were kept at room temperature for seed germination in the laboratory. Germination counts were recorded daily for a period of 15 days and root and shoot length was recorded with a measuring scale.

2.5. Growth performance in amended leaf powder 5, 10, 20 and 40gm Andrographis leaf powder was mixed into 1kg soil to get a leaf-soil mixture of the concentration 0.5, 1, 2 and 4% (w/w) in 6" diameter earthen pots. 10 seeds were sown per pot three replicate were taken for all concentration, and 3 replication treatments with pure soil were also sated to serve as control. For the estimation of carbohydrate, protein and chlorophyll content 9 plants were selected from each the treatment after 30 days of sowing and sprayed with 100ml of treatment solution per plant daily for five days. On the sixth day, the estimation was made from the freshly plucked leaves following the procedure given carbohydrate, protein and chlorophyll content were studied.

2.6.Determination of total chlorophyll content

Chlorophyll was extracted using dimethyl sulphoxide following the method of Hiscox and Israelstan [10] and estimated using the equation of Arnon [11]. It was expressed using in terms of dry weight of the tissue as suggested by Daizy and Kohli [12].

2.7. Determination of total protein content The content of water-soluble protein was determined following the method given by Lowry et al. [13]. For this, 200mg of leaves each of treated or control plants were crushed in 5ml of distilled water, and centrifuged at 15000rpm for about 30min. From the supernatant, estimation of protein was done and amount measured against known concentration of bovine serum albumin used as standard. There were three replicated for each treatment including control.

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2.8. Determination of carbohydrate content The methodology employed by Loweus [14] was followed for this purpose. (a) Water soluble carbohydrates- to 5mg dry powdered material was added 5ml of pure water. It was kept in boiling water bath for 5 minutes and centrifuged; the supernatant was used as a water soluble fraction. (b) Acid soluble carbohydrates- to the residue left as above were added 5ml of 6 N HCl. This was kept in a boiling water bath for 20 minutes and centrifuged. The supernatant was used as an acid soluble fraction. 2.9. Statistical analysis Computerized statistical analysis was done through ANOVA (Analysis of Variance) to assess the significance of difference between various treatments. 3. Result and Discussion The plant Allelo-chemicals may interfere the growth, development and performance of a plant through their direct or indirect action on metabolism of plants. Many physiological and chemical activities altered by these chemical interference. Nevertheless, it is difficult to determine the primary mechanism involved in the actions of these chemicals. It is believed that a variety of mechanisms of action must exist for different allelo-chemicals or herbicides. Even a specific compound may affect several metabolic functions and as a result, it is seldom possible to sort out primary effects from the secondary ones. In addition, the uncertainty in interpreting the observed effects in isolated enzymes to other bio-chemical effects in intact plant system also exists. No doubt, allelo-chemicals or herbicides (natural or synthetic) act on plants though enzymatically controlled reactions. Aqueous leachates of A. paniculata affected the germination percentage and seedling growth of test plants. Higher concentration of the treatment adversely affect the germination percentage of C. murale (Table 1). Root length and shoot length also adversely affected by the treatment as compared to control. The maximum reduction in root length and shoot length (60.51% and 59.75% respectively) was observed at 4%. Thus it may be concluded that the ameliorative effect of applied A. paniculata aqueous leachates on growth parameters leading to the observed decrease in seedling growth with increase the concentration of treatment solution.

Table: 1: Allelopathic effect of aqueous leachates of Andrographis paniculata on germination and seedling growth of C. murale

In case of acid and water soluble carbohydrates, acid soluble and water soluble carbohydrates were increased by the treatment solution compared to the control. The amount of acid soluble and water soluble carbohydrate in control set was 52.85mg/ g dry weight and 56.85 mg/g dry weight respectively. The maximum effect was observed in Aqueous leachates (38.94%) treatment solution (Table 2). Protein content was dramatically reduced by the treatments compared to the control. The minimum protein content (32.14%) was calculated in Aqueous leachate treatment. The allele-chemicals either block protein synthesis by attacking at the molecular level or they hydrolyze the existing one. To solve this mystery, researches needed at the molecular level. The amino acids found in plants as results of extensive investigation by numerous workers are glysine alanine, valine, leucine, phenyl, alanine, tyrosine, tryptophan, cysteiens, methionine, praline, hydroxyl-proline, aspartic acid glutamic acid, hidtidine, arginine and lysine [15,16].

In case of chlorophyll content, almost half, maximum chlorophyll content of C. murale was decreased by the aqueous leachates as compared to the 3.18µg/ mg fresh weight in control setup. Chlorophyll contents were not much affected by Chloroform fraction but the difference was statistically significant (Table 2). One thing is very clear that A. paniculata leaves allelochemicals exert a very negative influence on the acid soluble and water soluble carbohydrate of C. murale. It

Treatment C. murale

Germination Root length Shoot length

Control 100±1.11 7.37±0.08 7.90±0.09

0.5% 95±1.06 6.83±0.08 7.36±0.08

1% 92±1.02 5.38±0.06 5.99±0.07

2% 87±0.97 3.72±0.04 4.13±0.05 4% 76±0.084 2.91±0.03 3.18±0.04 LSD at 5% 2.00 0.125 0.136 LSD at 1% 2.91 0.182 0.198

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is very well depicted by an increased amount of carbohydrate content due to the plant being under stress. Its influence mainly by aqueous leachates that is in its glycosidic form. The results are in conformity with the results of various authors in different plants [17,18,19,20,21,22]. The chlorophyll content decreases dramatically as compared to the control and in this case to the aqueous leachates proved to be much more effective than the other treatments. The leachates from Andrographis paniculata when tested on carbohydrate content of C. murale explicitly showed the inhibitory tendency. Thereby, confirming the phytotoxic property against the C. murale.

Table: 2: Effect of aqueous leachates and organic extract fractions derived from the leaves of A. paniculata on total carbohydrate content, protein content and chlorophyll content of the leaves of C. murale

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Treatment Solutions

C. murale Acid soluble

carbohydrates (mg/gdry wt.)

Water soluble Carbohydrates (mg/g dry wt.)

Total protein content (mg/g dry wt.)

Total chlorophyll content (µg/mg

fresh wt.) Control 52.85 ± 0.65 56.85 ± 0.31 56.18 ± 0.17 3.18± 0.13 Aqueous leachates AL (1%g/ml fresh wt.)

73.43 ± 0.12 72.85 ± 0.37 38.12 ± 0.73 1.79 ± 0.16

Petroleum ether Fraction PF(0.09%w/v)

66.18 ± 0.15 68.27 ± 0.15 40.33 ± 0.76 2.76 ± 0.42

Methanolic fraction MF(0.09%w/v)

55.78± 0.12 61.19± 0.36 43.24 ± 0.23 2.17 ± 0.19

Chloroform fraction CF(0.09%w/v)

67.21 ± 0.92 68.13 ± 0.39 47.11 ± 0.31 2.83 ± 0.73

Water fracrtion WF(0.09%w/v)

63.18 ± 0.54 60.37 ± 0.37 38.16 ± 0.12 2.67 ± 0.49

LSD at 5% 0.617 0.162 0.522 0.427 LSD at 1% 0.878 0.231 0.743 0.608

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