Summer Research Fellowship Programme of India's Science Academies

Analysis of Citrus Maxima (Burm.) Merr. Essential oil, studies on allelopathic effect of its major constituents and morphological observations of its encapsulates

Sucheta Raha

Student, J. K. College, Ketika, Purulia, West Bengal 723101

Prof. Ravinder K. Kohli

Vice Chancellor, Central University of Punjab, Mansa Road, Bathinda, Punjab 151001


Allelopathy is an important branch of applied plant science including biochemistry and ecology. Plants secrete certain allelochemicals, basically secondary metabolites, which affect the growth and physiology of other plants and thus can be harnessed for their potential as bioherbicides. In the present piece of work, an attempt has been made to extract the essential oil (EO) from the leaves of Citrus maxima (Burm.) Merr. through steam distillation. Chemical characterization of the oil was done through GC-MS analysis. The analysis revealed the presence of 21 compounds constituting 91.19% of the oil. The oil chiefly consisted of monoterpenes (oxygenated monoterpenes and hydrocarbon monoterpenes like limonene, beta-myrcene, alpha-pinene, beta-pinene, ortho-cymene, gamma-elemene, geraniol, thymol, citronellol etc). D-limonene content was found to be highest, followed by β- Myrcene and sesquiterpenes contributed least in the chemical composition of EO. Allelopathic effect of its major constituents like Limonene and myrcene was assessed against rice weed i.e., Echinochloa crus-galli L. Changes in certain parameters which serve as reliable indicators of allelopathic response, like seed germination, seedling length were measured and the total, chlorophyll content and cellular respiration were spectrophotometrically evaluated. Further, an attempt was made to provide physical stability to these constituents by encapsulating them inside biopolymers like cyclodextrin, gum Arabic and maltodextrin. Encapsulation of these two major components of EO was carried out by co-precipitation and spray drying methodmetos. The main purpose of encapsulation is to prevent volatilization of EO during field application. Scanning electron microscopic (SEM) studies of the encapsulates were carried out to check the surface morphology of the encapsulates.

Keywords: allelopathy, herbicides, GC-MS analysis, chlorophyll content, cellular respiration, SEM


Abbreviations Full forms
EO Essential Oil
βCD β - Cyclodextrin
L βCDLimonene+ β - Cyclodextrin
M βCDMyrcene+ β - Cyclodextrin
GAMD Gum Arabic + Cyclodextrin
LGAMDLimonene +Gum Arabic+Cyclodextrin
MGAMDMyrcene+ Gum Arabic+Cyclodextrin
SEM Scanning Electron Microscopy
GC-MS Gas Chromatography-Mass spectrometry


In our agro ecosystem continual interaction between the biotic and abiotic components takes place. Allelopathic interactions mainly takes place within the biotic components & may be positive or negative in nature. Positive allelopathic interactions improve crop production, enhance genetic diversity, whereas negative interactions deteriorate crop quality and quantity.

Weeds are unwanted invaders, which are often needed to be eliminated. Allelopathy is actively used in the field of weed science to selectively inhibit the weed plants keeping the desired crops unharmed.

During last three decades, allelopathy has become an important branch of applied sciences including chemical ecology (Singh et al., 2003). Allelopathic interactions include the action of certain target specific allelochemicals. The allelochemicals produced as by-products of primary metabolites and are termed as secondary metabolites, having low molecular weight, are metabolically non-functional, mostly related with defence mechanism of plants. Alkaloids, flavonoids, betalins, glucosionolates, terpenoids which can be again categorised into mono, sesqui, di, tri -terpenes etc are few of them.

Allelochemicals are biochemical that are used as weedicide because of their shorter half-life in environment.

Weed control by allelopathic mechanism can be done by:

Intercropping of Allelopathic Crops with Other Crops to Restrict Weeds

Liebman and Dyck (1993) stated that including allelopathic plants in a crop rotation or as part of an intercropping system may provide a non-herbicide mechanism for weed control. They found few studies that focused on use of allelopathy in rotations, but management of allelopathic cover crops for weed control has been extensively investigated (Bullock, 1992).

Crop Residues Could be Useful in Weed Suppression

Crop residues mulching can be opted for organic and sustainable weed management of different crops. Barker and Bhowmik (2008) studied the effect of residues from corn (Zea mays L.), soybean (Glycine max Merr.), and sunflower (Helianthus annuus L.) on weed control and on crop productivity of tomato (Lycopersicon esculentum Mill.) and summer squash (Cucumis pepo L. var. melopepo Alef.). Study was conducted to evaluate the influence of different crop residues (Chaffed herbage of four crops viz., sorghum, sunflower, rice and maize was mixed in different combinations) on weeds dynamics and productivity of maize under semi-arid conditions (Mahmood et al, 2015). They observed that rice + sunflower + maize mulch produced almost similar results to chemical herbicides metolachlor + atrazine in term of weed suppression and crop yield, and therefore, and can be successfully employed in organic weed management programs in maize.

Allelopathic Interaction of One Weed Species over Other Weeds Could be Utilized

For example, Yeo and Fisher in 1970 observed that Canadian pondweed (Elodea canadensis Michaux) and curly pondweed (Potamogeton crispus L.) can be eliminated from drainage canal with the introduction of needle spikerush (Eleocharis acicularis [L.] Roem. & Schult.) Allelochemicals from some weedy species can be extracted, purified, and used directly like synthetic herbicides. For example, extraction of parthenin from ragweed parthenium (Parthenium hysterophorus L.) (Batish et al., 2002b) and artemisinin from Artemisia sp. (Duke et al., 2000a).

Allelochemicals can be Extracted From Aromatic Plants

Plants like Mentha, Citrus, Neem etc and could be explored for their herbicidal effect against obnoxious weeds.

Like allelopathic effects of orange peel (Citrus sinensis L.) essential oil decreased both qualitative and quantitative growth of seedlings of test plants Euphorbia heterophylla L. and Ipomoea grandifolia (Riberio et al. 2012). Treatment of weed Phalaris minor Retz., or little seed canary grass with Eucalypt oil showed chlorosis, necrosis even death of the weeds in a concentration dependent manner (Batish et al. 2007).

Application of synthetic herbicides, mechanical weeding, and other modern means are used to reduce infestation of weeds and improve crop productivity. However, certain hurdles like increased labour cost, development of herbicide resistant varieties, low durability of synthetic allelochemicals in the field and their adverse effects on health and environment narrowed down their scope and demand. Therefore, researchers are harnessing the potential of natural products like Eos for weed management in agroecosystems because of their non-toxic and biodegradable nature. Scientists today believe that the innovative approach of using allelochemicals should be employed in the field for future sustainable agriculture and horticulture. However, Eos themselves are volatile in nature and therefore they need to physically stabilized. Taking these facts into consideration, the present work was restricted towards the extraction of EO, chemical characterization, phytotoxicity assessment of major compounds of EO and their encapsulation using biopolymers. The main question in this context arises about the need of encapsulation.

The main reason for encapsulation is chemically unstable bioactive agents and susceptibility to oxidative degradation and loss of volatile compounds in exposure to oxygen, metal ions, light, moisture, heat etc (Bakry et al., 2016).

Microencapsulation can be considered good option in order to sustain the biological and functional characteristics of EOs and their constituents. Microencapsulation can be defined as a process where the material or compound to be encapsulated (solid, liquid or gaseous substance) is embedded inside the encapsulating material or wall material like polymers to form small capsules (Gharsallaoui et al., 2007). Controlled release of the core material through microcapsule takes place by diffusion under suitable conditions (Fang and Bhandari, 2010). Encapsulates may be of various shapes and sizes viz. simple matrix, multicore, multiwall etc. and can prepared by various techniques like spray drying and coacervation (Devi and Maji 2009). Microencapsulation of EO mainly focusses on determining functional properties and potential application of encapsulated compounds, its industrial and agricultural approaches (Bakry et al., 2016).


Effect of EO on E. crus-galli L

Kueh et al (2019) analysed the effect of Melaleuca cajuputi extract on barnyard grass, Echinochloa crus-galli where 0.05m concentration of extract exhibited allelopathic effect. The main component of extract was caryophyllene as detected by GC-MS analysis. Laosinwattana et al (2018) investigated the chemical composition and herbicidal effect of EO from Tagetes erecta leaves on pre and post emergent activities of E. crus-galli. GC-MS determ.

Sharma et al (2019) explored phytotoxicity and cytotoxicity of Hyptis suaveolens EO on rice (Oryza sativa) and its weed, E. crus-galli, under laboratory conditions. GC-MS analysis showed monoterpenoid α-phellandrene (22.8%), α-pinene (10.1%) and limonene (8.5%) as the major chemical constituents of EO. They determined various parameters like chlorophyll content, seed germination, seedling growth, cell viability, cytogenetic analysis and results showed that the EO had complete inhibitory effect due to its mitodepressive activity.

Laosinwattana et al (2018) investigated the chemical composition and herbicidal effect of EO from Tagetes erecta leaves on pre and post emergent activities of E. crus-galli. GC-MS determined the major components of EO which were monoterpenes and sesquiterpenes by nature. In pre-emergent stage, strong inhibition took place due to α-amylase activity whereas in post emergent condition, foliar applied EO had immense inhibitory activity which decreased chlorophyll and carotenoid content. Studies on membrane integrity, lipid peroxidase, seedling germination and growth supported the fact that T. erecta EO can be a sustainable source of herbicide.

Fagodia et al (2017) studied phytotoxic and cytotoxic effects of Citrus aurantiifolia EO after determining its major components limonene and citral via GC-MS analysis. Dose response studies were carried out on E. crus-galli and other two weeds proving the allelopathic potential of C. aurantiifolia EO.

Abdelgalei et al (2014) did GC-MS analysis of hydrodistilled EO from twenty plant species, and examined the inhibitory effects of EO on seed germination and seedling growth of E. crus-galli L. EOs of Myrtus communis, Artemisia monosperma, Vitex agnus-castus and Pelargonium graveolens showed the highest inhibition of root and shoot growth where root growth inhibition was greater than that of shoot growth suggesting that EO may serve as a natural herbicide.

Physicochemical Analysis of EO Encapsulates

Co-precipitation method

Several researchers explored the effectiveness of microencapsulation and determined that it can increase aqueous solubility and molecular stability against varying heat, light and oxidation conditions and decrease volatility (Babaoglu et al., 2017; Hill et al., 2013; Li et al., 2011; Ponce Cevallos et al., 2010; Rakmai et al., 2017). Cyclodextrins are cyclic oligosaccharides having an external hydrophilic surface and internal hydrophobic core, central part entraps the guest molecule (oil) and hydrophilic region serve as wall material which binds to water or hydrophilic head of other wall materials (Jadhav et al., 2013, Cheong et al., 2016). Parameters such as size, chemical structure, physical properties method of preparation of the guest molecules, type of cyclodextrin used etc decide the formation of inclusion complex between a guest molecule and CDs (Abarca et al., 2016; Del Valle, 2004). β-CD is selected among various α, β, γ etc kind of cyclodextrins because of its cost effectiveness and EO entrapping capability (Waleczek et al.,2003).​

Yuan et al (2019) characterized lavender essential oil via FT-IR, TG-DTA method and encapsulated it in hydroxypropyl-β-cyclodextrin. Results determined that lavender EO was successfully encapsulated hydroxypropyl-β-cyclodextrin cavity plus it increased thermal stability supporting its processing and storage.

Rakmai et al (2017) characterized the physico-chemical properties and bio-efficacies of hydroxypropyl-β-cyclodextrin (HPβCD) encapsulated black pepper EO via FT-IR, UV-vis spectroscopy analysis, phase solubility study and morphological examination. Result showed that EO was successfully entrapped in HPβCD which protects the active components from the effect of light and increase stability of encapsulated EO by 18 to 24%.

Rakmai et al (2018) studied the antioxidant and antimicrobial properties of hydroxypropyl-β-cyclodextrin after characterizing the encapsulates on the basis of morphological analysis, FT-IR, UV-vis spectroscopy and phase solubility analysis. Morphological examinations showed that active compounds were successfully encapsulated in HPβCD concluding limonene the major component of guava oil, had an encapsulation efficiency of 91.8%. They stated aqueous solubility of EO can be increased by increasing HBβCD concentrations.

Encapsulation with sodium alginate and assessment of antimicrobial and antioxidant activity of clove oil was studied by Radunz et al (2019) where Sodium alginate showed high efficiency of clove essential oil encapsulation. Encapsulated particles displayed lower antioxidant activity and greater inhibitory action in comparison to unencapsulated oil.

Synthesis of β-cyclodextrin modified chitosan nanoparticles via ionic gelation method for controlled release of Cinnamomum zeylanicum essential oil was reported by Matshetshe et al (2018) which concluded by the UV-vis and FTIR spectral studies the presence of EO and β-CD and high encapsulation efficiency of 58.02 %

Barbieri et al (2018) evaluated the effect of the Lippia graveolens EO after encapsulating it with β- and γ-cyclodextrins. Characterization was done through SEM, XRD, TGA, FTIR showing that EO of different Mexican oregano chemotypes formed complexes with both β and γ cyclodextrins. Monoterpene β-caryophyllene showed strongest affinity with β-cyclodextrin whereas carvacrol thymol showed attraction towards γ cyclodextrins.

Lin et al (2018) tried improving stability of thyme EO solid liposome by using βCD as a cryoprotectant Techniques namely FTIR, UV-vis spectroscopy, SEM, DSC provided evidence that EO was successfully encapsulated and liposome can be protected by the addition of β-cyclodextrin as cytoprotectant during freeze-drying process.

Piletti et al (2019) showed microencapsulation of garlic oil with βCD against Escherichia coli and Staphylococcus aureus together with providing a thermal stability to the EO. FTIR analysis showed interaction of βCD molecules with EO. Encapsulation process protected substrates from degradation fixed the molecules of garlic oil at higher temperatures which is above the volatilization temperature.

Shrestha et al (2018) encapsulated oil from Melaleuca alternifolia by mixing solid amorphous βCD with tree tea oil reducing the drying time of the powder and increasing surface oil in product.

Studies on antifungal and physicochemical properties of inclusion complexes based on βCD and essential oil derivatives was done by Herrera et al (2019) showing different levels of affinity between active compound and the cavity of b-CD.

Spray drying method

Spray drying is an inexpensive and widely used technique. Carrier material, Gum Arabic and Maltodextrin plays a vital role in encapsulation process (Fang and Bhandari, 2010; Ozkan and Bilek, 2014). Gum Arabic is a non-toxic odourless, colourless, tasteless substance with low viscosity and good emulsifying ability obtained from the exudates of Acacia sp. tree (Chranioti and Tzia, 2014). It is an edible biopolymer with complex chemical composition consisting of macromolecules rich in carbohydrate and low protein content, having good emulsifying and film forming capacities. (Montenegro et al., 2012, Silva et al., 2013). Maltodextrins with dextrose equivalence of 10-20 are flavourless, easily biodegradable, water soluble, have low viscosity, and lower emulsifying ability. These are basically hydrolysed starches from corn, wheat, potato etc (Sturm et al., 2019)

Sturm et al (2019) aimed to encapsulate of non-dewaxed propolis by freeze-drying and spray-drying using gum Arabic, maltodextrin and inulin as coating materials. In the study gum Arabic was useful for encapsulation efficiency and yields and acted as a stabilizer. HPLC data confirmed that freeze-drying was more efficient for both total phenols and encapsulation efficiency than spray-drying. Water dispersibility of spray dried encapsulates were higher. Propolis were successfully encapsulated by both techniques according to DSC measurements.

Chew et al (2018) microencapsulated refined kenaf seed oil and calculated the potential of β-cyclodextrin combination with gum Arabic and sodium caseinate as carrier material. Wall materials increased the solubility of kenaf seed oil in water, Gum Arabic had greater particulate density in compared to sodium caseinate. Study showed that oil was well encapsulated with encapsulation efficiency above 90%.

Oregano EO was encapsulated in Arabic gum, maltodextrin and modified starch by spray drying technique and studied physiochemically by Partheniadis et al (2019). Spray dried powder showed about 98.3% and 97.9% encapsulation efficiencies.

Phenolic compounds of spent coffee grounds were retained into gum Arabic and maltodextrin encapsulates prepared by spray drying and freeze-drying techniques by Ballesteros et al (2017). He concluded that maltodextrin provides highest retention percentages of phenolic compounds and flavonoids within the matrix and also the best functional properties for the encapsulated sample. Best encapsulation efficiency (62%-73%) was gained by using 100% maltodextrin as wall material and freeze-drying technique.

Henao et al (2018) aimed to access microparticles via spray drying method using gum Arabic, maltodextrin and a modified starch as carrier compounds to increase stability of lutein. Their formulation protected the lutein extract at least 20 days from the storage like environment. Encapsulation efficiency of 91.94% was reached using gum Arabic as it has amazing film forming properties.

Microencapsulation of raspberry anthocyanins using Gelatin (GE) and Gum Arabic (GA) emulsion was performed by Shaddel et al (2018) FTIR results showed that not all GA and GE took part in coacervation process.

Castel et al (2019) evaluate the potential of Brea gum (BG) and inulin in the microencapsulation of corn oil in comparison with gum Arabic and concluded that combination of BG and inulin is an alternative wall material for microencapsulation of hydrophobic compounds in replacement of GA.

Mohammed et al (2017) microencapsulated Nigella sativa oil using spray drying method and examined its moisture content, solubility, efficiency etc. They proposed that the wall material concentration positively affects the encapsulation efficiency in terms of the oil retained, higher the concentration, more efficient is the encapsulation. HPLC analysis showed microencapsulated oil had an effective amount of thymoquinone about 5.45mg/ml trapped in it.

de Souza et al (2017) proposed to encapsulate the phenolic compounds present in the cinnamon extract by complex coacervation using polymeric pairs formed between gelatin and five different polysaccharides with Gum Arabic as one of them and characterize the encapsulated extract particles. They concluded the spray drying process of cinnamon extract caused losses in the phenolic compounds in comparison to the liquid extract. FTIR analysis showed the oil was actively encapsulated in the polymers and was highly solubility in water.

Zhou et al (2019) investigated a way of protecting Ganoderma lucidum spore oil by microencapsulation with spray drying technique, after evaluating different wall materials they selected gum Arabic and maltodextrin, which showed encapsulation efficiency of 83.57%. Results designated that using GA and MD as wall materials could efficiently protect the spore oil and increase its oxidation stability.


Gaining knowledge about the progress in research in the field of allelopathy during rescent times was the main objective of conducting this research. Togetherly, determining the chemical composition of the volatile oil, to assess the phytotoxic effects of 2 main components of Citrus oil against Echinochloa crus-galli through a series of laboratory experiments estimating different biochemical parameter, stabilizing its effect by encapsulation with the aid of modern instruments.

The main objectives are:

  • To learn extraction of allelochemicals, specifically essential oil from plant tissue through steam distillation technique.
  • Characterization of EO through GC MS.
  • To determine the effect of allelochemicals through the estimation of different parameters like chlorophyll extraction and estimation following Hiscox and Israelstam (1979) modified by Rani and Kohli (1991), determination of cellular respiration (Steponkus and Lanphear, 1967).
  • Learning various techniques like co-precipitation and spray drying for encapsulation of essential oil so that they can be used as herbicides.
  •   Surface morphology study of encapsulated material via SEM.



1. Plant material Mature, fresh leaves of Citrus maxima were collected from 4-5 year old plants from the campus of Central University of Punjab (30 ̊ 10 ' 17 " N 74 ̊ 57 ' 58 " E), Bathinda, India for the study purpose. Seeds of test weeds were procured from Punjab Agriculture University, Ludhiana, India.

2. Chemicals

a. For chlorophyll estimation

Dimethyl Sulfoxide (DMSO)

b. For estimation of cellular respiration

Preparation of phosphate buffer solution

  • 0.87g of K2HPO4 (1M) is dissolved in 5ml of distilled water.
  • 0.68g of KH2PO4 (1M) is dissolved in 5ml of distilled water.
  • 4.01ml of K2HPO4 solution is mixed with 0.99ml of KH2PO4 to form a 5ml solution which is again mixed with 45ml water to make a 0.1M solution.

Preparation of TTC solution:

  • 300g of Triphenyl Tetrazolium Chloride is dissolved in the Phosphate buffer solution and stored in dark place.

c. Encapsulation via co-precipitation method

β-Cyclo Dextrin(βCD) solution (2%): prepared by dissolving 20 g of βCD in 1:2 mixture of ethanol and distilled water.

d. Encapsulation via spray drying method

Maltodextrin, Gum Arabic.

(Loba chemicals Pvt. Ltd.)

3. Glassware: Petridishes, centrimeter scale, test tubes, beakers micropipetts, conical flasks (100ml), measuring cylinder (10 ml, 50ml), bakelite screw capped glass vials of Borosil, glass rod, etc.

4. Instruments: steam distillation apparatus, Digital balance, Shimadzu QP 2010 mass spectrophotometer, growth chamber, UV Spectrophotometer, hot air oven, homogenizer, hot plate magnetic stirrer, Scanning Electron Microscope, Spray dryer

5. Miscellaneous: Filter papers, tissue paper, aluminium foil, cotton, cellotapes, glass marker, plastic trays, test tube rack, Morter pestle, spatula, forceps, distilled water etc.

Essential Oil Extraction

Citrus leaves (Fig. 1a) were collected and one Kilogram (Fig. 1b) of it mixed with small quantity of water inside the distillation apparatus (Fig. 1c). The mixture inside the apparatus was heated at 300℃ for 3 hours. The oil (Fig. 1d) thus obtained was collected from the nozzle of the condenser. The oil was stored in an airtight screw capped glass vial at 4℃ for determining the composition and further characterization.

Twig with fruits ofCitrus maxima
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Weighing leaves of C. maxima
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Steam distillation apparatus for EO extraction
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citrus oil.png
Extracted Oil
    Stepwise EO extraction procedure 

    GC-MS analysis of EO

    Chemical composition of citrus EO was determined by Shimadzu QP 2010 mass spectrophotometer (Fig. 2) following the parameters given in table 1 & 2.

    Parameters programmed for GC- MS analysis
    GC-2010 programme GCMS-QP2010 Ultra programme
    *Column Oven Temp. 40.0 °C Ion Source Temp. 200.00 °C
    Injection Temp. 250.00 °C Interface Temp. 260.00 °C
    Injection Mode Split Solvent Cut Time 4.50 min
    Flow Control Mode Pressure Detector Gain Mode Relative
    Pressure 66.7 kPa Detector Gain 1.12 kV +0.00 kV
    Total Flow 10.6 mL/min Threshold 0
    Column Flow 1.24 mL/min    
    Linear Velocity 40.2 cm/sec    
    Purge Flow 3.0 mL/min    
    Split Ratio 5.1    
    *Oven temperature programme
    Rate Temperature Hold time
    - 40.0 3.00
    4:00 220.0 5.00
    15:00 250.0 5.00
      GC -MS instrument used to analyse the chemical composition of Essential Oil

      Seed Germination Studies

      Seeds of Echinochloa crus-galli were used as a test weed to determine the herbicidal activity of 2 main components D-limonene and β-Myrcene of Citrus maxima EO. Each petri dish was lined up with 2 layers of Whatman filter paper and moistened with 5 ml of distilled water. Following International Seed Testing Association Rules, 25 seeds were placed in each dish and three replicates were maintained (all total 27 petri dishes). Four different concentrations of D-Limonene and β-Myrcene were applied on the inner lid of petri dish (0.5, 1.0, 2.5, 5 µl/petri dish). A similar set without EO served as a control. The Petri dishes were sealed with adhesive tapes so that EO doesn’t volatilize. The experimental set was kept in growth chamber under standardised conditions of temperature (25℃), photoperiod (12/12 light/dark) and 40% humidity. After seven days of trial set up, the following parameters were consideration to examine the effect of D- Limonene and β-Myrcene.

      • Seed germination percentage
      • Root and shoot length
      • Total Chlorophyll content
      • Respiratory percentage

      Seed Germination Percentage

      After seven days, germinated seeds on each petri dish was counted. Centimetric scale was used for measurement of root and shoot length of germinated seedlings. Following formula was used for the calculation of germination percentage:

      Germination percentage = C-T/C *100

      where, C represents control and T represents the treated seeds.

      Estimation of Chlorophyll Content (Fig. 6)

      Procedure: Hiscox and Israelstam (1979) modified by Rani and Kohli (1991)

      •  25mg of tissue of the test plant materials (separately both dry and fresh weight basis) were taken in test tubes.
      • 2 ml of DMSO were added to each test tube having fresh shoots.
      • The mouth of the test tubes was covered and kept in hot air oven for 1 hour at 60℃.
      • After cooling at room temperature absorbance measured through UV Spectrophotometer at 645 nm and 663 nm and the optical density (OD) values were noted using a DMSO as blank.
      • Weight of 25 mg of overnight dried shoots were noted.


      The calculations of dry weight content were done on dry weight basis.

      Total chlorophyll content(µg/mL) = 6.45 × A663+ 17.72 × A645 = X

      Total Chlorophyll content (µg/mg) = (X × volume of DMSO)/Dry weight of tissue

      Estimation of Cellular Respiratory Percentage (Fig. 7)

      Procedure: (Steponkus and Lanphear, 1967)

      • 1.5 ml of phosphate buffer is poured into each test tube containing root tissue of test samples.
      • Samples incubated at room temperature, for 18 hours in dark.
      • After incubation, TTC solution was drained off from test tubes and rinsed twice in distilled water (Fig. 8).
      • 1.5 ml of ethanol was added to each sample, test tubes covered and kept in hot air oven at 80℃ for 20 minutes.
      • After cooling, absorbance was recorded at 530 nm.


      The cellular respiratory ability was expressed as a percent with respect to control.

      Encapsulation Process

      Encapsulation of Citrus EO via co-precipitation method (Fig. 9)


      • The βCD mixture was heated at 60℃ for 1 hour.
      • For preparing the 2% encapsulates, 45 ml of polymer + 1 ml of EO + 4 ml of ethanol was taken.
      • The mixture of EO and ethanol was added drop by drop into the polymer solution.
      • Thereafter, the solution was homogenised for 1 hour.
      • In a similar manner control was prepared (didn’t involve EO and ethanol).
      • The homogenised solution was further kept at 4℃.
      • After 24 hours the precipitates were recovered by filtration.
      • Final weight of the precipitates was noted down.

      Encapsulation of Citrus EO by Spray drying method (Fig. 10)


      • Spray drying was carried out by encapsulating Citrus EO inside Gum Arabic and Maltodextrin.
      • 2% solution of polymer was prepared by adding 10 g of Gum Arabic and 10 g of Maltodextrin (1:1) in 200ml of distilled water.
      • The solution was heated at 40℃ till the solution became clear.
      • For preparing 2% encapsulates 45ml of biopolymer + 1 ml of EO + 4 ml ethanol was taken.
      • In a similar manner control was prepared (didn’t involve EO and ethanol).
      • The mixture of EO and ethanol was added dropwise into the polymer solution.
      • Thereafter, the solution was homogenised for 1 hour.
      • The homogenised solution was subjected to spray dryer.
      • The dried powder was collected and weight was calculated.

      Scanning Electron Microscopy (SEM)

      Surface morphology of prepared encapsulates was analysed by SEM. The model was Zeiss Merlin Compact. The samples were coated with a thin film of gold and analysed at different magnifications (Fig. 11 & 12 )


      Chemical Composition of EO extracted from Citrus maxima

      Oil extracted from Citrus maxima was pale yellow with lemon-like odour. Average oil yield was recorded to be 0.00178% (v/w). Extracted oil possessed solubility in organic solvents like ethanol, acetone, dimethyl sulfoxide (DMSO) and n-hexane.

      GC–MS analyses revealed the presence of 21 components constituting 91.19% of the oil (Table 3 ). The oil chiefly consisted of monoterpenes (oxygenated monoterpenes and hydrocarbon monoterpenes) like alpha-pinene, beta-pinene, citronellol, limonene, beta-myrcene, o-cymene, geraniol, 3-carene, alpha terpineol, alpha-thujene). Sesquiterpenes contributed least in the chemical composition of EO (Fig. 3 & 4 ). Two most abundant constituents of the test sample were D-Limonene and β-Myrcene.

      Chemical composition of EO Extracted from C. maxima
      Sl. No. Compounds Area% Method of identification
      01 Alpha – Pinene 0.80 MS
      02 Beta – Pinene 1.08 MS
      03 3 – Carene 0.30 MS
      04 Beta – Myrcene 19.36 MS
      05 Gamma – Terpinene 8.24 MS
      06 Beta -Ocimene 5.17 MS
      07 O – Cymene 0.49 MS
      08 Caryophyllene 0.41 MS
      09 Terpinen - 4 – ol 1.18 MS
      10 Gamma – Elemene 0.33 MS
      11 L- alpha – Terpineol 0.88 MS
      12 Geranyl acetate 0.13 MS
      13 Citronellol 0.50 MS
      14 Geraniol 0.71 MS
      15 Thymol 0.37 MS
      16 Alpha – Thujene 9.48 MS
      17 Alpha – Terpinene 0.41 MS
      18 D – Limonene 34.87 MS
      19 E – Citral (Neral) 5.54 MS
      20 Z -Citral (Geranial) 0.82 MS
      21 Beta – Sinesal 0.12 MS
      Annotation 2019-05-17 205700.png
        Quantitative analysis of EO
        peak report tic_1.png
          Peak Report

          Total identified (%) = 91.19%


          Homologous findings with respect to the chemical composition of Citrus EO were also reported by Fagodia et al (2017), Martins et al (2017) and El Sahi et al (2019).

          Effect of D-Limonene and β-myrcene on Seed Germination and Seedling Growth of E. crus-galli L.

          Effect of EO constituents on seed germination

          The performed experiment showed that the germination of E. crus-galli L. was not affected significantly on exposure to D-Limonene and β-Myrcene. α-Amylase activity helps in breaking down complex starch into consumable glucose chains thus helping in seed germination and root and shoot growth. Restriction to this activity probably causes inhibition of the other factors studied (Kato-Noguchi et al., 2010).

          Effect of EO constituents on root growth of E. crus-galli L. (Fig. 5)

          Root growth inhibition was prominent in the performed experiment. Limonene concentration at 5µL showed greatest inhibition on root development. With an increase in concentration of EO constituents, the root length gradually decreased from 3.56% - 95.17% in limonene treated seeds and 38.57% -53.88% in Myrcene treated seeds. The reduction in root growth was proportional to the increase in concentration of Limonene and Myrcene. Thus, these terpenoids can act as weed supressing agents. Inhibition on root growth was more than the effect noticed on shoot growths after treatment with EO (Table 4).

          Effect of EO constituents on root growth of E. crus-galli L.
          Control 4.77±0.14a
          Limonene 0.5 4.6±0.61a
          Limonene 1 3.46±0.43b
          Limonene 2.5 3.13±0.27b
          Limonene 5 0.23±0.13c
          Myrcene 0.5 2.93±0.24b
          Myrcene 1 2.66±0.13b
          Myrcene 2.5 2.36±0.18b
          Myrcene 5 2.2±0.11b

          Data have been analysed using SPSS software version 18.0. Different letters for each EO constituent indicate significant difference between control and treatments whereas same letters indicate non-significant effect.


          Mode of action of the essential oils has not been investigated in the current study but, the probable cause of decrease in root length is due to:

          • Hindered DNA synthesis on the growing root tips which inhibited the meristematic cell division (Romagni et al. 2000; Nishida et al. 2005)
          • Disruption of membrane integrity (Tworkoski 2002; Singh et al. 2009)
          • Induction of oxidative stress by essential oil derivatives, resulting electrolyte leakage from root tissue (Scrivanti et al., 2003; Singh et al., 2006, Saad et al., 2014)


          C. aurantiifolia EO caused ∼88% inhibition in root, Limonene caused 30% and complete inhibition of P. minor seeds was noticed in treatment with citral as examined by Fagodia et al (2017). Allelochemical compounds from the environment are likely absorbed by roots first thus greater inhibition in roots are observed (Abdelgaleil et al. 2014).

          Effect on EO constituents on Shoot Growth

          A significant reduction in shoots at all concentrations of D-Limonene and β-Myrcene was observed in comparison to the control as shown in table 5. The shoot lengths reduced gradually with highest shoot inhibition in Myrcene 5. Root inhibitory percentage was reduced gradually from 11% to 54% when exposed to myrcene treatment, though limonene treated seeds showed limited shoot inhibition from 32.87% to 48.73%. It is noteworthy to mention that the leaf colour of treated seeds by Limonene 1, 2.5 were changed to pale yellow and Myrcene 2.5, 5 to dark yellow colour.

          Effect of EO constituents on shoot growth of E. crus-galli L.
          SAMPLE SHOOT LENGTH (Mean ± SE)
          Control 6.3±0.85a
          Limonene 0.5 4.23±0.17b
          Limonene 1 3.90±0.09c
          Limonene 2.5 3.86±0.14c
          Limonene 5 3.23±0.10c
          Myrcene 0.5 5.56±0.13a
          Myrcene 1 4.76±0.14b
          Myrcene 2.5 3.73±0.23c
          Myrcene 5 3.00±0.28c

          Data have been analysed using SPSS software version 18.0. Different letters for each EO constituents indicate significant difference between control and treatments whereas same letters indicate non-significant effect.


          The degree of inhibition depends on type of essential oil, its concentration, species response etc increasing with increase in EO concentration. (EL Sawi et al., 2019)

          Monoterpenes having phytotoxic properties may cause anatomical and physiological changes in plant seedlings leading to accumulation of lipid globules in cytoplasm, reduction in number of cytoplasmic organelles like mitochondria, disruption of mitochondrial membrane or inhibition of DNA synthesis. (Tabana et al., 2013; Bouajaj et al., 2014).


          In studies conducted by Fagodia et al (2017) Citrus aurantiifolia, citral oil caused significant reduction in the coleoptile growth of E. crus-galli at and above the concentration of 0.25mg/ml. ≥0.50 mg/ml of Limonene significantly inhibited coleoptile growth. Reduction in shoot length with respect to control in E. crus-galli was approximately 40%, 59% and 13%. Moreover, reduction in shoot length was observed by Riberio et al. (2012) in cases of Euphorbia heterophylla and Ipomoea grandifolia seedlings in presence of Citrus sinensis EO.

          limo treatment crop.png
          Effect of different concentrations of  Limonene on seedling growth
            myrc trtmnt crop.png
            Effect of different concentrations of Myrcene on seedling growth
              Pictures of germinated seeds of E. crus-galli L. on exposure to Limonene and Myrcene.

              Estimation of chlorophyll

              Chlorophyll content of the shoots was estimated using UV spectrophotometer. A significant reduction in chlorophyll content was observed when treated with Limonene, chlorophyll of the leaves decreased from 31.70% to 66.89% at 0.05-5µl concentration of EO with respect to control. Myrcene treatment at various concentrations from 0.05-5 µl reduced chlorophyll content by 40.77% to 82.92% (Table 6).

              Effect of EO constituents chlorophyll content of test weed
              S. No. Sample Name Chlorophyll Content (µg/mg)
              1 Control 2.87a
              2 Limonene 0.5 1.96a
              3 Limonene 1 1.65ab
              4 Limonene 2.5 1.34bc
              5 Limonene 5 0.95c
              6 Myrcene 0.5 1.70b
              7 Myrcene 1 1.12b
              8 Myrcene 2.5 0.75c
              9 Myrcene 5 0.49d
              chloro bt crop.jpg
              Shoots dipped in DMSO
                chloro crop.jpg
                Chlorophyll extracted after heating
                  Experimental results for chlorophyll extraction


                  Inhibition of photosynthetic machinery of plant results in deterioration of plant growth. The probable reason for decreased chlorophyll content could be attributed to

                  • Chlorophyll synthesis inhibition.
                  • Enhanced chlorophyll degradation.
                  • Interference with leaf air diffusibility and other related parameters (Polova and Vicherkova, 1986).


                  Batish et al. (2006) estimated amounts of chlorophyll a and b and total chlorophyll content of 2 weeds and 2 crop plants after treating them with eucalyptol EO using this method.

                  Estimation of cellular respiration

                  A sharp decrease in cellular respiration was observed 66.49%-11% from concentration 0.5 to 2.5µL of Limonene assuming the control as 100%. At higher concentration of limonene(5µL) increase in respiration over control by 50 % is observed. In myrcene treatment decease in respiratory percentage occurs at 0.5 µL, and then increase from 2.61% to 53.40% increase in cellular respiration takes place when treated with 1 to 5 µL concentrations of Myrcene (Table 7).

                  Effect of EO constituents on respiration of test seedlings
                  1 Control 100
                  2 Limonene 0.5 33.508
                  3 Limonene 1 51.309
                  4 Limonene 2.5 89.005
                  5 Limonene 5 150.26
                  6 Myrcene 0.5 71.73
                  7 Myrcene 1 102.62
                  8 Myrcene 2.5 106.28
                  9 Myrcene 5 153.40
                  resp bt0.jpg
                  Roots dipped in TTC solution
                    Resp crop.jpg
                    Red formazon in final step of experiment
                      Expeimental results of Respiration Study
                      resp bt0.jpg
                      Roots dipped in TTC solution
                        Resp crop.jpg
                        Red formazon in final step of experiment
                          Expeimental results of Respiration Study


                          TTC captures electrons from mitochondrial electron transport chain and forms a red colour formazon, providing an indirect measurement of cellular respiration. It provides a visible idea of viability (Maness et al., 1999, Batish et al., 2007).

                          It may be interpreted that the greater respiratory activity could be shown by the plants by increasing the metabolic rate under low stress condition.


                          However, the decreased respiration rate leads to severe metabolic dysfunction. Respiratory loss affects the plant metabolism, alters the synthesis of macromolecules and subsequently results in plant growth retardation (Penuelas et al., 1996). Moreover, Abrahim et al (2000) reported monoterpenes as uncoupling agents of oxidative phosphorylation.

                          Encapsulation through Co-Precipitation and Spray Drying Method

                          Encapsulates of β-CD, GAMD controls along with the EO treated powders were weighed after preparation, which showed the following result:

                          Weight of prepared microparticles
                          Sl. NO. SAMPLE NAME WEIGHT (gm)
                          1 GAMD 2.47
                          2 LGAMD 2.81
                          3 MGAMD 2.37
                          4 β-CD 3.37
                          5 Lβ-CD 4.11
                          6 Mβ-CD 4.14
                          Heating the solution
                            Homogenizing the solution
                              Encapsualates left for drying
                                Encapsulates collected
                                  Preparation of βCD encapsulates via co-precipitation method
                                  spray dryer crop1.jpg
                                  Spray dryer
                                    Deposition of encapsulates
                                      temp. prograames.jpg
                                      Spray dryer programme settings
                                          Preparation of GAMD encapsulates via spray drying method

                                          Scanning Electron Microscopy

                                          Scanning Electron Microgram images of β-CD, GAMD controls, LimβCD, MyrCD, LimGAMD, MyrGAMD showed the morphological and physical appearance of the encapsulates.

                                          The encapsulates of β-CD, LimβCD and MyrCD, had rough surfaces with irregular shapes and size. The crystalline β-CD particles aggregated into clusters in presence of D-limonene and β-myrcene. Lin et al (2018) coated solid liposomes with cyclodextrins and lipids (2:1) which showed tendency of agglomeration and fusion, the agglomerating property decreased with increasing concentration of β-CD. β-CD encapsulated terpenoids showed slight difference in structures in comparison to pure β-CD capsules. Piletti et al (2019) obtained similar results encapsulating garlic oil in β-CD, treated capsules had smaller diameter (5-10µm) than the pure ones. With increase in temperature, the EO may increase porosity of the capsules and volatilize. Spray drying process yields a very few wrinkled capsules (Mohammed et al., 2017). Particle size is dependent upon the spray drying conditions amongst which inlet temperature is the most important one, larger encapsulates are produced by increased inlet temperature and low difference between inlet and outlet air temperatures (Ng et al. 2013). Morphology of GAMD, LimGAMD, and MyrGAMD capsules were more or less rounded along with pitted walls. Spherical shape is the characteristic property of spray dried particles as previously shown by Sturm et al (2019), encapsulates of Gum Arabic and maltodextrin with propolis as core material were prepared by them and the micrograph images (2500x) showed mean particle diameter in the dry powdered form were twice in size of the water dispersed particles. They also reported spherical shape of the spray dried particles, slender in size (majority at 1µm) which was also observed in the present work. Diameter of the encapsulates studied are <10µm.

                                          Comparatively larger sized particles with a diameter less than 30µm were reported by Ballesteros et al (2017), encapsulated phenolic extracts of spent coffee grounds via spray dryer at 100℃ showed some dehydrated aspects with controls with spherical or irregular shape.Observed under a SEM to characterize the evidence of inclusion formation compared with the free HPβCD

                                            Magnified β-CD
                                                Magnified Lβ-CD 
                                                    Magnified Mβ-CD
                                                      β-CD encapsulates under SEM characterization
                                                            Magnified LGAMD
                                                              GAMD encapsulates under SEM observation


                                                              Chemical composition of Citrus maxima via GC-MS analysis showed the presence of monoterpenes like limonene and myrcene as major constituents. Application of monoterpenes on E. crus-galli L. didn’t produce any effect on seed germination. However, higher concentrations of limonene and myrcene caused a significant reduction in root and shoot length of test weed. Chlorophyll content was also affected to a slighter extent whereas lower concentrations of monoterpenes showed decrease in cellular respiration and higher concentrations displayed an increase in cellular respiration to combat stress. It may be concluded that application of Eos/constituents can be a promising source for bioherbicide development and for weed eradication in agroecosystems. Encapsulation is an emerging technique to provide physical stability to EO/constituents since they are volatile and are sensitive to air and light exposure. In future, encapsulated EO/constituents may be applied in agricultural fields for weed killing purpose instead of synthetic herbicides.


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                                                              I wish to record my deep sense of gratitude to my supervisor Prof.  Ravinder K. Kohli for his guidance, generous help, encouragement and for providing all the facilities available at CUP, Bathinda.

                                                              I am extremely thankful to IAS, INSA, NASI for allowing me to participate in the summer research fellowship programme.

                                                              I remain ever grateful to all the Faculties, Research scholars and Staff of the Department of Environmental Science and Technology, especially to Mrs. Shilpa Sharma for her sincere guidance and help during handling of the instruments and preparation of the report. 

                                                              I am also grateful to Dr. Sanjeev Thakur, HoD of Plant Sciences and Dr. Sunil Mittal HoD of Environmental Science and Technology for their constant support throughout the working period.

                                                              I would also like to express my deep sence of regards and gratitude to Dr. Shantanu Chattopadhyay , Principal, J.K. college and Dr. Sujit Ghosh HoD in Botany for their encouragement and kind permission to join this summer research fellowship programme.

                                                              I am thankful to the Librarian for his active cooperation during library works. Last but not least, I acknowledge the care and cooperation of Hostel warden and friends during my stay at Bathinda.

                                                              Written, reviewed, revised, proofed and published with