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UDC 633.85:582.929.3:631.51
EFFECT OF PLANTING DENSITY AND SOWING DATE ON THE ESSENTIAL OIL CONTENT AND COMPOSITION OF LEMON VERBENA (LIPPIA CITRIODORA)
Amin Farnia, Ali Nematian, Researchers Department of Agronomy, Islamic Azad University, Borujerd, Iran E-mail: alinematian1982@gmail.com
Mohammad Ali Shariati, Researcher Islamic Azad University, Tehran, Iran E-mail: shariatymohammadali@gmail.com
ABSTRACT
Lemon verbena leaves are used as the plant for injection, aromatic, digestive and antispasmodic properties. Lemon verbena leaves has a mild sedative and reputation for relieving abdominal discomfort. In order to examine the influence of planting density and sowing date on the essential oil Neral, Geranial and Limonene in Lippia citriodora, an experiment was performed in Rasht experimental farm, Gilan province, 2011. The experiment was done as a factorial on the basis of randomized complete blocks design in three replications. Density treatments at three levels: 2, 4, 6 plants /m2 and sowing treatment of March 24th, April 8th and April 24th. The chemical composition was analyzed by Gas chromatography (GC) and Gas chromatography/mass spectrometry (GC/MS). 37 compounds were identified in the Essential oil of Lippia citriodora. The most important compounds in plant lemon verbena were Neral, Geranial and Limonene. Regarding the obtained results, planting density and sowing date treatments had a significant effect on the rate of essential oil and the highest amounts of Neral, Geranial and Limonene were measured as 23.25, 33.68 and 5.84 in the 4 plant/m2 and planting on March 24th.
KEY WORDS
Lippia citriodora; Sowing date; Planting density; Essential oil.
The genus Lippia (Verbenaceae) brings together about 200 species which are native to South and Central America and Africa. These compounds have widespread uses in foods, cosmetics and household products (Catalan and Lampasona, 2002; Santos-Gomes et al., 2005). The essential oils and leaves are used in perfume industry and producing drinks with different flavors, respectively and for food preparations(Pascual et al., 2001).In the United States, Lippia citriodora is listed in Generally Regarded As Safe (GRAS) for human consumption in alcoholic beverages(Gomes et al., 2006). Lippia citriodora synonyms are Aloysia citriodora Palau (Aloysia triphylla) and Verbenaceae which is a native species in Argentina, Chile and Peru and grows throughout Latin America as well as North Africa (Morocco), southern Europe and parts of Asia (Carnat et al., 1999; Botta, 1979; Rotman and Mulgura Romero, 1999). Lippia citriodora is an aromatic perennial shrub that is valued for its medicinal use (as a tea or tincture) as a digestive aid, antimicrobial, antispasmodic, analgesic, diuretic plant; besides, it is used as a treatment for cold, insomnia and anxiety (Botta, 1979; Carnat et al., 1999; Rotman and Mulgura Romero, 1999). Antibacterial and antioxidant activities of Lippia citriodora have been found in essential oils, tea and tinctures (Bilia et al., 2008; Cowan, 1999; Pereira and Meireles, 2007; Bully et al., 2002; Ohno et al., 2003; Sartoratto et al., 2004). Extracts of leaf essential oils are also used extensively in cosmetics and flavoring industries.
Today, due to revealing the lateral complication of chemical drugs, the use of herbal drugs is increasing. Whereas very low researches have been applied on enhancement of medicinal plants production, presenting the appropriate planting methods is very important for increasing the quantity and quality of medicinal plants(Farooqi et al., 2001). The planting
product of a medicinal plant is cost-effective when its secondary metabolites measure would be reached to an appropriate amount. The purpose of trading production of medicinal plants is obtaining further amount of biomass in area containing high rate of active substances (Default et al., 2003). One of the most important peripheral factors for growth of medicinal plants that have a major effect on the quantity and quality of active substances may refer to planting density and sowing date.
In the study applied on the effect of sowing date on the chicory root yield, the researcher concluded that the earlier sowing date will increase the desiccated root and fructose yield (Schittenhem, 2001). Circella et al(1993) performed a study on Ajowan, observed that the planting rows up to 50 cm are suitable for branching and growing growth. While studying the influence of different densities on the yield and quality of Artemisia essential oil, it was observed that the density had significant effect on the yield and essential oil of this plant and within the density of 15 plant/m2, the highest amount of Artemisia essential oil and compound (Ayanoglu & Mert, 2002). Experiments with spacing Thymus vulgaris have shown that, while wider spacing led to the highest level of biomass and yield of essential oil plant cultivation dense significantly increased the yield of essential oil per unit area(Clark and Menary,1979;Shalaby Razin,1992).
The density was shown to have a global effect on Mentha piperita essential oil yield per hectare, giving higher yields in the mid-range 10 to 30 cm densities, deserved simply to the increase in biomass, as the concentration of essential oil did not differ significantly between spacings(Rissanen et al., 2002). The quality of essential oil of lemon verbena is largely determined by the merger of the two isomers of citral (Neral and Geranial) and Limonene. Studies have shown that these compounds are present in all chemotypes of Lippa citriodora, but their concentrations vary considerably according to source of origin, parts of the plant sampled, and stage of plant development (Argyropoulou et al.,2007; Gil et al., 2007). Production of high quality aromatic herbs requires an excellent raw material, which implies good agricultural procedures, collection date correct, appropriate industrial processing and storage conditions, all measures that could affect the sensory quality of the final product (Rodrigo et al., 2009). Perusing the references implies that so far no study has been applied on the effect of different climatic factors such as planting density and sowing date on lemon verbena in Iran. Considering the importance of this plant in the society health and pharmaceutical, food, cosmetic and hygienic industries, as well as studying the factors affecting essential oil upraising, this research has been implemented in Gilan Province, Rasht County.
MATERIALS AND METHODS
Planting. This study has been applied with the objective of measuring the lemon verbena essential oil rate in different sowing dates and planting densities as the factorial on the basis of randomized complete blocks design in 3 replications, in Gilan Province, Rasht County on a experimental farm 450 square meters in area, each patches including 5 planting lines 4m in length, the interval between two planting lines 50cm (the area of each patch is 10 square meters and patch length is 4m and patch width is 2.5m). The climatic specifications and testing area soil have been provided in tables 1 and 2. The test treatments included sowing date for three dates of March 24th, April 8th and April 24th, and planting density for 2, 4 and 6 plant/m2; in density 2, the interval between 2 cuttings is 100cm, in density 4, the interval between 2 cuttings is 50cm, and in density 6, the interval between 2 cuttings is 33.3cm. At first, the field is plowed then was flattened and patched. Firstly, some health, juicy and same-sized cuttings 30cm in length and 5mm in diameter were provided fro the lower parts of the plant. The cuttings have been placed in greenhouse mode after treating by Indole Butyric Acid hormone (concentration of 100 ppm) in sand bed. They were rooted after about one month. The rooted cuttings were cultivated with relative densities on the related dates. The weeds were controlled manually in several times. During the test, no pests and diseases were observed.
Table 1 - Climatic characteristics of experimental farm
Longitude Latitude Mean precipitation Mean humidity Maximum temperature Minimum temperature
Eastern ,36'°49 Northern ,18'°37 1357 mm 81.9% 20.5 11.3
Table 2 - Chemical and physical characteristics for soil of experimental farm (at the soil depth of 0 - 30 cm)
Soil texture pH EC (ds/m) Water absorption % Organic Carbon % Total Nitrogen % Absorptive Phosphorus % (p.p.m) Absorptive Potassium % (p.p.m) Clay % Sand % Silt %
Loamy- clay 7.2 1.05 59 4.26 0.13 19.73 289 33 37 25
Collecting. Collections had been made early in the morning of days of sunshine 120 days after cultivation. Two leaves were collected from the middle node through five stems of two plants in each replicate(20 sheets in total). The leaves were placed into packages with aluminum foil and stored in a refrigerator for transport to the laboratory for analysis. The sheets were utilized to measure surface area and were dried for two days at 60°C.
Extracting. Essential oil extraction from growing body is performed via distillation by water and Clevenger. For this purpose, 40 gr of desiccated root of each treatment (3 replicates) is weighted and grinded thoroughly and put in Clevenger for 4 hours in 500 ml of water in order to extract its essential oil (European Pharmacopoeia, 2005).
GC/GC MAS Chromatography. For identification and measurement of lemon verbena essential oil constituents, gas chromatograph and gas chromatograph connected to the mass spectroscope with the following specifications were used:
GC: Gas chromatograph Shimadzu model A column DB-5 30m in length and 0.25mm in diameter, stationary phase layer thickness 0.25^m, column thermal planning from 50 to 280°c with the temperature upraise of 3°c per minute, detector FID with 270°c temperature, helium carrying gas with the pressure of 3kg/cm2.
GC/MS: Gas chromatograph connected to the mass spectroscope Saturan model 3400 column DB-5 30m in length and 0.25mm, stationary phase layer thickness 0.25^m, column thermal planning from 50 to 250°c with the temperature upraise of 4°c per minute, injection chamber temperature of 360°c, ionization energy of 170 electron volt , helium carrying gas. Identification of spectrums aiding their inhibition index and upon injection of normal hydrocarbons (C9-C27) under equal mode was calculated by injecting the essential oils and aiding a program in Basic language(Bos et al., 2000). The quantitative calculation (determination of composition rate) was performed via Area normalization method and response factor related to the spectrums.
The data of this study was analyzed by statistical software MSTAT-C software and the mean values were compared by Duncan method.
RESULTS OF RESEARCH
Planting density. The summary of analysis of variance indicated that the effect of different ranges of density on the essential oil rate is significant within 0.01(table 3). As well as, the result of comparing the mean values by Duncan test showed that the highest rate of Neral with the mean value of 21.58 and highest rate of Geranial with the mean value of 30.6, and highest rate of Limonene with the mean value of 4.9 have been obtained for 4 plant/m2 (table 4). No significant difference observed between the density D1 and D2 in both tested traits, and have been placed in a statistical group.
Sowing date. The summary of analysis of variance indicated that the effect of sowing date ranges on the essential oil rate is significant within 0.01(table 3). Upon comparing the mean values by Duncan test, it was concluded that the highest rate of Neral, Geranial,
Limonene with the mean value respectively equal to 22.35, 32.7 and 5.53 was observed in sowing date treatment Si(April 24th) (table 4).
Table 3 - Variance analysis results of plant density and sowing date on the characteristics of lemon
verbena
S.O.V df Square Means
Essensiol oil Neral Geranial Limonene
Replication 2 0.008ns 78.5ns 84.6ns 312.8ns
Density 2 1.512** 165413.2** 62573.6** 40368.2**
Sowing date 2 1.573** 179353** 52848.9** 411154.3**
Density*Sawing date 4 0.027** 3571.6** 490304.8** 1839.5**
Error 16 115.5 102.6 117.5 167.4
C.V% 4.2 5.2 4.8 5.7
ns,* and **: Non significant ,and significant at the 5% and 1% levels of probability, respectively
Table 4 - Mean comparisons of various density and sowing date levels on Essensioal oil, Neral,
Geranial, Limonene of lemon verbena
2 Density (plant/m ) Measured Features
Essensiol oil Neral Geranial Limonene
D1 1.85a 21.58a 30.62a 4.9a
D2 1.31a 20.72a 29.8a 4.1a
D3 1.08b 18.2b 22.85b 3.21b
Sowing date
S1 1.73a 22/35a 32.7a 5.53a
S2 1.42b 18.93b 29.94b 4.91b
S3 1.02c 14.82c 17.8c 3.12c
Means with the same letters in each column are not significantly different.
Di: 2(Plant/m2), D2: 4(Plant/m2), D3: 6(Plant/m2), Si = March 24th, S2 = April 8th and S3 = April 24th
Interaction between planting density and sowing date. The summary of ANOVA table implies the significance of interaction between planting density and sowing date affecting all tested traits within 0.01 (table 3). Comparison of mean values by Duncan test showed that the highest rate of Neral, Geranial, Limonene was obtained in treatment D2S1 (density of 4 plant/m2 and sowing date April 24th) (table 5).
Table 5 - Mean comparison of density and sowing date interaction for the features of Essensioal oil,
Neral, Geranial, Limonene
Plant density*Sowing date Measured Features
Essensioal oil % Neral % Geranial % Limonene %
D1S3 2 de 19.32 e 24.6 e 3.96 de
D2S3 1.3 e 19.76 ef 25.4 e 3.81 e
D3S3 1.1 f 18.5 g 18.3 g 3.1 f
D1S2 2.7 bc 21.34 b 30.41 b 4.93 b
D2S2 2.5 cd 20.96 b 29.83 c 4.85 b
D3S2 2.3 d 19.6 d 20.42 f 4.12 d
D1S1 2.9 b 22.23 ab 32.3 a 5.21 ab
D2S1 3.2 a 23.25 a 33.63 a 5.84 a
D3S1 2.5 c 20.42 c 29.2 dc 4.34 c
Table 6 - Lemon verbena essential oil compounds
Row Compounds RIa Area,%
1 5-hepten-2-one-6-methyl 993 1.5
2 1,8-cineol 1034 1.8
3 p-pinene oxide 1152 0.3
4 3-Elemene 1337 0.6
5 (E)-caryophyllene 1424 3.2
6 Geraniol 1263 0.9
7 T-cadinol 1657 0.2
8 a-humulene 1454 0.2
9 5-cadinene 1522 0.6
10 a-zingiberene 1497 0.9
11 Geranyl propanoate 1479 0.3
12 trans-p-mentha 1(7),8-dien-2-ol 1186 0.3
13 Linalool 1104 0.4
14 Nerol 1238 0.7
15 Neral 1255 23.5
16 Geranial 1285 33.2
17 iso-Isopulegol 1165 0.2
18 -pinene a 932 0.2
19 trans-p-ocimene 1048 3.2
20 cis-sabinene hydrate 1073 0.2
21 caryophyllene oxide 1586 2.6
22 Bicyclogermacrene 1502 4.3
23 3-octanol 998 0.2
24 Limonene 1031 5.3
25 (E)-Nerolidol 1567 1.7
26 Spathulenol 1582 0.8
27 a-curcumene 1514 4.1
28 Sabinene 973 0.6
29 1- Octen-3-ol 982 0.2
30 Rosefuran epoxide 1177 0.2
31 a-terpineol 1197 0.8
32 Eugenol 1360 0.2
33 Germacrene D 1484 5.3
34 Geranyl acetate 1386 1.1
35 allo-Aromadendrene 1462 0.3
36 a-cedrene 1413 0.3
37 a-copaene 1375 0.3
a Retention indices on BP-20 column; (experimental)
DISCUSSION
Upon reviewing the references, it is concluded that there are rather reliable information on the pharmaceutical-medical and health effects of lemon verbena is available but the just finite information on the planting and agricultural management of this plant is available (Bandoni et al., 2008).
The peripheral factors are effective on the quality and quantity of active substances of pharmaceuticals such as planting density and sowing date. The biosynthesis of active substances of lemon verbena is dependent on the light regimes and plant breathing (Letchamo et al., 2004). Therefore, in density D1 and D2, due to lower ghosting and higher light absorption by the lower parts of Canopy, the maximum active substance was obtained. In density D1 and D2, more light is received by the plant and this light increasing has positive effect on the essential oil value, and in density D3, due to the ghosting the essential oil value is reduced. These results are in compliance with the observations(petrropoulos, 2004) of parsley and research applied by Schittenhelm(2001) on chicory. For optimum use of peripheral factors such as water, foodstuffs and prevention from emerging the intensive competition, the number of shrubs in the area unit must be appropriate. The appropriate density of shrub is a density on account of which all peripheral factors are used completely
by the lemon verbena, yet the competition inside and outside of shrub to be minimum, in order to obtain the maximum value of essential oil with the high quality. Due to the quality and climatic status appropriateness for increasing the biosynthesis of active substance, sowing date April 24th is the better sowing date for this trait. So that the thermal mode of this sowing date comparing to the warmer days, at the time of harvest intends to increase the active substance. Upon increasing the temperature during the plant growth and growing period shortening, reduction of leaf area and reduction of photosynthesis area will reduce the active substance. Timely cultivation of lemon verbena increases the growth period so that leads to maximum leaf production and ultimately increasing the active substance (Rocha et al., 2011). At this date, due to the plant’s whole use of raining thoroughly, better competition with weed, less infection by pests and diseases and suitable climatic status at the season beginning, the rate of above essential oil has been obtained higher than other sowing dates. The delay in planting results in germination delay and reduction of plant growth speed (Zehtab Salmasi et al., 2002). This is consistent with the results found by Argyropoulou et al. (2007).
Similar results have been shown to enhance the production of essential oil of thyme, lavender, mint Australian and Hungarian perppermint and Tasmania(Clark and Menary 1979; Shalaby and Razin 1992; Topalov and Zheljazkov, 1996; Rissanen et al., 2002; Arabaci et al., 2007).
Based on the results of our study should be planted lemon verbena (4 plant/m2 & March 24th) in order to optimize the percentage of essential oils from leaves.
ACKNOWLEDGEMENTS
Thanks for Dr. Ghooshchi and Dr. Ariapour for support during this research.
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