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Changes in the essential oil content and composition of palmarosa (Cymbopogon martini) harvested at different stages and short intervals in two different seasons
Industrial Crops and Products 69 (2015) 348–354
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Changes in the essential oil content and composition of palmarosa (Cymbopogon martini) harvested at different stages and short intervals in two different seasons Pandu Sastry Kakaraparthi a,∗ , K.V.N.S. Srinivas a , J. Kotesh Kumar a , A. Niranjana Kumar a , Dharmendra K. Rajput a , S. Anubala b a b
CSIR-Central Institute of Medicinal and Aromatic Plants, Research Centre, Boduppal, Hyderabad 500092, India Natural Products Laboratory, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 23 January 2015 Accepted 11 February 2015 Keywords: Palmarosa Geraniol Geranylacetate Relative humidity
a b s t r a c t The essential oil of palmarosa (Cymbopogon martini Roxb. Wats.), is one of the industrially important essential oils. Since the content and composition of essential oils is known to depend on extrinsic and intrinsic factors, including climate and season of harvest, knowledge of the optimal harvesting time is necessary for production of quality essential oil. Hence, experiments were conducted to study the variation in the essential oil composition of palmarosa harvested twelve times at short intervals during a six month period from October 2012 to March 2013. Chemical profiling of the essential oils was attempted by GC/GC–MS analysis, which revealed the presence of eleven compounds in the essential oil. The compounds are myrcene, cis--ocimene, trans--ocimene, linalool, neral, geraniol, geranial, geranylacetate, caryophyllene, geranyl isobuterate, and farnesol. A significant increase in the geraniol content and a significant decrease in the geranylacetate were noticed with passage of time. Maximum and minimum temperatures exhibited significant positive correlation with geraniol and showed significant negative relation with geranylacetate. Geraniol and geranylacetate in the essential oil were inversely related. Ideal harvesting time is around 70–80 days taking in to consideration of oil content, oil yield/plant and the content of geraniol and geranylacetate in the essential oil. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The genus Cymbopogon (Family: Poaceae) includes about 140 species and most of them were reported from Africa (52), followed by India (45), Australia, South America (six each), Europe (4), North America (2), and the rest are distributed throughout South Asia (Jagadish Chandra, 1975). High value essential oils produced from Cymbopogon find extensive use in perfumery, cosmetics, and in the pharmaceutical industries. Cymbopogon species displays a wide variation in morphological attributes and essential oil compositions at inter and intra-specific levels. Cymbopogon flexuosus, Cymbopogon nardus var. nardus, Cymbopogon citratus, Cymbopogon pendulus, Cymbopogon winterianus, and Cymbopogon martinii var. motia and sofia are the economically important species of the genus
∗ Corresponding author. Tel.: +91 9490755616. E-mail addresses: [email protected], [email protected] (P.S. Kakaraparthi). http://dx.doi.org/10.1016/j.indcrop.2015.02.020 0926-6690/© 2015 Elsevier B.V. All rights reserved.
(Rao, 1997; Gupta and Jain, 1978; Kumar et al., 2000; Verma et al., 2009). In Southeast Asia, Indian palmarosa oil is widely used, in many commercial cosmetics and toiletry products (Mallavarapu et al., 1998; Duarte et al., 2007; Muller et al., 2009). Chemical studies of the palmarosa oil reveals that it contains monoterpenes, sesquiterpenes, and alcohols like geraniol, geranylacetate, farnesol, nerolidol, geranial, limonene, terpinene, myrcene, caryophyllene, humulene, selinenes, linalool, and fatty acid 16-hydroxypentacos14-(z)-enoic acid (Mallavarapu et al., 1998; Raina et al., 2003). Geraniol and geranylacetate are extensively used in pharmaceutical, cosmetics, flavoring, and many other preparations. Geraniol, the major chemical constituent of palmarosa oil has characteristic rose-like odor and the taste (at 10 ppm) is described as sweet floral rose-like, citrus with fruity, waxy nuances (Burdock, 2010). This monoterpene alcohol is a widely used as fragrance material (it is present in 76% of deodorants on the European market, included in 41% of domestic and household products, and in 33% of cosmetic formulations based on natural ingredients (Rastogi et al., 1996,
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1998, 2001). Geraniol is also an effective plant based insect repellent (Barnard and Xue, 2004) and antimicrobial agent (Bard et al., 1988). Stage and season of harvest, weather, and soil conditions influence the content and composition of active plant principles in the essential oils (Verma et al., 2010, 2012; Sastry et al., 2014) and knowledge of the optimal harvesting time is necessary for production of quality essential oil. Hence, experiments were conducted with an objective to study the seasonal variation in the essential oil composition of palmarosa harvested at short intervals of time during a six month period.
2. Materials and methods 2.1. Experimental site and design of the experiment Seasonal variation in the essential oil composition of palmarosa was studied in a field experiment during a six month period from October 2012 to March 2013 at the research farm of Central Institute of Medicinal and Aromatic Plants (CIMAP), Research Centre, Boduppal, Hyderabad, Telangana, India. The experimental site is located at an altitude of 542 m above sea level with a geographical bearing of 78◦ 8 E longitude and 17◦ 32 N latitude. The mean annual rainfall of this region is generally 750 mm. The soil is a red sandy loam (alficusto chrept) with pH 8.27 (1.25 soil to solution ratio), EC-1.21 ds/m, organic C-0.58%, available N (215.40 kg/ha), available P (10.30 kg/ha), and exchangeable K (103.08 kg/ha). The experimental field was ploughed, harrowed, and leveled with tractor drawn implements before starting the nursery. Seeds of CSIR–CIMAP variety ‘Trishna’ were sown in nursery and healthy well grown seedlings were transplanted in rows following a row spacing of 60 cm between rows and 60 cm between plants in 4.8 m × 6.0 m plots. The crop was planted during first week of June 2012. A fertilizer dose of 100:40:40 Kg/ha of N: P: K was applied to the crop. Uniform doses of P and K were applied during ploughing. Nitrogen was applied in four splits. The crop was managed as per standard practices under irrigated conditions in 40 plots cultivated uniformly under similar conditions. The plots were kept weed free. During the last week of September 2012, the crop was harvested uniformly in all the plots. Between first week of October and end of December 2012, the crop was harvested four times at twenty days interval in different plots. The days of harvesting constituted the treatments and the treatments were replicated five times in a randomized complete block design. This is designated as the first phase. The field plots were again harvested commonly up to a height of 20 cm above ground level by the last week of December 2012. During the ninety days period from January 2013 onwards to the end of March 2013, the crop was harvested eight times at ten days interval (at 10, 20, 30, 40, 50, 60, 70 and 80 days) up to the end of March 2013. The dates of harvesting constituted the treatments and the treatments were replicated five times. This is designated as second phase. Observations were recorded at each harvest on the morphological characters, essential oil content, and composition in both the phases. During the second phase a separate set of observations was also recorded apart from the routine observations. Starting from sixty days which corresponds to the initiation of green inflorescence stage the crop was harvested six times at five days interval up to seed setting stage. This is in addition to the regular schedule in the second phase. The harvest dates (60, 65, 70, 75, 80, and 85 days) constituted the treatments and the treatments were replicated three times. The harvest dates correspond to different stages of inflorescence development which are presented in Fig. 1. The oil samples were subjected to GC analysis.
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2.2. Observation on morphometric traits The herb was harvested from ten randomly selected plants in each treatment plot in all the replications. Data were recorded for six morphometric traits viz., plant height and number of tillers/plant, number of leaves/plant, leaf length, leaf width, leaf area, and fresh weight of clump. The plants were dried till uniform weight at 60 ◦ C and the dry weight of plant/clump weight was also recoded. 2.3. Essential oil extraction/distillation The aerial parts of palmarosa were collected from ten random plants in each plot. For the extraction of essential oils, freshly collected herbage was subjected to hydro-distillation using a Clevenger-type apparatus for 4 h. The essential oils obtained were dried over anhydrous sodium sulphate and stored at 4 ◦ C until the GC analysis was carried out. The crop was harvested four times in the first phase (October–December) and eight times in the second phase (January–March). The oil content and quality were observed at all the harvested days. The treatments were replicated for five times. 2.4. Gas Chromatography (GC) analysis The essential oils were analyzed on a Varian CP-3800 model gas chromatograph with Galaxy software system equipped with flame ionization detector (FID) and an electronic integrator. Separation of the compounds was achieved employing a Varian CP-Sil 5CB capillary column (ID: 50 m X 0.25 mm; film thickness 0.25 m). Nitrogen was used as the carrier gas at a constant flow rate of 0.4 ml/min. The column temperature was programmed from 120 ◦ C (held for 2 min.) to 240 ◦ C (held for 5 min.) at a rate of 8 ◦ C/min. The injector and detector temperature were set at 250 ◦ C and 300 ◦ C, respectively. Samples of 0.2 L were injected with a 20:100:20 split ratio. Retention indices were generated with a standard solution of n-alkanes (C6 –C19 ). The composition was reported as a relative percentage of the total peak area without FID response factor correction. 2.5. Gas Chromatography–Mass Spectrometry (GC–MS) analysis GC–MS analysis was carried out on a SHIMADZU GCMS-QP2010 PLUS using a Zebron ZB5MS capillary column (ID: 30 m X 0.32 mm; film thickness 0.25 m). The column initially held at 90 ◦ C for 4.5 min, then heated to 150 ◦ C at a rate of 7 ◦ C/min and to 170 ◦ C at a rate of 10 ◦ C/min, held for 8 min. Injector and detector temperatures were kept at 250 ◦ C. Helium was used as carrier gas at 86.1 KPa (12.48 psi). Mass detection was performed by an electron ionization mode with ionization energy of 70 eV and ion source temperature of 250 ◦ C. 2.6. Chemical compounds identification The identification of the essential oil constituents was based on comparison of their retention indices relative to homologous series of n-alkanes (C6 –C19 ; Poly Science; Niles, USA) and published data. Chemical constituents were further confirmed by correlating the GC data with GC–MS data and compared to the NIST mass spectral library. 2.7. Statistical analysis Analysis of variance was performed to determine the effect of different times of harvest on morphological traits, essential yield and quality parameters using statistical software IRRISTAT [IRRI,
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Stage of harvest Stage 1: Stage 2: Stage 3: Stage 4: Stage 5: Stage 6:
Harvest date Identification 60 Green inflorescence emergence,25% of inflorescence in flowering stage 65 Pale yellow colour inflorescence, 50% of inflorescence in flowering stage Yellow colour inflorescence, 75% of inflorescence in flowering stage and seed 70 setting started Yellow colour inflorescence, 100% of inflorescence in flowering stage and early 75 seed setting in 30% flowers 80 Whitish brown inflorescence, Seed setting stage in complete inflorescence 85 Reddish brown inflorescence, Mature seed formation stage
Fig. 1. Different stages of inflorescence development starting from 60 days.
Manila, Philippines]. Means were compared using least significant differences [LSDs] at 5% probability levels. 3. Results
October–December period. During the second phase (Table 1) oil content increased from 0.30% at 10 DH to 0.85% at 80 DH. Essential oil yield/clump increased significantly with time in both the phases (0.22–2.24 and 0.02–2.18 g/clump, respectively, in the first and second phases).
3.1. Morphological characters as influenced by different times of harvesting 3.2. Essential oil composition at different times of harvesting 3.1.1. Plant height, number of tillers/clump, leaf length, leaf width and fresh weight/clump The data (Table 1) indicated a significant increase in the plant height (88.4-–151.6 cm), number of tillers/clump (43.6–-97.6), number of leaves/clump (60.7-–270.5), leaf length (21.0-–32.0 cm), and leaf width (1.0–-2.2 cm) during October–December period. Similarly, significant increases were also observed in plant height, number of tillers/clump, number of leaves/clump, leaf length, and leaf width during January–March period is presented in Table 1. Fresh weight (44.0–-264.4 g) showed significant increase with time in the first phase (Table 1) and also in second phase (5.9–-256.0 g/clump, Table 2). Similarly, earlier experiments also showed a significant improvement in the fresh mass accumulation, dry mass accumulation, and leaf area in Cymbopogon species (Castro et al., 2007). 3.1.2. Essential oil content and oil yield/clump Essential oil content (Table 1) is increased from 0.50% at 20 days harvest (DH) to 0.85% at 80 DH in the first phase during
In the essential oil, eleven compounds were identified (Table 2) and they are myrcene (0.1–-0.2%), cis--ocimene (0.2–-0.3%), trans-ocimene (1.3–-1.4%), linalool (2.1–-2.5%), neral (0.1%), geraniol (66.2–76.9%), geranial (0.1–-0.4%), geranylacetate (14.9–-24.6%), caryophyllene (0.3-–0.5%), geranyl isobuterate (0.1%), and farnesol (0.9–-1.3%) during first phase. Similar pattern was noticed in the second phase also (Table 2). The data in Table 2 indicated a significant increase in the geraniol content up to 80 DH (66.2-–76.9%) during October–December period. Similarly, an increase in geraniol content was noticed up to 80 DH during January–March period (Table 2). Geranylacetate followed increase decrease pattern up to 60 DH in the first phase and up to 30 DH during second phase. Thereafter, it decreased significantly. During both phases a slight increase in linalool content was noticed. Myrcene, trans--ocimene, linalool, geranial, and farnesol showed an increasing pattern, cis--ocimene, and caryophyllene showed decreasing pattern during first phase and a variable pattern during the second phase.
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Table 1 Morphological characters, oil content and oil yield/plant in palmarosa at different days of harvest during October 2012-–March 2013. Harvest date
Plant height (cm) No. of tillers/clump
No. of leaves/clump
Leaf length (cm)
Leaf width (cm)
Oil content Fresh weight/clump (g) (%,w/w)
Oil yield/clump (g)
October–-December 2012 88.4 20 40 119.8 147.6 60 151.6 80 LSDa (P = 0.05)b 7.6 c 4.1 C.V.(%)
43.6 71.1 91.5 97.6 7.2 3.6
60.7 202.9 240.6 270.5 15.9 3.2
21.0 28.0 31.0 32.0 3.0 7.4
1.0 1.4 1.9 2.2 0.1 4.4
44.0 75.7 241.6 264.4 11.2 5.3
0.50 0.65 0.80 0.85 0.06 6.74
0.22 0.49 1.93 2.24 0.07 6.11
January–-March 2013 10 52.2 20 91.0 30 119.6 40 119.6 50 120.2 60 125.8 130.6 70 138.4 80 LSDa (P = 0.05)b 10.3 6.3 C.V.(%)c
27.0 37.3 41.4 43.5 43.5 53.4 68.8 83.1 8.0 5.4
42.5 56.9 117.2 105.5 138.1 210.9 271.8 328.2 19.7 4.4
19.0 24.1 28.0 29.1 29.9 30.0 31.0 32.7 4.4 10.9
1.0 1.3 1.3 1.6 1.7 1.7 1.8 1.9 0.2 7.8
5.9 19.9 72.5 75.4 132.0 186.0 231.0 256.0 9.6 5.4
0.30 0.50 0.65 0.65 0.80 0.80 0.85 0.85 0.22 22.681
0.02 0.10 0.47 0.49 1.06 1.49 1.96 2.18 0.02 0.016
a b c
LSD: least significant difference. P = 0.05, probability level at 5%. C.V.(%): coefficient of variability.
3.3. Essential oil composition in herb harvested at five days interval The details about different stages of harvesting are presented in Fig. 1 and the data pertaining to the major components of the essential are presented in Fig. 2. Geraniol, the major constituent in the essential oil increased from 69.4% to 77.7% at 75 DH and later decreased to 59.3% at 85 DH. Geranylacetate decreased from 20.8% to 13.0% between 60 DH and 85 DH. The linalool content of the oil showed an inconsequential pattern (3.2–-1.6–-2.7). 3.4. Influence of weather parameters on the essential oil composition In this experiment, the content of the different constituents of the essential oil at different times were correlated with weather parameters over a period of 180 days is presented in Table 4.
The data pertaining to the weather parameters is presented in Fig. 3. Maximum temperature exhibited significant positive correlation with myrcene, cis--ocimene, trans--ocimene, linalool, geraniol, and showed significant negative relation with geranylacetate. Minimum temperature exhibited significant positive correlation with trans--ocimene and geraniol (Table 3). Relative humidity exhibited significant negative correlation with myrcene, cis--ocimene, trans--ocimene, and showed significant positive relation with geranylacetate. Sunshine hours showed significant positive correlation with caryophyllene only (Table 3). The individual chemical constituents of the essential oil noticed at different times of harvest during the 180 days study were also correlated among themselves (Table 4). Geranylacetate showed significant negative correlation with myrcene, cis--ocimene, trans--ocimene, linalool, and geraniol. The correlation between neral and geranial was positive and significant.
Table 2 Essential oil composition of palmarosa variety ‘Trishna’ at different times of harvest during October 2012–December 2012. Harvest date
Chemical composition of essential oil (%) Myrcene cis--Ocimene trans-ˇ-Ocimene Linalool
Neral Geraniol Geranial Geranylacetate Caryophyllene Geranyl isobutyrate Farnesol
October-–December 2012 0.1 20 40 0.2 0.2 60 0.2 80 LSDa (P = 0.05)b 0.1 22.4 C.V.(%)c
0.3 0.3 0.2 0.2 0.1 15.9
1.3 1.3 1.3 1.4 0.1 6.3
2.2 2.1 2.1 2.5 0.1 4.3
0.1 0.1 0.1 0.1 0.1 32.8
67.7 66.2 72.2 76.9 5.8 5.6
0.1 0.4 0.4 0.4 0.1 12.4
24.6 25.6 19.9 14.9 5.9 19.0
0.5 0.5 0.4 0.3 0.1 9.9
0.1 0.1 0.1 0.1 0.1 35.4
0.9 0.9 1.1 1.3 0.1 5.8
January-–March 2013 0.2 10 0.1 20 0.1 30 0.2 40 0.2 50 0.2 60 0.3 70 0.2 80 a b LSD (P = 0.05) 0.1 c 19.3 C.V.(%) RInp 984
0.4 0.2 0.2 0.2 0.3 0.2 0.5 0.4 0.1 11.1 1028
1.7 1.2 1.2 1.3 1.6 1.4 3.1 2.3 0.2 10.0 1041
2.1 2.0 2.0 1.6 1.8 2.9 2.4 2.9 0.3 8.6 1105
0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.1 19.2 1222
73.5 75.3 71.4 74.6 76.0 76.9 76.0 78.3 1.9 1.7 1238
0.3 0.5 0.5 0.4 0.4 0.3 0.2 0.4 0.1 9.1 1240
16.0 17.2 20.9 16.9 13.1 13.9 12.1 11.7 1.3 5.8 1364
0.7 0.3 0.3 0.6 0.9 0.6 0.6 0.4 0.1 8.5 1422
0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.1 31.3 1491
0.9 1.3 1.3 1.6 1.7 0.9 1.8 1.2 0.1 6.9 1698
RInp : retention index; non-polar column. a LSD: least significant difference. b P = 0.05: probability level at 5%. c C.V.(%): coefficient of variability.
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100.0
Content in essential oil, %
90.0 80.0 70.0 60.0 Linalool
50.0
Geraniol
40.0
Geranyl acetate
30.0
Total
20.0 10.0 0.0 60
65
70
75
80
85
Harvest date Fig. 2. Composition of the essential oil of herb harvested at five days interval between 60 and 85 days.
Table 3 Correlation between essential oil constituents of palmarosa variety ‘Trishna’ and weather parameters during October 2012–March 2013. Constituent
Maximum temperature (◦ C)
Myrcene 0.54* 0.55* cis--Ocimene 0.66* trans--Ocimene 0.63* Linalool −0.15 Neral Geraniol 0.65* Geranial −0.27 −0.77* Geranylacetate Caryophyllene 0.01 Geranyl isobutyrate 0.12 Farnesol 0.14
Minimum temperature (◦ C)
RH (%) Sunshine hours
0.26 0.37 0.50* 0.47 −0.10 0.53* −0.10 −0.51* −0.27 0.37 0.27
−0.79* −0.69* −0.68* −0.46 0.16 −0.40 0.39 0.70* −0.30 0.19 0.05
Maximum temperature (°C)
Minimum temperature (°C)
RH (%)
Sunshine hours
100.0 90.0
0.19 −0.11 −0.22 −0.06 0.07 0.08 0.07 −0.17 0.56* −0.39 −0.25
80.0 70.0
60.0 50.0 40.0 30.0
RH: relative humidity. * Significant (P = 0.05).
20.0 10.0
4. Discussion
0.0 March
February
January
December
The morphological characters viz., plant height, number of tillers/clump, number of leaves/clump, leaf length, leaf width, fresh weight, oil content, and oil yield increased significantly with advancement in the time of harvesting during both the phases (Table 1). Significant improvement in the oil yield with age was due to increase in the weight of herb. Immature leaves are biogenetically active to synthesize and accumulate essential oil substantially and earlier experiments also indicated a significant improvement in the accumulation of fresh and dry mass in aromatic grasses (Castro
November
October
4.1. Influence of harvest date on morphological characters
Fig. 3. Mean monthly weather para meters during the expe rimental period.
et al., 2007). This was substantiated when leaves were analyzed for essential oil content and composition at different developmental stages in citronella Java (Luthra et al., 1991; Sastry et al., 2014). Also in the present study, leaf expansion is associated with significant improvement in the essential oil content and yield.
Table 4 Correlation between essential oil constituents of palmarosa variety ‘Trishna’ harvested during 1 October 2012–March 2013. Chemical constituent
cis-- Ocimene
trans-- Ocimene
Linalool
Neral
Geraniol
Geranial
Geranylacetate
Caryophyllene
Geranyl isobutyrate
Farnesol
Myrcene cis--ocimene trans--ocimene Linalool Neral Geraniol Geranial Geranylacetate Caryophyllene Geranyl isobutyrate
0.86*
0.80* 0.96*
0.45 0.46 0.46
0.17 −0.06 −0.12 −0.04
0.33 0.31 0.42 0.48 −0.24
−0.03 −0.20 −0.29 −0.02 0.90* −0.35
−0.69* −0.65 −0.69 −0.57* 0.03 −0.80* 0.21
0.07 −0.09 −0.09 −0.35 −0.13 0.00 −0.28 −0.23
−0.21 −0.12 0.02 −0.30 −0.17 −0.09 −0.15 0.25 −0.08
−0.14 0.04 0.11 −0.42 −0.37 0.03 −0.39 −0.02 0.39 0.67*
*
Significant (P = 0.05).
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The fresh weight of herb (44.0–264.4 g/plant), oil content (0.5–0.85%) and oil yield/plant (0.22–2.24 g/plant) was high during October–December season compared to January–March season (fresh weight:5.9–256 g/plant, oil content:0.3–0.85% and oil yield:0.02–2.18 g/plant). The higher yields during October–December season were due to moderate temperature and humidity throughout the crop period (maximum temperature: 30–33 ◦ C and minimum temperature: 14–20 ◦ C) compared to January–March wherein the temperatures increased from 29.4 to 37 ◦ C during day and from 12.6 to 21 ◦ C during night. Moderate temperatures ranging around 30 ◦ C during day and 17 ◦ C during night contributed to higher herb and oil yields. This suggests that these temperature ranges are more ideal for production of better quality oil. The significant increases in the fresh weight of herb, and oil yield/clump with advancement in date of harvesting up to 80 days indicate that the crop can be harvested between 70 and 80 days when the fresh weight was highest. 4.2. Influence of harvest date on the essential oil composition Significant increase in the content of geraniol (varied from 66.2–76.9% during first phase and from 73.5–78.3% during second phase), and a significant decrease in geranylacetate (24.6–14.9% during first phase and 16.0–20.9–11.7% during second phase) were noticed during the time course of the study (Table 2). Myrcene, trans--ocimene, linalool, geranial, and farnesol showed an increasing pattern while cis-ˇ-ocimene and caryophyllene showed decreasing pattern during first phase and a variable pattern during the second phase. In the five day harvest experiment, it was observed that after 85 days geraniol content was reduced to 59.3% (77.2% at 70 DH, 77.7% at 75 DH and 70.8% at 80DH). Geranylacetate decreased from 20.8% to 13.0% between 60 DH and 85 DH. The major constituents of the essential oil reached maximum by 75 days and there was a rapid decline in the essential oil constituent especially geraniol after 75 days. With increase in harvest date seed setting increased and the major constituents in essential oil decreased. By 75 days the seed set started in thirty per cent of the inflorescences and by 80 days seed setting was noticed in 100% of the inflorescences. Mature seed were noticed by 85 days. Since, desired levels of geraniol and geranylacetate concentrations associated with higher herb and oil yields were noticed between 70 and 80 days and this period is recommended as ideal harvest time. The practice is to harvest the crop when 50% of the inflorescences in the crop are in flowering stage around 65–70 days to get 5–6 harvests in a calendar year. This experiment clearly indicates that harvesting between 70 and 80 days when both the geraniol and geranylacetate contents are optimum combined with higher essential oil yield hence this is an economically viable practice. In palmarosa, the period from the opening of the spikelets to complete maturity in the intact plant under field conditions was approximately one month. During inflorescence development in palmarosa, the composition of essential oil indicated a constant decrease in the geranylacetate percentage, with corresponding increase in the geraniol percentage. It was also observed that the geranylacetate proportion in the oil was relatively less as compared to geraniol at all stages of inflorescence development (Dubey et al., 2000, 2003). Similarly in citronella also, with leaf expansion, the quantities of citronellal, geraniol, and citronellol in the essential oil increased and the amounts of geranylacetate, and citronellylacetate decreased with time after transplanting or first harvest (Luthra et al., 1991; Sastry et al., 2014).
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4.3. Influence of weather on essential oil composition The variation in the weather parameters are presented in Fig. 3. Maximum temperature was lowest 29.1 ◦ C in January and highest 37.1 ◦ C in the last week of March during the experimental period. Geraniol exhibited significant positive correlation with both maximum and minimum temperatures while geranylacetate exhibited significant negative correlation with both maximum and minimum temperatures. Geraniol the major constituent is largely influenced by day and night temperatures where as geranylacetate is influenced by the relative humidity also besides temperatures. Decrease in geranylacetate with increase in temperatures might have facilitated increase in geraniol content since both are inversely related. Biosynthesis and accumulation of essential oil and geraniol were found to increase with maturity, while the trend was reverse in case of geranylacetate. It was reported that Geranyl acetate esterace (GAE) controls and regulates the level of geraniol (G) and geranylacetate (GA) in palmarosa. The enzyme GAE which is mainly involved in the conversion of GA–G. The portions of GA in essential oils declined corresponding to an increase in the G level. The GAE activity significantly varied during the leaf developmental stages studied. The GAE activity was comparatively higher during the early stages and young leaves, than in their later stages of leaf development. GAE had optimum content at temperature at 30 ◦ C and pH at 8.5. In addition, different regulatory controls operating at organ, cellular, subcellular, enzyme/isoenzyme levels also play a role in controlling the level of GA and G (Dubey and Luthra, 2001; Dubey et al., 2003; Deepak and Luthra, 2009). GAE activity markedly varies as a function of inflorescence development in palmarosa (C. martinii). The GAE activity steadily decreases with an increase in the age of inflorescence with the highest value in the immature stage of inflorescence. In our study, we also observed that when average maximum temperature is around 30 ◦ C, the geraniol content is high and geranylacetate content is low. Variations in the oil composition due to season and geographic localisation were found to be not significant (Silvestre et al., 1997) in Eucalyptus globulus Labill. subspp. globules, whereas in case of evening primrose (Oenothera spp.), oil content at seed maturity was positively correlated with both mean daily temperature and mean daily incident solar radiation in cv. Merlin. Strong negative correlations existed between the final ␥-linolenic acid, content of the oil, and climatic variables during the final phase of oil accumulation. Temperature was probably the primary determinant of the final ␥linolenic acid content (Andrew et al., 2000). Similarly, the essential oil compositions of aromatic plants were found to be dependent on their genetic structure, climatic factors, and the agronomical practices (Sangwan et al., 2001; Telci et al., 2006). Maximum and minimum temperatures and relative humidity influenced the composition of minor components also in the essential oil. Minor constituent trans--ocimene showed significant positive correlation with maximum temperature and minimum temperature. This indicates the significance of diurnal variation of temperature in determining the composition of essential oils. Relative humidity exhibited significant negative correlation with myrcene, cis--ocimene, trans--ocimene, and showed significant positive relation with geranylacetate indicating the significance of humidity. Literature indicates variable response between day temperature and essential oil composition. Day temperature showed a dominant influence on the essential oil composition compared to other weather parameters in case of rose scented geranium (Pelargonimum spp., Rao et al., 1995), in contrast, it was also reported that the commercially desired constituent, citronellal was
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higher at 27/21 ◦ C than at 32/27 ◦ C in all four citronella selections (Cymbopogon nardus (L.) Rendle.) indigenous to Sri Lanka. These alcohols were maximum during August but they decreased during September and first part of October (Nizharadze and Bagaturiya, 1981). One variety of citronella (Medini) was found to be geraniol rich during summer compared to winter (Verma et al., 2009). Variations in the chemical composition of natural essential oils are quite a common phenomenon and can be possibly due to various causes like origin, weather conditions, chemotype/genotype, developmental stage of the collected plant materials and season of harvest (Verma et al., 2009, 2010, 2012). 4.4. Inter-relationships between essential oil constituents The individual chemical constituents of the essential oil noticed at different times of harvest during the 180 days study were also correlated among themselves (Table 4). Geranylacetate showed significant negative correlation with myrcene, cis--ocimene, trans--ocimene, linalool, and geraniol. The correlation between neral and geranial was positive and significant. In earlier studies in palmarosa (Dubey et al., 2003), the essential oil analysis indicated a constant decrease in the geranylacetate percentage with corresponding increase in the geraniol percentage. In citronella crop, geraniol content was positively correlated with citronellol and citronellal contents (Luthra et al., 1991), in another study, geraniol exhibited a very high negative correlation with citronellol in one of the varieties of citronella (Rao et al., 1995). This once again confirms the hypothesis that essential oil content and composition varies between varieties, and also varies with time, and changes in weather parameters (Sastry et al., 2014). 5. Conclusions The changes in the chemical composition of the essential oil of palmarosa were studied for a period of six months at short harvest intervals. A significant increase in the plant height, number of tillers, number of leaves, leaf length, leaf width, leaf area, fresh weight, and dry weight were recorded with passage of time. Oil yield per plant also increased significantly. A significant increase in the geraniol content of the essential oil (66.2–76.9%) up to 80 days after harvest was noticed, whereas geranylacetate decreased significantly from 24.6 to 14.9% (20–80 days after harvest) during the first phase. Similar pattern in case of major constituent geraniol was observed in second phase also. Maximum temperature exhibited significant positive correlation with myrcene, cis--ocimene, trans--ocimene, linalool, geraniol, and showed significant negative relation with geranylacetate. Minimum temperature exhibited significant positive correlation with trans--ocimene, and geraniol. Harvesting between 70 and 80 days produced good quality essential oil. Acknowledgements The authors are thankful to the Director, CSIR–CIMAP, Lucknow and Director, CSIR–IICT for the encouragement and facilities. References Andrew, F., Fieldsend, J., Morison, I.L., 2000. Climatic conditions during seed growth significantly influence oil content and quality in winter and spring evening primrose crops (Oenothera spp.). Ind. Crops Prod. 12, 137–147. Bard, M., Albrecht, M.R., Gupta, N., Guynn, C.J., Stillwell, W., 1988. Geraniol interferes with membrane functions in strains of Candida and Saccharomyces. Lipids 23, 534–538.
Barnard, D.R., Xue, R., 2004. Laboratory evaluation of mosquito repellents against Aedes albopictus, Culex nigripalpus, and Ochlerotatus triseriatus (Diptera: Culicidae). J. Med. Entomol. 41, 726–730. Burdock, G.A., 2010. Geranio, Fenaroli’s Handbook of Flavor Ingredients, 6th ed. CRC Press, pp. 733–734. Castro, H.G., Barbosa, L.C.A., Leal, T.C.A.B., Souza, C.M., Nazareno, A.C., 2007. Growth, content and composition of the essential oil of Cymbopogon nardus (L.). Revista Brasileira de Plantas Medicinais 9, 55–61. Deepak, G., Luthra, R., 2009. Geranyl acetate esterase controls and regulates the level of geraniol in Lemongrass (Cymbopogon fl exuosus Nees ex Steud.) Mutant cv. GRL-1 Leaves. Z. Naturforsch. 64c, 251–259. Duarte, M.C.T., Leme, E.E., Delarmelina, C., Soares, A.A., Figueira, G.M., Sartoratto, A., 2007. Activity of essential oils from Brazilian medicinal plants on Escherichia coli. J. Ethnopharmacol. 111, 197–201. Dubey, V.S., Luthra, R., 2001. Biotransformation of geranylacetate to geraniol during palmarosa (Cymbopogon martinii Roxb. Wats. var. motia) inflorescence development. Phytochemistry 57, 675–680. Dubey, V.S., Mallavarapu, G.R., Luthra, R., 2000. Changes in the essential oil content and its composition during palmarosa (Cymbopogon martinii (Roxb.) Wats. Var. motia) inflorescence development. Flavour Frag. J. 15, 309–314. Dubey, V.S., Bhalla, R., Luthra, R., 2003. An esterase is involved in geraniol production during palmarosa inflorescence development. Phytochemistry 63, 257–264. Gupta, B.K., Jain, N., 1978. Cultivation and utilization of genus aromatic Cymbopogon in India. Indian Perfumer 22, 55–68. Jagadish Chandra, K.S., 1975. Cytogenetical Evolution in Some Species of Cymbopogon Cited in Advancing Frontiers in Cytogenetic. Hindustan Publishing Corporation. Kumar, S., Dwivedi, S., Kukreja, A.K., Sharma, J.R., Bagchi, G.D., 2000. Cymbopogon: The Aromatic Grass Monograph. CIMAP Publication, Lucknow, India. Luthra, R., Singh, N., Sharma, S., 1991. Changes in monoterpene content accompanying development of Cymbopogon winterianus Jowitt leaves. J. Essent. Oil Res. 3, 349–354. Mallavarapu, G.R., Rao, B.R.R., Kaul, P.N., Ramesh, S., Bhattacharya, A.K., 1998. Volatile constituents of the essential oils of the seeds and the herb of palmarosa (Cymbopogon martinii (Roxb.) Watson var. motia Burk.). Flavour Frag. J. 13, 167–169. Muller, G.C., Junnila, A., Butler, J., Kravchenko, V.D., Revay, E.E., Weiss, W.R., Schlein, Y., 2009. Efficacy of the botanical repellents geraniol: linalool and citronella against mosquitoes. J. Vector Ecol. 34, 2–8. Nizharadze, A.N., Bagaturiya, N.Sh., 1981. Effect of harvesting times of geraniums on the composition of essential oils. Maslozhirovaya Promyshlennost 6, 17–18. Raina, V.K., Srivastava, S.K., Aggarwal, K.K., Syamasundar, K.V., Khanuja, S.P.S., 2003. Essential oil composition of Cymbopogon martinii from different places in India. Flavour Frag. J. 18, 312–315. Rao, E.V.S.P., Rao, S.G., Ramesh, S., 1995. Seasonal variation in oil content and its composition in two chemotypes of scented geranium (Pelargonium sp.). J. Essent. Oil Res. 7, 159–163. Rao, B.L., 1997. Scope for Development of New Cultivars of Cymbopogon as a Source of Terpene Chemicals. National Institute of Science and Communication, New Delhi, India. Rastogi, S.C., Johansen, J.D., Menne, T., 1996. Ingredients based cosmetics. Content of selected fragrance sensitizers. Contact Dermatitis 34, 423–426. Rastogi, S.C., Johansen, J.D., Frosch, P., Menne, T., Bruze, M., Lepoittevin, J.P., Dreier, B., Andersen, K.E., White, I.R., 1998. Deodorants on the European market: quantitative chemical analysis of 21 fragrances. Contact Dermatitis 38, 29–35. Rastogi, S.C., Heydorn, S., Johansen, J.D., Basketter, D.A., 2001. Fragrance chemicals in domestic and occupational products. Contact Dermatitis 45, 221–225. Sangwan, N.S., Farooqi, A.H.A., Shabih, F., Sangwan, R.S., 2001. Regulation of essential oil production in plants. Plant Growth Regul. 34, 3–21. Sastry, K.P., Srinivas, K.V.N.S., Kumar, J.K., Kumar, A.N., Rajput, D.K., Sarma, V.U.M., 2014. Variation in the essential oil content and composition of Citronella (Cymbopogon winterianus Jowitt.) in relation to time of harvest and weather conditions. Ind. Crops Prod. 61, 240–248. Silvestre, A.J.D., Cavaleiro, J.A.S., Delmond, B., Claude, F., Guy, B., 1997. Analysis of the variation of the essential oil composition of Eucalyptus globulus Labill. from Portugal using multivariate statistical analysis. Ind. Crops Prod. 6, 27–33. Telci, I., Bayram, E., Yılmaz, G., Avcı, B., 2006. Variability in essential oil composition of Turkish basils (Ocimum basilicum L.). Biochem. Syst. Ecol. 34, 489–497. Verma, R.S., Laiq-Ur-Rahman Verma, R.K., Chauhan, A., Singh, A., Kukreja, A.K., Khanuja, S.P.S., 2009. Qualitative performance of Java citronella (Cymbopogon winterianus Jowitt) cultivars in Kumaon Himalaya. J. Med. Aromat. Plant Sci. 31, 321–325. Verma, R.S., Rahman, L., Verma, R.K., Chanotiya, C.S., Chauhan, A., Yadav, A., Yadav, A.K., Singh, A., 2010. Changes in the essential oil content and composition of Origanum vulgare L.: during annual growth from Kumaon Himalaya. Curr. Sci. 98, 1010–1012. Verma, R.S., Padalia, R.C., Arya, V., Chauhan, A., 2012. Aroma profiles of the curry leaf Murraya koenigii (L.) Spreng. Chemo types: variability in north India during the year. Ind. Crops Prod. 36, 343–349.
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Changes in the essential oil content and composition of palmarosa (Cymbopogon martini) harvested at different stages and short intervals in two different seasons Pandu Sastry Kakaraparthi a,∗ , K.V.N.S. Srinivas a , J. Kotesh Kumar a , A. Niranjana Kumar a , Dharmendra K. Rajput a , S. Anubala b a b
CSIR-Central Institute of Medicinal and Aromatic Plants, Research Centre, Boduppal, Hyderabad 500092, India Natural Products Laboratory, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 23 January 2015 Accepted 11 February 2015 Keywords: Palmarosa Geraniol Geranylacetate Relative humidity
a b s t r a c t The essential oil of palmarosa (Cymbopogon martini Roxb. Wats.), is one of the industrially important essential oils. Since the content and composition of essential oils is known to depend on extrinsic and intrinsic factors, including climate and season of harvest, knowledge of the optimal harvesting time is necessary for production of quality essential oil. Hence, experiments were conducted to study the variation in the essential oil composition of palmarosa harvested twelve times at short intervals during a six month period from October 2012 to March 2013. Chemical profiling of the essential oils was attempted by GC/GC–MS analysis, which revealed the presence of eleven compounds in the essential oil. The compounds are myrcene, cis--ocimene, trans--ocimene, linalool, neral, geraniol, geranial, geranylacetate, caryophyllene, geranyl isobuterate, and farnesol. A significant increase in the geraniol content and a significant decrease in the geranylacetate were noticed with passage of time. Maximum and minimum temperatures exhibited significant positive correlation with geraniol and showed significant negative relation with geranylacetate. Geraniol and geranylacetate in the essential oil were inversely related. Ideal harvesting time is around 70–80 days taking in to consideration of oil content, oil yield/plant and the content of geraniol and geranylacetate in the essential oil. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The genus Cymbopogon (Family: Poaceae) includes about 140 species and most of them were reported from Africa (52), followed by India (45), Australia, South America (six each), Europe (4), North America (2), and the rest are distributed throughout South Asia (Jagadish Chandra, 1975). High value essential oils produced from Cymbopogon find extensive use in perfumery, cosmetics, and in the pharmaceutical industries. Cymbopogon species displays a wide variation in morphological attributes and essential oil compositions at inter and intra-specific levels. Cymbopogon flexuosus, Cymbopogon nardus var. nardus, Cymbopogon citratus, Cymbopogon pendulus, Cymbopogon winterianus, and Cymbopogon martinii var. motia and sofia are the economically important species of the genus
∗ Corresponding author. Tel.: +91 9490755616. E-mail addresses: [email protected], [email protected] (P.S. Kakaraparthi). http://dx.doi.org/10.1016/j.indcrop.2015.02.020 0926-6690/© 2015 Elsevier B.V. All rights reserved.
(Rao, 1997; Gupta and Jain, 1978; Kumar et al., 2000; Verma et al., 2009). In Southeast Asia, Indian palmarosa oil is widely used, in many commercial cosmetics and toiletry products (Mallavarapu et al., 1998; Duarte et al., 2007; Muller et al., 2009). Chemical studies of the palmarosa oil reveals that it contains monoterpenes, sesquiterpenes, and alcohols like geraniol, geranylacetate, farnesol, nerolidol, geranial, limonene, terpinene, myrcene, caryophyllene, humulene, selinenes, linalool, and fatty acid 16-hydroxypentacos14-(z)-enoic acid (Mallavarapu et al., 1998; Raina et al., 2003). Geraniol and geranylacetate are extensively used in pharmaceutical, cosmetics, flavoring, and many other preparations. Geraniol, the major chemical constituent of palmarosa oil has characteristic rose-like odor and the taste (at 10 ppm) is described as sweet floral rose-like, citrus with fruity, waxy nuances (Burdock, 2010). This monoterpene alcohol is a widely used as fragrance material (it is present in 76% of deodorants on the European market, included in 41% of domestic and household products, and in 33% of cosmetic formulations based on natural ingredients (Rastogi et al., 1996,
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1998, 2001). Geraniol is also an effective plant based insect repellent (Barnard and Xue, 2004) and antimicrobial agent (Bard et al., 1988). Stage and season of harvest, weather, and soil conditions influence the content and composition of active plant principles in the essential oils (Verma et al., 2010, 2012; Sastry et al., 2014) and knowledge of the optimal harvesting time is necessary for production of quality essential oil. Hence, experiments were conducted with an objective to study the seasonal variation in the essential oil composition of palmarosa harvested at short intervals of time during a six month period.
2. Materials and methods 2.1. Experimental site and design of the experiment Seasonal variation in the essential oil composition of palmarosa was studied in a field experiment during a six month period from October 2012 to March 2013 at the research farm of Central Institute of Medicinal and Aromatic Plants (CIMAP), Research Centre, Boduppal, Hyderabad, Telangana, India. The experimental site is located at an altitude of 542 m above sea level with a geographical bearing of 78◦ 8 E longitude and 17◦ 32 N latitude. The mean annual rainfall of this region is generally 750 mm. The soil is a red sandy loam (alficusto chrept) with pH 8.27 (1.25 soil to solution ratio), EC-1.21 ds/m, organic C-0.58%, available N (215.40 kg/ha), available P (10.30 kg/ha), and exchangeable K (103.08 kg/ha). The experimental field was ploughed, harrowed, and leveled with tractor drawn implements before starting the nursery. Seeds of CSIR–CIMAP variety ‘Trishna’ were sown in nursery and healthy well grown seedlings were transplanted in rows following a row spacing of 60 cm between rows and 60 cm between plants in 4.8 m × 6.0 m plots. The crop was planted during first week of June 2012. A fertilizer dose of 100:40:40 Kg/ha of N: P: K was applied to the crop. Uniform doses of P and K were applied during ploughing. Nitrogen was applied in four splits. The crop was managed as per standard practices under irrigated conditions in 40 plots cultivated uniformly under similar conditions. The plots were kept weed free. During the last week of September 2012, the crop was harvested uniformly in all the plots. Between first week of October and end of December 2012, the crop was harvested four times at twenty days interval in different plots. The days of harvesting constituted the treatments and the treatments were replicated five times in a randomized complete block design. This is designated as the first phase. The field plots were again harvested commonly up to a height of 20 cm above ground level by the last week of December 2012. During the ninety days period from January 2013 onwards to the end of March 2013, the crop was harvested eight times at ten days interval (at 10, 20, 30, 40, 50, 60, 70 and 80 days) up to the end of March 2013. The dates of harvesting constituted the treatments and the treatments were replicated five times. This is designated as second phase. Observations were recorded at each harvest on the morphological characters, essential oil content, and composition in both the phases. During the second phase a separate set of observations was also recorded apart from the routine observations. Starting from sixty days which corresponds to the initiation of green inflorescence stage the crop was harvested six times at five days interval up to seed setting stage. This is in addition to the regular schedule in the second phase. The harvest dates (60, 65, 70, 75, 80, and 85 days) constituted the treatments and the treatments were replicated three times. The harvest dates correspond to different stages of inflorescence development which are presented in Fig. 1. The oil samples were subjected to GC analysis.
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2.2. Observation on morphometric traits The herb was harvested from ten randomly selected plants in each treatment plot in all the replications. Data were recorded for six morphometric traits viz., plant height and number of tillers/plant, number of leaves/plant, leaf length, leaf width, leaf area, and fresh weight of clump. The plants were dried till uniform weight at 60 ◦ C and the dry weight of plant/clump weight was also recoded. 2.3. Essential oil extraction/distillation The aerial parts of palmarosa were collected from ten random plants in each plot. For the extraction of essential oils, freshly collected herbage was subjected to hydro-distillation using a Clevenger-type apparatus for 4 h. The essential oils obtained were dried over anhydrous sodium sulphate and stored at 4 ◦ C until the GC analysis was carried out. The crop was harvested four times in the first phase (October–December) and eight times in the second phase (January–March). The oil content and quality were observed at all the harvested days. The treatments were replicated for five times. 2.4. Gas Chromatography (GC) analysis The essential oils were analyzed on a Varian CP-3800 model gas chromatograph with Galaxy software system equipped with flame ionization detector (FID) and an electronic integrator. Separation of the compounds was achieved employing a Varian CP-Sil 5CB capillary column (ID: 50 m X 0.25 mm; film thickness 0.25 m). Nitrogen was used as the carrier gas at a constant flow rate of 0.4 ml/min. The column temperature was programmed from 120 ◦ C (held for 2 min.) to 240 ◦ C (held for 5 min.) at a rate of 8 ◦ C/min. The injector and detector temperature were set at 250 ◦ C and 300 ◦ C, respectively. Samples of 0.2 L were injected with a 20:100:20 split ratio. Retention indices were generated with a standard solution of n-alkanes (C6 –C19 ). The composition was reported as a relative percentage of the total peak area without FID response factor correction. 2.5. Gas Chromatography–Mass Spectrometry (GC–MS) analysis GC–MS analysis was carried out on a SHIMADZU GCMS-QP2010 PLUS using a Zebron ZB5MS capillary column (ID: 30 m X 0.32 mm; film thickness 0.25 m). The column initially held at 90 ◦ C for 4.5 min, then heated to 150 ◦ C at a rate of 7 ◦ C/min and to 170 ◦ C at a rate of 10 ◦ C/min, held for 8 min. Injector and detector temperatures were kept at 250 ◦ C. Helium was used as carrier gas at 86.1 KPa (12.48 psi). Mass detection was performed by an electron ionization mode with ionization energy of 70 eV and ion source temperature of 250 ◦ C. 2.6. Chemical compounds identification The identification of the essential oil constituents was based on comparison of their retention indices relative to homologous series of n-alkanes (C6 –C19 ; Poly Science; Niles, USA) and published data. Chemical constituents were further confirmed by correlating the GC data with GC–MS data and compared to the NIST mass spectral library. 2.7. Statistical analysis Analysis of variance was performed to determine the effect of different times of harvest on morphological traits, essential yield and quality parameters using statistical software IRRISTAT [IRRI,
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Stage of harvest Stage 1: Stage 2: Stage 3: Stage 4: Stage 5: Stage 6:
Harvest date Identification 60 Green inflorescence emergence,25% of inflorescence in flowering stage 65 Pale yellow colour inflorescence, 50% of inflorescence in flowering stage Yellow colour inflorescence, 75% of inflorescence in flowering stage and seed 70 setting started Yellow colour inflorescence, 100% of inflorescence in flowering stage and early 75 seed setting in 30% flowers 80 Whitish brown inflorescence, Seed setting stage in complete inflorescence 85 Reddish brown inflorescence, Mature seed formation stage
Fig. 1. Different stages of inflorescence development starting from 60 days.
Manila, Philippines]. Means were compared using least significant differences [LSDs] at 5% probability levels. 3. Results
October–December period. During the second phase (Table 1) oil content increased from 0.30% at 10 DH to 0.85% at 80 DH. Essential oil yield/clump increased significantly with time in both the phases (0.22–2.24 and 0.02–2.18 g/clump, respectively, in the first and second phases).
3.1. Morphological characters as influenced by different times of harvesting 3.2. Essential oil composition at different times of harvesting 3.1.1. Plant height, number of tillers/clump, leaf length, leaf width and fresh weight/clump The data (Table 1) indicated a significant increase in the plant height (88.4-–151.6 cm), number of tillers/clump (43.6–-97.6), number of leaves/clump (60.7-–270.5), leaf length (21.0-–32.0 cm), and leaf width (1.0–-2.2 cm) during October–December period. Similarly, significant increases were also observed in plant height, number of tillers/clump, number of leaves/clump, leaf length, and leaf width during January–March period is presented in Table 1. Fresh weight (44.0–-264.4 g) showed significant increase with time in the first phase (Table 1) and also in second phase (5.9–-256.0 g/clump, Table 2). Similarly, earlier experiments also showed a significant improvement in the fresh mass accumulation, dry mass accumulation, and leaf area in Cymbopogon species (Castro et al., 2007). 3.1.2. Essential oil content and oil yield/clump Essential oil content (Table 1) is increased from 0.50% at 20 days harvest (DH) to 0.85% at 80 DH in the first phase during
In the essential oil, eleven compounds were identified (Table 2) and they are myrcene (0.1–-0.2%), cis--ocimene (0.2–-0.3%), trans-ocimene (1.3–-1.4%), linalool (2.1–-2.5%), neral (0.1%), geraniol (66.2–76.9%), geranial (0.1–-0.4%), geranylacetate (14.9–-24.6%), caryophyllene (0.3-–0.5%), geranyl isobuterate (0.1%), and farnesol (0.9–-1.3%) during first phase. Similar pattern was noticed in the second phase also (Table 2). The data in Table 2 indicated a significant increase in the geraniol content up to 80 DH (66.2-–76.9%) during October–December period. Similarly, an increase in geraniol content was noticed up to 80 DH during January–March period (Table 2). Geranylacetate followed increase decrease pattern up to 60 DH in the first phase and up to 30 DH during second phase. Thereafter, it decreased significantly. During both phases a slight increase in linalool content was noticed. Myrcene, trans--ocimene, linalool, geranial, and farnesol showed an increasing pattern, cis--ocimene, and caryophyllene showed decreasing pattern during first phase and a variable pattern during the second phase.
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Table 1 Morphological characters, oil content and oil yield/plant in palmarosa at different days of harvest during October 2012-–March 2013. Harvest date
Plant height (cm) No. of tillers/clump
No. of leaves/clump
Leaf length (cm)
Leaf width (cm)
Oil content Fresh weight/clump (g) (%,w/w)
Oil yield/clump (g)
October–-December 2012 88.4 20 40 119.8 147.6 60 151.6 80 LSDa (P = 0.05)b 7.6 c 4.1 C.V.(%)
43.6 71.1 91.5 97.6 7.2 3.6
60.7 202.9 240.6 270.5 15.9 3.2
21.0 28.0 31.0 32.0 3.0 7.4
1.0 1.4 1.9 2.2 0.1 4.4
44.0 75.7 241.6 264.4 11.2 5.3
0.50 0.65 0.80 0.85 0.06 6.74
0.22 0.49 1.93 2.24 0.07 6.11
January–-March 2013 10 52.2 20 91.0 30 119.6 40 119.6 50 120.2 60 125.8 130.6 70 138.4 80 LSDa (P = 0.05)b 10.3 6.3 C.V.(%)c
27.0 37.3 41.4 43.5 43.5 53.4 68.8 83.1 8.0 5.4
42.5 56.9 117.2 105.5 138.1 210.9 271.8 328.2 19.7 4.4
19.0 24.1 28.0 29.1 29.9 30.0 31.0 32.7 4.4 10.9
1.0 1.3 1.3 1.6 1.7 1.7 1.8 1.9 0.2 7.8
5.9 19.9 72.5 75.4 132.0 186.0 231.0 256.0 9.6 5.4
0.30 0.50 0.65 0.65 0.80 0.80 0.85 0.85 0.22 22.681
0.02 0.10 0.47 0.49 1.06 1.49 1.96 2.18 0.02 0.016
a b c
LSD: least significant difference. P = 0.05, probability level at 5%. C.V.(%): coefficient of variability.
3.3. Essential oil composition in herb harvested at five days interval The details about different stages of harvesting are presented in Fig. 1 and the data pertaining to the major components of the essential are presented in Fig. 2. Geraniol, the major constituent in the essential oil increased from 69.4% to 77.7% at 75 DH and later decreased to 59.3% at 85 DH. Geranylacetate decreased from 20.8% to 13.0% between 60 DH and 85 DH. The linalool content of the oil showed an inconsequential pattern (3.2–-1.6–-2.7). 3.4. Influence of weather parameters on the essential oil composition In this experiment, the content of the different constituents of the essential oil at different times were correlated with weather parameters over a period of 180 days is presented in Table 4.
The data pertaining to the weather parameters is presented in Fig. 3. Maximum temperature exhibited significant positive correlation with myrcene, cis--ocimene, trans--ocimene, linalool, geraniol, and showed significant negative relation with geranylacetate. Minimum temperature exhibited significant positive correlation with trans--ocimene and geraniol (Table 3). Relative humidity exhibited significant negative correlation with myrcene, cis--ocimene, trans--ocimene, and showed significant positive relation with geranylacetate. Sunshine hours showed significant positive correlation with caryophyllene only (Table 3). The individual chemical constituents of the essential oil noticed at different times of harvest during the 180 days study were also correlated among themselves (Table 4). Geranylacetate showed significant negative correlation with myrcene, cis--ocimene, trans--ocimene, linalool, and geraniol. The correlation between neral and geranial was positive and significant.
Table 2 Essential oil composition of palmarosa variety ‘Trishna’ at different times of harvest during October 2012–December 2012. Harvest date
Chemical composition of essential oil (%) Myrcene cis--Ocimene trans-ˇ-Ocimene Linalool
Neral Geraniol Geranial Geranylacetate Caryophyllene Geranyl isobutyrate Farnesol
October-–December 2012 0.1 20 40 0.2 0.2 60 0.2 80 LSDa (P = 0.05)b 0.1 22.4 C.V.(%)c
0.3 0.3 0.2 0.2 0.1 15.9
1.3 1.3 1.3 1.4 0.1 6.3
2.2 2.1 2.1 2.5 0.1 4.3
0.1 0.1 0.1 0.1 0.1 32.8
67.7 66.2 72.2 76.9 5.8 5.6
0.1 0.4 0.4 0.4 0.1 12.4
24.6 25.6 19.9 14.9 5.9 19.0
0.5 0.5 0.4 0.3 0.1 9.9
0.1 0.1 0.1 0.1 0.1 35.4
0.9 0.9 1.1 1.3 0.1 5.8
January-–March 2013 0.2 10 0.1 20 0.1 30 0.2 40 0.2 50 0.2 60 0.3 70 0.2 80 a b LSD (P = 0.05) 0.1 c 19.3 C.V.(%) RInp 984
0.4 0.2 0.2 0.2 0.3 0.2 0.5 0.4 0.1 11.1 1028
1.7 1.2 1.2 1.3 1.6 1.4 3.1 2.3 0.2 10.0 1041
2.1 2.0 2.0 1.6 1.8 2.9 2.4 2.9 0.3 8.6 1105
0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.1 19.2 1222
73.5 75.3 71.4 74.6 76.0 76.9 76.0 78.3 1.9 1.7 1238
0.3 0.5 0.5 0.4 0.4 0.3 0.2 0.4 0.1 9.1 1240
16.0 17.2 20.9 16.9 13.1 13.9 12.1 11.7 1.3 5.8 1364
0.7 0.3 0.3 0.6 0.9 0.6 0.6 0.4 0.1 8.5 1422
0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.1 31.3 1491
0.9 1.3 1.3 1.6 1.7 0.9 1.8 1.2 0.1 6.9 1698
RInp : retention index; non-polar column. a LSD: least significant difference. b P = 0.05: probability level at 5%. c C.V.(%): coefficient of variability.
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100.0
Content in essential oil, %
90.0 80.0 70.0 60.0 Linalool
50.0
Geraniol
40.0
Geranyl acetate
30.0
Total
20.0 10.0 0.0 60
65
70
75
80
85
Harvest date Fig. 2. Composition of the essential oil of herb harvested at five days interval between 60 and 85 days.
Table 3 Correlation between essential oil constituents of palmarosa variety ‘Trishna’ and weather parameters during October 2012–March 2013. Constituent
Maximum temperature (◦ C)
Myrcene 0.54* 0.55* cis--Ocimene 0.66* trans--Ocimene 0.63* Linalool −0.15 Neral Geraniol 0.65* Geranial −0.27 −0.77* Geranylacetate Caryophyllene 0.01 Geranyl isobutyrate 0.12 Farnesol 0.14
Minimum temperature (◦ C)
RH (%) Sunshine hours
0.26 0.37 0.50* 0.47 −0.10 0.53* −0.10 −0.51* −0.27 0.37 0.27
−0.79* −0.69* −0.68* −0.46 0.16 −0.40 0.39 0.70* −0.30 0.19 0.05
Maximum temperature (°C)
Minimum temperature (°C)
RH (%)
Sunshine hours
100.0 90.0
0.19 −0.11 −0.22 −0.06 0.07 0.08 0.07 −0.17 0.56* −0.39 −0.25
80.0 70.0
60.0 50.0 40.0 30.0
RH: relative humidity. * Significant (P = 0.05).
20.0 10.0
4. Discussion
0.0 March
February
January
December
The morphological characters viz., plant height, number of tillers/clump, number of leaves/clump, leaf length, leaf width, fresh weight, oil content, and oil yield increased significantly with advancement in the time of harvesting during both the phases (Table 1). Significant improvement in the oil yield with age was due to increase in the weight of herb. Immature leaves are biogenetically active to synthesize and accumulate essential oil substantially and earlier experiments also indicated a significant improvement in the accumulation of fresh and dry mass in aromatic grasses (Castro
November
October
4.1. Influence of harvest date on morphological characters
Fig. 3. Mean monthly weather para meters during the expe rimental period.
et al., 2007). This was substantiated when leaves were analyzed for essential oil content and composition at different developmental stages in citronella Java (Luthra et al., 1991; Sastry et al., 2014). Also in the present study, leaf expansion is associated with significant improvement in the essential oil content and yield.
Table 4 Correlation between essential oil constituents of palmarosa variety ‘Trishna’ harvested during 1 October 2012–March 2013. Chemical constituent
cis-- Ocimene
trans-- Ocimene
Linalool
Neral
Geraniol
Geranial
Geranylacetate
Caryophyllene
Geranyl isobutyrate
Farnesol
Myrcene cis--ocimene trans--ocimene Linalool Neral Geraniol Geranial Geranylacetate Caryophyllene Geranyl isobutyrate
0.86*
0.80* 0.96*
0.45 0.46 0.46
0.17 −0.06 −0.12 −0.04
0.33 0.31 0.42 0.48 −0.24
−0.03 −0.20 −0.29 −0.02 0.90* −0.35
−0.69* −0.65 −0.69 −0.57* 0.03 −0.80* 0.21
0.07 −0.09 −0.09 −0.35 −0.13 0.00 −0.28 −0.23
−0.21 −0.12 0.02 −0.30 −0.17 −0.09 −0.15 0.25 −0.08
−0.14 0.04 0.11 −0.42 −0.37 0.03 −0.39 −0.02 0.39 0.67*
*
Significant (P = 0.05).
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The fresh weight of herb (44.0–264.4 g/plant), oil content (0.5–0.85%) and oil yield/plant (0.22–2.24 g/plant) was high during October–December season compared to January–March season (fresh weight:5.9–256 g/plant, oil content:0.3–0.85% and oil yield:0.02–2.18 g/plant). The higher yields during October–December season were due to moderate temperature and humidity throughout the crop period (maximum temperature: 30–33 ◦ C and minimum temperature: 14–20 ◦ C) compared to January–March wherein the temperatures increased from 29.4 to 37 ◦ C during day and from 12.6 to 21 ◦ C during night. Moderate temperatures ranging around 30 ◦ C during day and 17 ◦ C during night contributed to higher herb and oil yields. This suggests that these temperature ranges are more ideal for production of better quality oil. The significant increases in the fresh weight of herb, and oil yield/clump with advancement in date of harvesting up to 80 days indicate that the crop can be harvested between 70 and 80 days when the fresh weight was highest. 4.2. Influence of harvest date on the essential oil composition Significant increase in the content of geraniol (varied from 66.2–76.9% during first phase and from 73.5–78.3% during second phase), and a significant decrease in geranylacetate (24.6–14.9% during first phase and 16.0–20.9–11.7% during second phase) were noticed during the time course of the study (Table 2). Myrcene, trans--ocimene, linalool, geranial, and farnesol showed an increasing pattern while cis-ˇ-ocimene and caryophyllene showed decreasing pattern during first phase and a variable pattern during the second phase. In the five day harvest experiment, it was observed that after 85 days geraniol content was reduced to 59.3% (77.2% at 70 DH, 77.7% at 75 DH and 70.8% at 80DH). Geranylacetate decreased from 20.8% to 13.0% between 60 DH and 85 DH. The major constituents of the essential oil reached maximum by 75 days and there was a rapid decline in the essential oil constituent especially geraniol after 75 days. With increase in harvest date seed setting increased and the major constituents in essential oil decreased. By 75 days the seed set started in thirty per cent of the inflorescences and by 80 days seed setting was noticed in 100% of the inflorescences. Mature seed were noticed by 85 days. Since, desired levels of geraniol and geranylacetate concentrations associated with higher herb and oil yields were noticed between 70 and 80 days and this period is recommended as ideal harvest time. The practice is to harvest the crop when 50% of the inflorescences in the crop are in flowering stage around 65–70 days to get 5–6 harvests in a calendar year. This experiment clearly indicates that harvesting between 70 and 80 days when both the geraniol and geranylacetate contents are optimum combined with higher essential oil yield hence this is an economically viable practice. In palmarosa, the period from the opening of the spikelets to complete maturity in the intact plant under field conditions was approximately one month. During inflorescence development in palmarosa, the composition of essential oil indicated a constant decrease in the geranylacetate percentage, with corresponding increase in the geraniol percentage. It was also observed that the geranylacetate proportion in the oil was relatively less as compared to geraniol at all stages of inflorescence development (Dubey et al., 2000, 2003). Similarly in citronella also, with leaf expansion, the quantities of citronellal, geraniol, and citronellol in the essential oil increased and the amounts of geranylacetate, and citronellylacetate decreased with time after transplanting or first harvest (Luthra et al., 1991; Sastry et al., 2014).
353
4.3. Influence of weather on essential oil composition The variation in the weather parameters are presented in Fig. 3. Maximum temperature was lowest 29.1 ◦ C in January and highest 37.1 ◦ C in the last week of March during the experimental period. Geraniol exhibited significant positive correlation with both maximum and minimum temperatures while geranylacetate exhibited significant negative correlation with both maximum and minimum temperatures. Geraniol the major constituent is largely influenced by day and night temperatures where as geranylacetate is influenced by the relative humidity also besides temperatures. Decrease in geranylacetate with increase in temperatures might have facilitated increase in geraniol content since both are inversely related. Biosynthesis and accumulation of essential oil and geraniol were found to increase with maturity, while the trend was reverse in case of geranylacetate. It was reported that Geranyl acetate esterace (GAE) controls and regulates the level of geraniol (G) and geranylacetate (GA) in palmarosa. The enzyme GAE which is mainly involved in the conversion of GA–G. The portions of GA in essential oils declined corresponding to an increase in the G level. The GAE activity significantly varied during the leaf developmental stages studied. The GAE activity was comparatively higher during the early stages and young leaves, than in their later stages of leaf development. GAE had optimum content at temperature at 30 ◦ C and pH at 8.5. In addition, different regulatory controls operating at organ, cellular, subcellular, enzyme/isoenzyme levels also play a role in controlling the level of GA and G (Dubey and Luthra, 2001; Dubey et al., 2003; Deepak and Luthra, 2009). GAE activity markedly varies as a function of inflorescence development in palmarosa (C. martinii). The GAE activity steadily decreases with an increase in the age of inflorescence with the highest value in the immature stage of inflorescence. In our study, we also observed that when average maximum temperature is around 30 ◦ C, the geraniol content is high and geranylacetate content is low. Variations in the oil composition due to season and geographic localisation were found to be not significant (Silvestre et al., 1997) in Eucalyptus globulus Labill. subspp. globules, whereas in case of evening primrose (Oenothera spp.), oil content at seed maturity was positively correlated with both mean daily temperature and mean daily incident solar radiation in cv. Merlin. Strong negative correlations existed between the final ␥-linolenic acid, content of the oil, and climatic variables during the final phase of oil accumulation. Temperature was probably the primary determinant of the final ␥linolenic acid content (Andrew et al., 2000). Similarly, the essential oil compositions of aromatic plants were found to be dependent on their genetic structure, climatic factors, and the agronomical practices (Sangwan et al., 2001; Telci et al., 2006). Maximum and minimum temperatures and relative humidity influenced the composition of minor components also in the essential oil. Minor constituent trans--ocimene showed significant positive correlation with maximum temperature and minimum temperature. This indicates the significance of diurnal variation of temperature in determining the composition of essential oils. Relative humidity exhibited significant negative correlation with myrcene, cis--ocimene, trans--ocimene, and showed significant positive relation with geranylacetate indicating the significance of humidity. Literature indicates variable response between day temperature and essential oil composition. Day temperature showed a dominant influence on the essential oil composition compared to other weather parameters in case of rose scented geranium (Pelargonimum spp., Rao et al., 1995), in contrast, it was also reported that the commercially desired constituent, citronellal was
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higher at 27/21 ◦ C than at 32/27 ◦ C in all four citronella selections (Cymbopogon nardus (L.) Rendle.) indigenous to Sri Lanka. These alcohols were maximum during August but they decreased during September and first part of October (Nizharadze and Bagaturiya, 1981). One variety of citronella (Medini) was found to be geraniol rich during summer compared to winter (Verma et al., 2009). Variations in the chemical composition of natural essential oils are quite a common phenomenon and can be possibly due to various causes like origin, weather conditions, chemotype/genotype, developmental stage of the collected plant materials and season of harvest (Verma et al., 2009, 2010, 2012). 4.4. Inter-relationships between essential oil constituents The individual chemical constituents of the essential oil noticed at different times of harvest during the 180 days study were also correlated among themselves (Table 4). Geranylacetate showed significant negative correlation with myrcene, cis--ocimene, trans--ocimene, linalool, and geraniol. The correlation between neral and geranial was positive and significant. In earlier studies in palmarosa (Dubey et al., 2003), the essential oil analysis indicated a constant decrease in the geranylacetate percentage with corresponding increase in the geraniol percentage. In citronella crop, geraniol content was positively correlated with citronellol and citronellal contents (Luthra et al., 1991), in another study, geraniol exhibited a very high negative correlation with citronellol in one of the varieties of citronella (Rao et al., 1995). This once again confirms the hypothesis that essential oil content and composition varies between varieties, and also varies with time, and changes in weather parameters (Sastry et al., 2014). 5. Conclusions The changes in the chemical composition of the essential oil of palmarosa were studied for a period of six months at short harvest intervals. A significant increase in the plant height, number of tillers, number of leaves, leaf length, leaf width, leaf area, fresh weight, and dry weight were recorded with passage of time. Oil yield per plant also increased significantly. A significant increase in the geraniol content of the essential oil (66.2–76.9%) up to 80 days after harvest was noticed, whereas geranylacetate decreased significantly from 24.6 to 14.9% (20–80 days after harvest) during the first phase. Similar pattern in case of major constituent geraniol was observed in second phase also. Maximum temperature exhibited significant positive correlation with myrcene, cis--ocimene, trans--ocimene, linalool, geraniol, and showed significant negative relation with geranylacetate. Minimum temperature exhibited significant positive correlation with trans--ocimene, and geraniol. Harvesting between 70 and 80 days produced good quality essential oil. Acknowledgements The authors are thankful to the Director, CSIR–CIMAP, Lucknow and Director, CSIR–IICT for the encouragement and facilities. References Andrew, F., Fieldsend, J., Morison, I.L., 2000. Climatic conditions during seed growth significantly influence oil content and quality in winter and spring evening primrose crops (Oenothera spp.). Ind. Crops Prod. 12, 137–147. Bard, M., Albrecht, M.R., Gupta, N., Guynn, C.J., Stillwell, W., 1988. Geraniol interferes with membrane functions in strains of Candida and Saccharomyces. Lipids 23, 534–538.
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