Chemical and Nutritional Compounds of Different Parts of Lemongrass ( Cymbopogon citratus (DC) Stapf.) Cultivated in Temperate Climate of Poland

: Lemongrass ( Cymbopogon citratus (DC) Stapf.) is a perennial plant indigenous to semi-tropical regions of Asia and cultivated in other semi-tropical countries. The present study aimed to examine the key chemical constituents of various parts of lemongrass cultivated in the temperate climate of Poland. The content of essential oil and its composition were determined in 4 plant parts: leaves (part C), overground shoots (part B), underground shoots (part A), and roots (part R). Moreover, the content of dry weight, chlorophyll, polyphenols and macro- and microelements was determined in the edible parts (excluding roots). The essential oil from the aerial part predominantly contained neral (> 30%) and geranial (> 40%), which is consistent with the data reported in literature; the main component of essential oil (EO) from the underground part was elemol (65%); interestingly, such a high concentration of it was found for the first time. The concentration of chlorophyll was found to be higher in leaves, as compared to parts B and A. The highest level of potassium, magnesium, zinc and sodium was found in part A while of calcium and copper in leaves. The quality of lemongrass raw materials grown in temperate climates did not differ significantly from those obtained in warmer regions. The study findings confirmed the usefulness of leaves as a raw material for the preparation of infusions (higher concentration of pigments, polyphenols and EO) and of near-ground parts of a plant as a culinary material (a higher content of macroelements at lower concentrations of green pigments and dry weight).


Introduction
Cymbopogon citratus DC Stapf. is a perennial plant native to tropical and semi-tropical regions of Asia and cultivated in South and Central America, Africa and other tropical countries. The plant forms extensive clusters composed of several or more blades growing from the rhizome connected with the fibrous root system of lemongrass. Plantations are used for 3-4 years and leaves can be cut twisted leaves forming pseudo-stems . However, such a harvest runs out the plantation as the entire plant is dug out. The parts mentioned above are used as a culinary additive for various oriental dishes, including spicy, meaty, poultry, seafood and curry vegetable dishes. They give lemon taste and smell, emphasising the orientality of dishes. They are particularly popular in Thai cuisine, yet gain popularity worldwide, including Poland. Therefore, an attempt was made to test the chemical composition of raw materials of lemongrass cultivated under the climate conditions of Poland, where lemongrass can be cultivated only as an annual plant due to low winter temperatures. The content of essential oil and its composition were determined in 4 plant parts leaves, overground shoots, underground shoots and roots . The EOs were also tested for their antioxidant activity. In the edible parts excluding roots , the concentrations of dry weight, chlorophylls, polyphenols, macro-and microelements were determined. The knowledge regarding the content of active substances in individuals parts of lemongrass cultivated in Poland should enable to determine the optimal harvesting management in the annual cropping system; the entire plant with the underground part can be dug out or the plant can be cut over the ground at a chosen level. Our findings were compared with the literature data regarding to chemical composition of lemongrass cultivated in Egypt 3, 6 , India 7, 8 , Iraq 9 , Nigeria 10 , Angola 11 , Ethiopia 2, 12 , Wenezuela 13 , Brasil 14 . The plants were collected in September, being in the 29 BBCH Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie stage at that moment, i.e. fully developed vegetative stage, fully tillering, with the maximum of shoots formed, but before the formation of the flower bud 15,16 . The entire plants with the underground part were dug out; their length and weight were determined, subsequently their roots were cut off part R . The aerial part was divided into individual shoots and cut according to colouring, starting with the near-ground part. The fragments were marked as part A, part B and part C, respectively Fig.  1 .
The mass of individual plant parts, percentage in the entire plant with roots and in the aerial part after root removal were determined. The length measurements were performed using a calibration measure to the accuracy of 1 mm; the mass of individual parts was determined with a calibration weighing scale to the accuracy of 0.01 g.
The content of dry weight DW in the samples was determined by over drying at 105 for at least 4 h. All mass measurements were performed to the accuracy of 0.001 g. Analysis was carried out in triplicates for each object.
Further determinations, i.e. total polyphenols, essential oil, chlorophylls, micro-and macroelement content, were performed for the samples of individual C. citratus aerial parts part A, B and C , dried in a laboratory drier with forced air circulation at 35 . Additionally, the content and composition of essential oil were analysed in roots part R .

Total polyphenols
Air-dried plant material was used for determinations. Water extracts were prepared pouring 200 mL of boiled water temp. 80 to 2.0 g samples, receiving a dilution proportion of 1:100. The content of total polyphenols TPC was determined in such solutions using the Folin-Ciocalteu total phenolic assay 17 , with gallic acid as a reference standard. Absorbance of the samples was measured with a UV-Vis spectrophotometer UV-2600, Shimadzu, Japan at 725 nm. The results were expressed as gallic acid equivalent mg GA per 100 mL water extract .

Essential oils
The essential oils EOs of air-dried plant material were obtained by hydro-distillation for 3 h in a Deryng-type apparatus. The oils were stored in tightly sealed 1.5 mL amber vials at 4 prior to analysis.

GC/MS analysis
Analysis was performed with a Shimadzu GC-2010 Plus instrument coupled to a Shimadzu QP2010 Ultra mass spectrometer. Compounds were separated on a fused-silica capillary column ZB-5MS 30 m, 0.25 mm i.d. with a film thickness of 0.25 mm Phenomenex . The oven temperature program was initiated at 50 , held for 3 min, then increased at the rate of 8-250 /min, and held for 2 min. The spectrometers were operated in EI mode; the scan range was 40-500 amu, the ionization energy 70 eV, and the scan rate was 0.20 s per scan. The injector, interface, and ion source were kept at 250, 250, and 220 , respectively. Split injection was conducted with a split ratio of 1:20 and helium was used as a carrier gas at a 1.0 mL/min flow rate. The retention indices were determined in relation to a homologous series of n-alkanes C8-C20 under the same operating conditions.

DPPH assay
EOs dissolved in MeOH were applied in triplicate on a 96-well microliter plate. The methanolic DPPH 50 mM solution was added to the test samples, and MeOH was used as a control. The plates were shaken for 2 min and incubated for 30 min in darkness at room temperature. Reading was taken at a wavelength of 517 nm.

Macro-and micro-elements
After the plant material was dried, crushed, ground and wet mineralized in analytically pure HNO 3 , the content of sodium Na , potassium K , calcium Ca , magnesium Mg , copper Cu , manganese Mn , iron Fe and zinc Zn was determined by atomic absorption spectrometry AAS according to EN-ISO 6869:2000 18 , with the use of a SOLAR 939 Unicam spectrometer. The results were expressed as mg/kg of dry weight DW .

Chlorophylls
Chlorophyll a and b were determined using a UV-Vis spectrophotometer UVS-2800 Labomed Inc., USA , by reading the absorbance at 470, 645 and 662 nm. The content of chlorophylls was calculated according to Wellburn 19 . For chlorophylls determination, 0.4 g of air-dried lemongrass samples part A, B and C were homogenized and extracted with acetone, using a magnetic stirrer at 700 rpm for 15 minutes. After the separation of the supernatant, the extraction was repeated Straumite et al. 20 .

Statistical analysis
The analysis of all parameters in the samples of individual C. citratus parts was performed in triplicates. Statistica 9.0 StatSoft was applied to analyse data. One-way ANOVA, with a Tukey test was applied to evaluate the significant differences among means at a level of p 0.05 .

Biometric parameters
After harvesting, the plants were separated into individual shoots, which were cutting according to colouring. Starting with the near-ground part, the fragments were reaching length respectively: part A -3 cm, part B -12 cm 10-14 cm . The remaining part, i.e. leaves part C , was 48 cm long on average Table 2 . The underground shoots part A weighed 46.6 g on average, which constituted 21.4 of the total aerial weight and 14.6 of the total plant weight with roots. This part was the smallest separated fragment of the plant Table 2, Figs. 2 and 3 . The leaves part C were the largest part 46 of the aerial part and 32 of weight of the plant with roots. Moreover, the leaves were characterised by the highest 19.6 concentration of dry weight, as compared to other parts. The value of this parameter successively decreased together with the distance from the ground and was 17.5 for part A. Cultivating lemongrass under warmer climate conditions e.g. Ethiopia 12 , Egypt 6 , higher weights of plant are achievable, which was confirmed by the results reported by Zigene et al. 12 and Hamed et al. 6 .
The total polyphenol content TPC in an aqueous extract of C. citratus varied with the plant part Table 2 . The leaf extract was characterised by the highest TPC, i.e. 6.57 mg GAE/100 mL extract. The closer the ground the plant part, the lower TPC in the extract -in part A, decreasing to 2.39 mg GAE/100 mL water extract. Soares et al. 11 have reported the similar TPC for water extract 4.28 mg GAE/100 mL , but yet they have not differentiated the plant parts but the mode of extraction of leaves. According to many other authors, including Figueirinha et al. 21 , Tavares et al. 22 , Uraku et al. 10 , Costa et al. 23 , lemongrass leaves can be the source of polyphenols, a very well known biologically active compounds. However, their findings cannot be compared with our results since the analytical methods used were different.

Essential oils
The GC/MS analysis showed that the chemical composi-   tion of EOs from leaves part C was very similar to that of the EOs hydro-distilled from parts B and A, except for the presence of a sesquiterpene alcohol, elemol in part A Table  3 . Elemol was also the major component of the EO obtained from C. citratus roots 65 - Fig. 4. The most characteristic components detected in the EOs from leaves and stems were neral 30 and geranial 40 . The composition of EO obtained from the aerial parts of lemongrass did not differ from that of EO from the plants cultivated in other climate zones, e.g. India 7 , Pakistan 24 , Ethiopia 2 , Wenezuela 13 Egypt 6 . The presence and percentages of elemol in the essential oil from roots of other Cymbo-pogon species have already been described in the literature, e.g. 19.1-42.3 in EO from the underground parts of C. flexuosus 7 , 10.67 25 and 14.5 26 in C. winterianus, 9 27 and 4.8 28 in C. nardus 27 , 4.9 27 in C. schoenantus, and 9.43 29 in C. proximus. To our best knowledge, we were the first to identify such a high concentration of elemol in C. citratus essential oil.
In the DPPH assay, the EO from part A of C. citratus presented the best antioxidant effect with EC 50 0.7 mg/mL Table 4 . The EO obtained from leaves showed weaker antioxidant activity, and EC 50 was 1.6 mg/mL. EC 50 value for pure citral neral geranial was 3.0 mg/mL. The similar  30 ; their data, however, cannot be compared with our results as they obtained the essential oil from fresh plants.

Macro-and microelements
Analysis of individual lemongrass parts demonstrated diverse amounts of mineral constituents, except for iron, whose average content was 91-94 mg/kg, irrespective of the plant part Table 5 . The highest content of potassium, magnesium, and zinc was found in the part nearest to the ground part A and decreased successively with the dis-  Citral (neral + geranial) -3.0 A -underground shoots, B -overground shoots, C -leaves tance to the ground. A similar correlation was observed for sodium; nevertheless, the values for part A and B were comparable 58.4 and 55.3 mg/kg and were not statistically significant. As far as calcium and copper are concerned, the opposite correlation was found; leaves contained the highest amount of these elements 3020 and 8.51 mg/kg, respectively while the lowest amount was noted in part A 1650 and 6.1 mg/kg . The results for manganese were ambiguous, ranging from 160 mg/kg for leaves to 361 mg/ kg for part B; the statistical analysis, however, did not confirm the significance of these differences.
Although the mineral constituents are considered to be of low importance for the preparation of infusions, for which lemongrass leaves are used, they can be of nutritional value for culinary applications of near-ground fragments part A and B . Nevertheless, Salvador et al. 31 have demonstrated a high level of iron in C. citratus tea 105-106 mg/L , which was the highest value among other tested herbal teas prepared from Matricaria chamomilla L. 54 mg/L , Mentha piperita L. 43-58 mg/L or Camellia sinensis L. O. Kuntze 0.21-0.26 mg/L . Furthermore, as compared to the values presented, AL-Joburi 9 has reported a double content of Mg in lemongrass leaves cultivated in Iraq, a comparable content of K 11900-25300 mg/kg and copper, a slightly lower amount of iron 52.35-84.20 mg/ kg , and a significantly lower amount of zinc 23-58 mg/ kg , depending on growth regulators used on lemongrass plantation.

Chlorophylls
The highest amounts of pigments were observed in C. citratus leaves part C ; 27.15 and 8.97 mg/100 g for chlorophyll a and b, respectively Table 6 . In lemongrass parts closer to the ground, the content of pigments successively decreased, reaching 8.74 mg/100 g of chlorophyll a and 3.53 mg/100 g for chlorophyll b. The colour of plant material is important when the plant is used for the preparation of teas; generally, a clear, deep colour of infusions is desirable. When lemongrass is added to dishes soups or sauces , the colouring, otherwise, can be considered undesirable particularly the greenish one . The results regarding chlorophyll pigments confirmed that upper parts of plant leaves could be a good material for infusions while the lower parts of a slight concentration of pigments -as a flavour and aroma enhancer in meals. Both d Ávila et al. 14 and AL-Joburi 9 have reported even 10-fold higher values of all pigments analysed chlorophyll a as well as b ; however, their determinations were carried out in fresh leaves of C. citratus while in our study dry plant material was used. Unfortunately, the authors mentioned above did not determine the content of pigments in the individual parts of the plant. Our studies demonstrate the distribution of important bioactives, as essential oil as well as polyphenols and some nutritional substances in lemongrass cultivated in Poland. No other so comprehensive analyses of the plant material obtained from various parts of lemongrass plants are available in the literature. Our findings showed, that the different part of lemongrass could be used, depending on their further application. Moreover, based on our results, the  height of cutting as well as the method of harvesting can be regulated, according to the application planned, e.g. for drying, for teas, infusions, production of essential oil or culinary additives.

Conclusions
1 The individual parts of C. citratus harvested in its 29 BBCH stage are characterised by diverse composition. 2 Essential oils obtained from the aerial part of lemongrass are rich in monoterpene aldehydes, neral 30 and geranial 40 . The major component detected in the essential oil from roots was sesquiterpene alcohol, elemol 65 . 3 The concentration of pigments chlorophyll a and b determined in leaves part C was higher compared to that in lower parts B and A . 4 The concentration of mineral constituents is correlated with the distance from the ground; thus, the highest content of potassium, magnesium, zinc and sodium was detected in part A while of calcium and copper in leaves. The amounts of Fe were comparable in all parts of lemongrass. 5 Our study results confirm the usefulness of lemongrass leaves as a material for infusions higher concentration of pigments, polyphenols and essential oil and of near-ground parts for culinary purposes higher concentration of macronutrients at lower concentration of green pigments and dry weight . 6 The material obtained from C. citratus cultivated under Polish climate conditions shows that the content and composition of essential oil, mineral constituents and chlorophyll which are comparable to lemongrass cultivated in other world regions. 7 Our findings showed the distribution of important bioactive and nutritional compounds in C. citratus plants, allowing to obtain raw materials most optimally, depending on their further applications.