quality parameters of c citratus leaves during …

Applied Engineering in Agriculture

Vol. 25(4): 543‐547 � 2009 American Society of Agricultural and Biological Engineers ISSN 0883-8542 543

QUALITY PARAMETERS OF CYMBOPOGON CITRATUS LEAVES DURING AMBIENT STORAGE

A. P. Martinazzo, E. C. Melo, L. C. de A. Barbosa, N. de F. F. Soares, R. P. Rocha, L. L. Randuz, P. A. Berbert

ABSTRACT. The effects of storage in different packages on the essential oil content and humidity of Brazilian lemon grass(Cymbopogon citratus) leaves were studied. Lemon grass leaves were dried at 50°C to moisture content of 11% (d.b.) andstored for one year in three different plastic and paper packages. Oil content and its principal compounds were isolated bythe hydro‐distillation method and analyzed by GC‐FID as well as GC‐MS every two months. The results showed a reductionin the oil content as well as a decrease in citral and myrcene in the plant for all packages during storage. Water concentrationdid not significantly vary during the period in the three packages.

Keywords. Air drying, Chemical preservation, Storage quality, Lemon grass.

he use of medicinal plants is part of a competitivemarket, which includes pharmaceuticals, food,cosmetics, and perfumery markets. In pharmaceu‐tics, plant extracts are especially relevant due to the

use of their active substances as prototypes for medicine de‐velopment and as sources of raw material, to obtain bothpharmaco and adjuvant. Medicinal plants are also used to ob‐tain medicines made exclusively from plant extracts such asphytotherapeutic medicines, whose use is limited by variousfactors including: cultivation, harvest period, climatic fac‐tors, humidity, brightness, part of the plant, transportationmethod, storage, drying process and extraction processwhich may all modify the composition of the products, di‐rectly affecting safety and efficiency (Calixto, 2000; Schen‐kel et al., 2003).

The species Cymbopogon citratus (D.C.) Stapf, is widelycultivated in Brazil and utilized for medicinal purposes,especially as tea, and industries are investing due to itsessential oil. According to Gomes (2001), Brazilian indus‐tries use three methods of drying, including: solar drying,covered storage rooms without air control, and dryers with

Submitted for review in October 2008 as manuscript number FPE 7743;approved for publication by the Food & Process Engineering InstituteDivision of ASABE in April 2009.

The authors are Ana P. Martinazzo, Agricultural Engineer, Professor,Department of Agrobusiness Engineering, Universidade FederalFluminense, Av. dos Trabalhadores, Volta Redonda, Rio de Janeiro, Brazil;Evandro C. Melo, Agricultural Engineer, Professor, Department ofAgricultural Engineering, Luiz C. de A. Barbosa, Chemist, Professor,Department of Chemistry, Nilda de F. F. Soares, Food Engineer, Professor,Department of Science and Food Technology, Ronicely P. Rocha,Agronomist, Doctorate student in Agriculture Engineering, Department ofAgricultural Engineering, Universidade Federal de Viçosa, Rua P.H. Rolfs,Viçosa, Brazil; Lauri L. Randüz, Agronomist, Professor, Department ofAgricultural Engineering, Universidade Regional Integrada, Erechim,Brazil; and Pedro A. Berbert, Agricultural Engineer, Professor,Department of Agricultural Engineering, Universidade Estadual do NorteFluminense Darcy Ribeiro, Av. Alberto Lamego 2000, Brazil. Corresponding author: Evandro C. Melo, Department of AgriculturalEngineering, Universidade Federal de Viçosa, Rua P.H. Rolfs, Viçosa,Brazil; phone: 55‐31‐3899‐1873; fax: 55‐31‐3899‐2729; e‐mail:[email protected].

forced hot air, where the material is previously cut in 2‐ to4‐cm pieces. After this process, the product is packed inplastic and raffia double cardboard bags. Dry leaves aresubjected to oil extraction and are kept in plastic flasks. Someproducers utilize plastic‐lined Kraft paper packages.

Due to the lack of standards in the dry plant market, itcannot be guaranteed that the producer or benefactor hadproperly conducted storage and drying processes. Despitecolor and odor characteristics, others are not easily detectedwhich require detailed analyses in order to identify its mainchemical composition. To avoid such variation and maintainthe quality of phytotherapeutics, Brazilian Legislation aimsto standardize production, requiring that companies registertheir products and present, among other documents, aquality‐control report in which a description of the drying,stabilization, and conservation methods of drug plant must beincluded (Brasil, 2004).

This work was developed with respect to the promisingBrazilian market of medicinal plants and the need for specifictechnological knowledge in the pre‐process area. Theobjectives of this investigation were to evaluate the effects ofstorage on the essential oil and its active principle in dryleaves kept in different packages and for different storageperiods, under natural light and ambient temperature andhumidity.

MATERIALS AND METHODSPLANT MATERIAL

The specie (Cymbopogon citratus D.C. Stapf) utilized fordrying process and storage experimentation was cultivated atthe Phythotechnic Department's Experimental Area at thethe Federal University of Viçosa – UFV (Minas Gerais,Brazil). The harvest period was from August 2004 to August2005.

STORAGETo evaluate characteristics concerning storage, harvested

leaves were manually cut after a selection process to

T

544 APPLIED ENGINEERING IN AGRICULTURE

approximately 2 cm and submitted to drying in a gas dryer at50°C until reach 11% (d.b.) water content.

STORAGE PACKAGINGThree different packages were used for dry leaves storage,

as shown by figure 1.In order to reproduce the packaging used by producers of

aromatic and medicinal species from the state of Paraná, themain Brazilian producer of this specie, Packaging 1 wascomposed of polypropylene, wrapped with two Kraft paperpackages.

Packaging 2 was made of one polypropylene layer, chosendue to its common commercialization for wrapping dryplants in compounding pharmacies, supermarkets, andothers.

Packaging P1

Packaging P2

Packaging P3

Figure 1. Packages used to store dry Cymbopogon citratus leaves.

In order to improve Packaging 1, Packaging 3 wascomposed of two Kraft paper layers and wrapped with apolypropylene package.

Plastic packages were sealed with a thermal‐sealer andpaper Kraft packages were sealed with tape.

STORAGE CONDITIONSThe experiment design was entirely randomized with

three repetitions. The treatments consisted of a 3 × 7factorial, with three packaging types (P1, P2, P3) and sevenstorage periods (0, 2, 4, 6, 8, 10, 12 months).

Seventy‐two packages were made – nine as reservepackages – each containing 100 g of product, and they werearranged on a shelf at room conditions and under naturallight. Data temperature and relative humidity registers wereconducted by thermal‐hygrograph.

Every two months, three repetitions were performed foreach packaging and the product's moisture content, essentialoil content and chemical composition were evaluated.

DETERMINATION OF MOISTURE CONTENT

Moisture content of the samples was determined using thegravimetric method recommended by ASAE Standards(2000) for forage and similar plants. This was done byplacing 25 g of the product in a stove with forced aircirculation at 103 + 2°C for 24 h, each done in triplicate.

EXTRACTION OF ESSENTIAL OIL

The extraction of essential oil was conducted by hydrodis‐tillation, utilizing Clevenger equipment. Samples of 20 and90 g were utilized for dry and fresh leaves, respectively.Starting from ebullition, the total extraction period was90 min which was determined by preliminary tests. The massof oil was measured on an analytical scale and the resultswere expressed in oil percentage as a function of theproduct's dry matter.

QUALITATIVE ANALYSIS OF ESSENTIAL OIL COMPONENTSThe identification of individual components was done

using a Shimadzu GC 14A model gas chromatographcoupled to Shimadzu selective mass detector QP 5000 massspectrometer (GC‐EM). The chromatographic column usedwas the smelt silica capillar type with a DB‐5 stationaryphase, 0.25 �m thick by 30 m long with an internal diameterof 0.25 mm. Helium was used as the carrier gas at a flow rateof 1.0 mL/min. The temperature of the injector was 220°Cand the temperature of the detector was 240°C. Initialtemperature in the stove was kept at 60°C for 2 min, and wasincreased at a rate of 3°C per second until it reached themaximum temperature of 240°C. This temperature wasmaintained for over 30 min, totaling 91 min for analysis. Splitratio was 1:20 and the solvent cut period was 5 min. Only ionsat charge mass (m/z) ratios between 29 and 600 were detectedby the mass spectrometer.

The sample volume injected was 1 �L, at a concentrationof 10,000 ppm with hexane as a solvent.

The identification of components was conducted bycomparing mass spectrometer readings obtained from theequipment database and Kovats retention index for eachcomponent, as shown in equation 1 (Lanças, 1993).

545Vol. 25(4): 543‐547

⎟⎟

⎠

⎞

⎢⎢

⎝

⎛

′−′

′−′+=

+ RZ1)R(Z

RZRX

tLogtLog

tLogtLog100100NCIK (1)

whereIK = Kovats indexNC = number of carbons from hydrocarbon

immediately before the evaluated componentt′RX = retention time of evaluated componentt′RZ = retention time of hydrocarbon immediately before

the evaluated componentt′R(Z+1) = retention time of the hydrocarbon immediately

after the evaluated componentIn order to draw the hydrocarbon standard curve to

calculate the Kovats index, a solution of linear hydrocarbonwas prepared, varying from hexane to tetracosane. Twomilligrams of each hydrocarbon were weighed in the sameflask in order to prepare the solution. The final mass wassolubilized in 2 mL of hexane, producing a 1000‐ppmsolution in relation to each hydrocarbon. The solution wasanalyzed using a gas chromatograph, coupled with a massspectrometer, with the same operational conditions used foressential oil samples.

QUANTITATIVE ANALYSIS OF ESSENTIAL OIL COMPONENTSTo specify the quantity of essential oil components, a

Shimadzu GC‐17 A gas chromatograph, equipped with aflame ionization detector (FID) and a smelt silica capillarcolumn with a DB‐5 stationary base, 0.25 �m thick by 30 mlong and with an internal diameter of 0.25 mm. Nitrogen wasused for the purging gas at a flow rate of 1.33 mL/min. Theinitial temperature of the column was kept at 60°C for 2 min,and programmed to increase at 3°C per minute, until reachingthe maximum temperature of 240°C, where it was main‐tained for 61 min for analysis. Split ratio was 1:10 and thesolvent cut period was 5 min. Injection and detectiontemperatures were fixed at 250°C. The sample injectionvolume was 1 �L, at a concentration of 2000 ppm, usinghexane as a solvent. The components were quantified basedon the comparison of compound's retention period, whichwere similar in both techniques. The normalization methodwas used; the value of total peak areas is considered 100%and the percentage of each component was calculated usingthe area of each peak.

STATISTICAL ANALYSISQualitative factors were analyzed using the Sisvar 4.3�

program and averages were compared using the Turkey testat 5% probability.

In order to analyze the effects of quantitative factors on theconsidered characteristics, simple regression analysis wasutilized. The criteria to define a more adequate regressionmodel were: regression variance analysis (P<0.05); thedetermination coefficient (R2) and significance of regressionparameters. Computer programs used were Sisvar 4.3� andSigmaPlot 7.0�.

RESULTS AND DISCUSSIONTHE INFLUENCE OF STORAGE PROCESSES ON ESSENTIAL

OIL CONTENTFrom the variance analysis (table 1) of the effect of various

packaging and storage period on essential oil from dry leaves

Table 1. Degree of freedom (D.F.), mean square (M.S.) for analysis of variance (ANOVA).[a]

Source of Variations D.F. M.S. F

Packaging (p) 2 0.001541 0.466[b]

Storage time (t) 6 0.055578 16.805[c]

p × t 12 0.002318 0.701[b]

Error 42 0.003307

Total 62

CV[d] = 8,14%

[a] Dates from essential oil during Cymbopogon citratus leaves storage.[b] No significant.[c] Significant 5% F test.[d] Coefficient of variation.

of C. citratus kept in a non‐controlled room, it was observedthat only the storage period had a significant effect on theessential oil content of the samples.

After obtaining results from variance analysis, the analy‐sis of the factor of period was conducted by regression. Theobserved and calculated data of essential oil content, with theadjusting equation is shown by figure 2.

From figure 2 one can observe the decreasing linear effectof storage period on the essential oil content. Even though adecrease during storage occurred, the obtained value after12 months is within those established by Brazilian Pharma‐copoeia V (2003), which states that a plant drug specie mustbe constituted by a 0.5% minimal essential oil content in dryleaves. Therefore, the product can be commercialized asinstituted by Brazilian Legislation.

THE INFLUENCE OF STORAGE PROCESS ON CITRALCONCENTRATION

From the variance analysis (table 2) of packaging andstorage period's effect on citral concentration in the essentialoil, it was observed that only the storage period variablepresented significant effect on citral concentration in essen‐tial oil from the samples. Packaging type had no influence onthe latter.

Figure 2. Observed and estimated values of essential oil from dry Cymbo‐pogon citratus leaves productivity, stored in a non‐controlled room.


Table 2. Degree of freedom (D.F.), mean square (M.S.) for analysis of variance (ANOVA). Dates from citral essential oil during

Cymbopogon citratus leaves storage.

Source of Variations D.F. M.S. F[a]

Packaging (p) 2 0.000679 0.210n.s.

Storage time (t) 6 0.023904 7.389*

p × t 12 0.001254 0.388n.s.

Error 42 0.003235

Total 62

CV[b] = 9,15%

[a] n.s. No significant;* Significant 5% F test;

[b] CV Coefficient of variation.

Figure 3 shows the variations in oil concentration, citralconcentration in the oil and the percentage of citral of theproduct (dry leaves) during storage.

At the beginning of storage, the observed citral contentcorresponded to 86% of the total essential oil composition.As stored, there was a decrease in the essential oilconcentration, as described earlier, as well as citral in theplant.

From the final percentage of citral in the essential oil, amore intense degradation of other oil components wasobserved. At the end of the storage period, citral accountedfor 90% of the final total oil composition. The BrazilianPharmacopoeia V (Brasil, 2003) established that essential oilextracted from dry C. citratus leaves must be composed of atleast 60% of citral. It did not establish minimum limits forother components.

Misharina (2001) and Misharina et al. (2003), evaluatingessential oil from Majorana hortensis leaf samples andcoriander seeds (Coriandrum sativum) stored for 12 months,observed an increase in some components as others de‐creased due to chemical transformation of terpene com‐pounds, which are commonly modified when exposed tolight, oxygen, humidity, heat, etc.

The results obtained from previously quoted authors referto essential oil storage after extraction. However, in thisresearch, the evaluation of essential oil from C. citrates wascarried out during storage of leaves. From the obtainedresults, it can be noticed that essential oil of stored leavespasses through modifications during storage, which might be

Figure 3. Obtained values of citral and essential oil concentration in dryCymbopogon citratus leaves during storage in a non‐controlled room.

related to oxygen, humidity, heat and temperature influences.Because there was no percentage difference of citral inutilized packaging, it is believed that light did not influencethe chemical composition of essential oil, due to the fact thatpackaging 2 (polypropylene) did not protect the product fromlight.

Sakamura (1987), studying ginger (Zingiber officinale“Oshoga”) storage, observed a decrease in the essential oillevel, an increase in nerol and geraniol concentrations and areduction of geranial acetate. The author considered thatgeranial acetate had hydrolyzed into geraniol and the formerhad oxidized in geranial and nerol.

Taking quoted researches into consideration, a relation‐ship between the decrease of any component and increase ofcitral in essential oil was draw. However, no other relation‐ship observed could explain the obtained result. Therefore,variations found could be related to any of the components,making identification impossible.

INFLUENCE OF STORAGE ON MYRCENE PERCENTAGE

From the variance analysis (table 3) of packaging andstorage period on myrcene concentration in essential oil, itwas observed that only the storage period had a significanteffect on myrcene concentration in essential oil from thesamples.

The variation of oil concentration, myrcene percentage inoil and myrcene percentage of the product (dry leaves) duringstorage is shown by figure 4.

The graph shows that both myrcene concentrations inessential oil and in the dry stored plant decreased as afunction of time. For this compound there are no BrazilianLegislation requirements concerning its concentration inessential oil, probably due to its low concentration in thespecie and the fact that many times it is hard to establish aminimum concentration which guarantees pharmacologicalactivity of an isolated substance when it is part of a complexmixture such as essential oil. From obtained results, eventhough there are not minimum values established, it wasconcluded that from the sicth month of storage, underevaluated conditions, there was an accentuated decrease inmyrcene content, which could interfere on its scientificallyconfirmed analgesic activity.

MOISTURE CONTENT OF THE PRODUCT DURING STORAGE

The initial moisture content of the samples submitted tothe storage process was 11% (d.b.). Temperatures in thestorage room varied from 17.5°C to 31°C and relativehumidity from 52.9% to 86.24%.

Table 3. Degree of freedom (D.F.), mean square (M.S.) for analysis of variance (ANOVA). Dates from myrcene essential oil

during Cymbopogon citratus leaves storage.


Packaging (p) 2 0.000013 0.221n.s.

Storage time (t) 5 0.000348 5.917*

p × t 10 0.000032 0.542n.s.

Error 36 0.000059

Total 53

CV[b] = 7,76%

[a] n.s. No significant;* Significant 5% F test;

[b] CV Coefficient of variation.

547Vol. 25(4): 543‐547

Figure 4. Obtained values of myrcene and essential oil concentrationsfrom dry Cymbopogon citratus leaves during storage period in a non‐controlled room.

The variance analysis of packaging and storage period onmoisture content of leaves is showed in table 4.

It was observed that moisture content of the stored productin different packaging did not significantly vary and only thestorage period had a significant effect. Table 5 shows thevariations in moisture content of the product (dry leaves)during storage.

Even though there were slight changes in moisture contentduring storage, it was observed that all, under evaluatedconditions, were within established limits for medicinalspecies (8% to 14% d.b.), among different Pharmacopoeiasfrom several countries (Farias, 2003).

Table 4. Degree of freedom (D.F.), mean square (M.S.) for analysis of variance (ANOVA). Dates from moisture content (%, d.b.)

Cymbopogon citratus leaves storage.


Packaging (p) 2 1.145778 3.04n.s.

Storage time (t) 5 7.193488 19.11*

p × t 10 0.159706 0.42n.s.

Error 36 0.376445

Total 53

CV[b] = 5,85%[a] n.s. No significant;

* Significant 5% F test;[b] CV Coefficient of variation.

Table 5. Moisture content (%, d.b.) obtained from dry Cymbopogoncitratus leaves stored under room conditions for 12 months.

Storage Period (months) Moisture Content (%, d.b.)[a]

2 9.95 a

4 10.08 a

6 10.13 a

8 10.16 a

10 10.32 a

12 12.30 b[a] Values with the same letter are not significantly different for 5% by

Tukey test.

CONCLUSIONDuring 12 months of storage the essential oil concentra‐

tion of the dry product linearly decreased independent of thetype of packaging used, yet it staying within the establishedlimits by Brazilian Legislation for pharmaceutics products.

Citral and myrcene concentrations decreased as a functionof storage period. However, even though it decreased, citralconcentration in the product after 12 months was within theestablished limits by Brazilian Pharmacopoeia V (Brasil,2003). Water concentration in the product did not significant‐ly vary during storage period in the three packages.

ACKNOWLEDGMENTS

The authors thank the FAPEMIG, CAPES, and CNPq fortheir financing support, essential for conducting the project.

REFERENCESASAE Standards. 2000. 358.2. Moisture Measurement – Forages.

St. Joseph, Mich.: ASAE.Brasil. 2003. Farmacopéia Brasileira, 5. Atheneu, São Paulo.Brasil. 2004. Resolução RDC no 48, de 16 de março de 2004.

Diário Oficial da União, Brasília.Calixto, J. B. 2000. Efficacy, safety, quality control, market and

regulatory guidelines for herbal medicines (phytotherapeuticagents). Brazilian J. Med. and Biol. Res. 33(2): 179‐189.

Farias, M. R. 2003. Avaliação da qualidade de matérias‐primasvegetais. In Farmacognosia: da Planta ao Medicamento , eds.C. M. O. Simões et al., 263‐288. Editora da UFRGS/Editora deUFSC: Porto Alegre/Florianópolis.

Gomes, E. C. 2001. Aspectos do cultivo e beneficiamento docapim‐limão (Cymbopogon citratus (D.C.) Stapf.) no estado doParaná, Brasil. Visão Acadêmica 2(1): 11‐18.

Lanças, F. M. 1993. Cromatografia em fase gasosa, 112‐1151.Acta: São Carlos.

Misharina, T. A. 2001. Influence of the duration and conditions ofstorage on the composition of the essential oil from corianderseeds. Applied Biochem. and Microbiol. 37(6): 622‐628.

Misharina, T. A., A. N. Polshkov, E. L. Ruchkina, and I. B.Medvedeva. 2003. Changes in the composition of the essentialoil of marjoram during storage. Applied Biochem. andMicrobiol. 39(3): 353‐358.

Sakamura, F. 1987. Changes in volatile constituents of Zingiberofficinale rhizomes during storage and cultivation.Phytochemistry 26(8): 2207‐2212.

Schenkel, E. P., G. Gosmann, and P. R. Petrovick. 2003. Produtosde origem vegetal e o desenvolvimento de medicamentos. InFarmacognosia: da Planta ao Medicamento , eds. C. M. O.Simões et al., 371‐400. Editora da UFRGS/Editora de UFSC:Porto Alegre/Florianópolis.

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