Drying Potential of Leaves Oil of Zanthoxylum armatum DC from North India

: Background: The study on drying and its social acceptance has increased rapidly. Among different drying techniques, shade drying is one of the most feasible methods to keep intact from the decay of the main active components of the plant materials. Shade drying is an ancient drying method that increases durability, major constituents and activity of the plant material. Research was conducted to examine the drying potential of aromatic leaves oil of Zanthoxylum armatum DC. The fresh plant material was collected from Lohaghat, Champawat district of Uttarakhand and hydrodistilled before and after shade drying to assess the changes in the quality of volatile constituents by GC and GC-MS techniques. A two-tailed paired t-test was executed to assess the difference between drying treatments using MS-Excel. The ( )-β-ocimene, α-pinene and β-phellandrene. In the oil from dried material, the three predominant compounds were noted. A significant increase was observed in the percentage of β-phellandrene, undecanal and myrcene after shade drying (p<0.01). Five components absent in the fresh plant material appeared and one disappeared during the drying process.


INTRODUCTION
The drying technique has been playing a successful role in the storage and food safety since ancient times. Efforts on drying methodology should be made to raise the active components of the aromatic herbs. These active components are the major part of various drugs, spices, natural beauty products, aroma profiles and are responsible for anticancer, antiaging, anti-oxidant, anti-microbial, anti-inflammatory and other various activities [1]. Shades drying, sun-drying, airdrying, oven-drying, microwave-drying, fluidized-drying and freeze-drying are the most useful methods which influence dry-ing technology. Shade drying represents the most traditional and more commonly used method to preserve the colour, quality and activity response of the major compounds of essential oils as compared to other drying methods. Dried herbs have been good sources of essential micronutrients, the main ingredient of the dishes and a small amount is responsible for colour, sweet aroma and tangy flavors [2]. Dried herbs become more valuable worldwide with beneficial health impacts. Therefore, different drying methods need to be part of the research.
Zanthoxylum armatum DC, is one of the most famous winged prickly aromatic shrubs belonging to the family Rutaceae, which comprises about 150 genera distributed worldwide [3]. It is native of India, China, Korea and Japan, mostly grow in wastelands, mountains, valleys, forests at an altitude range of approximately 1000-2500m. This wild aromatic spiny shrub is often locally known as Tejphal, Timur, Tejowati, Nepal pepper and Toothache tree [4]. Zanthoxylum armatum leaves, fruits, bark, seeds and young sticks are well known for their therapeutic potential as a medicinal component and widely used for carminative, stomachache, rheumatism, toothache, antipyretic, sudorific and anthelmintic properties [5]. Earlier studies show that the complete plant parts, i.e., bark, seeds, leaves, root and fruits, are a great source of alkaloids, steroids, flavonoids, amides, lignans, terpenes, carbohydrates, proteins and essential oils [6]. The fruits and young sticks of Z. armatum are the main components of traditionally used herbal toothpaste and to keep gum strengthen and hygienic. The barks, as well as fruits, are also useful in various types of skin disease, wounds, eye and ear disease, cough, fever, etc. Dried ground leaves and seeds of Zanthoxylum species are used in spice, soup and flavor products [4,7]. The chemical composition of the essential oil of the same species might be different due to different environmental, seasonal, geographical distribution (i.e., different longitude and latitude), different physicochemical properties of soils, different time of harvesting and different plant parts at different stages.
There are several reports on antimalarial [8,9], essential oil composition and anti-inflammatory [10], anthelmintic [11], antifungal, antibacterial, antimicrobial [12 -14], anticancer [15,16] and antioxidant free radical scavenging [17] activities of extract and essential oil of Z. armatum. Variation in the essential oil composition of different drying methods on some aromatic plants of family Rutaceae has been reported on Murraya koenigii L [18,19]. from north India, Citrus reticulata (Kinnow), C. sinensis (Mussami) and C. paradisii (Grapefruit) from Pakistan [20]. To the best of our knowledge, no earlier reports on the drying effect of the leaves oil of Z. armatum have been found. Therefore, this study was undertaken to investigate the effect of drying on Z. armatum leaves essential oil. For the effective production of essential oil, knowledge of the suitable post-harvest technique is needed for high-quality products. This study reflects on the potential of a shade drying approach on Z. armatum.

Collection and Identification of Z. armatum
Fresh leaves of Zanthoxylum armatum were collected from the wild field of Lohaghat (Latitude 29.404178; Longitude 80.0943366; Altitude 5869 ft), Champawat district, Uttarakhand in the month of September 2018. A fraction of plant material was shade dried (20±5°C) until a constant weight was obtained. The identification of the plant was done at Botany Department, D. S. B. Campus, Nainital (Acc. No. AR01).

Extraction of Essential Oil
Fresh and shade dried plant materials were sliced into small parts and 500 g and 200 g of fresh (ZFL) and dried (ZDL) samples were extracted by using the hydrodistillation method in a Clevenger apparatus for 5 hours [18] and 2.5 mL and 0.6 mL oils were obtained, respectively. The oils were dried over anhydrous sodium sulphate and stored in glass vials in Biological Oxygen Demand (BOD) incubator prior to the analysis [19]. All the experiments were performed in three replicates.

Analysis of Extracted Oil
GC conditions are summarized in Table 1.
The GC/MS used was 2010 GC coupled with Shimadzu QP 2010 plus with thermal desorption system TD 20 having Rtx-5 capillary column (30m x 0.25mm with film thickness 0.25µm). The GC-MS was programmed in similar conditions to those of GC. Helium was used as a carrier gas and the injector temperature was 230°C. The injection volume was 0.2μL diluted oil in n-hexane with split mode (split ratio 1:40). MS was taken at 70eV with a mass range of 40-650amu [21].

Identification of the Volatile Components
The identification of the oil components was done on the basis of their Retention Index (RI) which was calculated with respect to n-alkane series (C 9 -C 33 ; Polyscience Corp., Niles IL) under similar GC conditions, MS Library (NIST:NIH version 2.1 and WILEY: 7 th edition), comparison with the existing MS literature data [22] and the relative amount of individual volatile component was calculated on the basis of GC peak area without using any response factor [21].

Statistical Analysis
The mean and standard deviation of triplicate values were calculated using MS-Excel and the analyzed data was presented as mean ± standard deviation (SD). Two-tailed paired t-test was executed to compare mean values of percentage of constituents between fresh and shade dried Z. armatum at a probability level of p<0.01 and p<0.05 using MS-Excel [2].

Essential Oil Yield of Z. armatum
The oil yield of fresh Z. armatum leaves (0.5%; v/w) was higher as compared to the shade dried (0.3%) plant material. The obtained essential oils were found to be pale yellow in colour. According to Weyerstahl et al. (1999), oil yield was acquired 0.5% from the fresh leaves of Z. alatum [23]. Luong et al. (2003) from Vietnam reported 0.52% oil yield of dried Z. alatum leaves [24]. In a report from China, among six air-dried plant materials (leaves and branches) of the genus Zanthoxylum, the highest oil yield (0.53%) was observed for Z. armatum DC [25].
The main components of dried plant material, particularly β-phellandrene and myrcene, are reported to show antimicrobial and antifungal activity [27,28]. During the drying process, the changes in the percentage of various volatile components are probably due to chemical reactions such as the breakdown of glycosylates, esterification, dehydration reactions, or oxidation reactions or may be the consequences of cell rupture [29].

CONCLUSION
In the present study, the essential oil composition of leaves of Z. armatum subjected to natural shade drying was compared with fresh oil using a two-tailed t-test. Finally, it can be concluded that shade drying showed a significant effect on essential oil yield and components. Shade drying decreased the oil content, percentage of α-pinene, (E)-β-ocimene and linalool. Therefore, efforts should be taken to illustrate the proper drying method for a particular plant species to increase the emerging market of drying.

ETHICAL STATEMENT
Fresh leaves of Zanthoxylum armatum were collected from the wild field of Lohaghat (Latitude 29.404178; Longitude 80.0943366; Altitude 5869 ft), Champawat district, Uttarakhand in the month of September 2018.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
Not applicable.

FUNDING
None.

CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.