A study of essential oil from an invasive Piper aduncum L

An invasive Piper aduncum is a plant species that potentially produces essential oil. The study aims to determine the percentage, phytochemical compounds, and anti-fungal properties of essential oil produced from the leaves and fruits of Piper aduncum. The study showed that the leaves and fruits of P. aduncum contain 0.30% and 0.33% of essential oil, respectively. Apiol is the most abundant phytochemical compound obtained in essential oil of leaves and fruits of P. aduncum with 57.10% and 66.31%, respectively. We conclude that essential oil obtained in this study is able to inhibit the growth of both Aspergillus niger and Cladosporium sp. but not for Fusarium oxysporum and Fusarium solani.


INTRODUCTION
Piper aduncum is firstly introduced to Indonesia as an ornamental plant in 1860, and now has been widely distributed throughout Indonesia (Hartemink, 2010;Tjitrosoedirdjo et al., 2016). The species is neotropic plant that grows to an altitude of 2,000 meters and generally in open and disturbed areas, such as secondary forest (Jan et al., 2002;Tjitrosoedirdjo et al., 2016), for example in Borneo land clearing for road construction is reported to have contributed to the spread of this species (Padmanaba and Sheil, 2014). Despite being an invasive plant, in Papua New Guinea this species is used for raw material of farming equipment, pesticides, fertilizers, construction of houses, and windbreaks (Siges et al., 2005).
Piper aduncum contains an essential oil which is reportedly able to prevent bites of the Aedes albopictus mosquito (Misni et al., 2009). In addition to being a mosquito repellent, the essential oil is widely used in the drug industry, as food flavor, in cosmetics and fragrances (Rizal et al., 2009). Essential oils are also known to inhibit fungal growth. Essential oil from lemons is known to inhibit the growth of Aspergillus parasiticum fungus, A. carbonarius, Cladosporium cladosporioides, Eurotium herbariorum and Penicillium chrysogenum (Dimic et al., 2014). Essential oil from Thymus vulgaris has also been known to inhibit the growth of the fungus Rhizopus oryzae (de Lira Mota et al., 2012).
Efforts to control Invasive Alien Plants (IAPs) are constrained by costs. However, some invasive plant species are considered having an economic value that can be created as part of control costs (Stafford & Blignaut, 2017). This study aims to determine the yield, phytochemical content and potential anti-fungal properties of essential oil obtained from the leaves and fruits of an invasive Piper aduncum. Also, this present study can hopefully provide a preliminary information to anyone who interested on the topic.

Study area
All the processes and antifungal testing of essential oil of P. aduncum were conducted at Laboratory of Applied Botany of Bali Botanical Garden, while sequencing of phytochemical was carried out at the Development Unit for Clean Technology -LIPI. The sample materials were the leaves and fruits of P. Aduncum obtained from Bali Botanical Gardens with registered number XV.B.229 ( Figure 1). We selected leaves which are not too young and not too old, while the fruits were mature (but not ripen yet). We considered picking period for sample materials to yield an optimum amount of essential oil.

Extraction of Essential Oil
A hundred grams of fresh leaves and fruits of P. aduncum were distilled using Schott Duran steam distiller. Heating was about five hours at temperature of 100°C. The samples were three times replication to reduce the data errors. The essential oil was separated with the water using Schott Duran separator funnel (Handa, 2008). The separated oil was then analysed using GC-MS. The percentage of essential oil was calculated using the following formula:

Analysis of Chemical Components
The essential oil samples were then analysed using Shimadzu GC-MS QP 2010. About 1 μL of the sample was injected into Rtx-5MS column (60mx0.25mm) with a film thickness of 0.25μm, the pre-programmed oven temperature was 50 -280°C with a temperature rise rate of 5°C/minute. The carrier gas used was helium with a pressure of 101kPa, a total flow rate of 46.5 mL/minute, a column flow rate of 0.85 mL/minute, cleaning flow rate of 3.0 mL/minute, and a split ratio of 1:50.

Antifungal Assay
The essential oil samples were tested for antifungal properties against several species of fungi, such as Aspergillus niger, Cladosporium sp. Fusarium solani and F. oxysporum using agar diffusion method. The method was carried out by laying moistened filter paper with essential oil on the media that had been planted with the fungus with three replications. Clear area formation was observed for three days. The formation of clear area on the surface indicated the presence of fungal death (Pratiwi, 2008).

Extraction of Essential Oil
The distillation of 100 gr of P. aduncum fruits produced 0.33 ml of essential oil, which is equivalent to 0.33% of the gross weight of the fruits, while 100 gr of P. aduncum leaves produced an average of 0.30 ml of essential oil or 0.30% of the gross weight of the leaves (Table 1).

Antifungal assay
Inhibition test on fungi showed an inhibition zone on the inoculated medium of Aspergillus niger and Cladosporium sp., but not on the inoculated medium of Fusarium oxysporum and F. Solani (Table 3). The formation of inhibition zones ( Figure  2) on the inoculated medium of A. niger and Cladosporium sp. showed that essential oil of P. aduncum inhibits the fungus growth.

DISCUSSION
Apiol and Myristicin found in the essential oil of P. aduncum fruits and leaves are toxic to insects (Lichtenstein & Casida, 1963;Lichtenstein et al., 1974;Fuhremann and Lichtenstein, 1979;Bernard et al., 1995;Walia et al., 2004), thus the presence of these compounds in high concentrations promotes an opportunity to use P. aduncum essential oil as a natural insect repellent. In addition to Apiol, in a large percentage, the essential oil of P. aduncum leaves also contains caryophyllene. This compound is often found in essential oils of various species of Piperaceae family member, including P. cernuum and P. regnellii (Costantin et al., 2001;da Silva et al., 2017). Some dominant compounds obtained in the fruits are also found in the leaves with different concentrations. The ratio of such compounds can be seen in Figure 3. This present study showed that the leaves and fruits of P. aduncum are both dominated by terpenoids, these compounds are known having many biological activities, such as antimicrobial, antifungal and antibacterial (Cavaleiro, 2015).
The inhibitory ability is likely to correlate with the presence of beta-Ocimene compounds. Cavaleiro et al. (2015) reported beta-Ocimene is a compound with antifungal properties. The compund is also reported having anti-inflammatory and antiviral properties (Terpene, 2018). On the contrary, ineffectiveness in inhibiting the growth of F. oxysporum and F. solani was probably because the nature of active ingredients has no wide spectrum, it means the essential oil only inhibits the growth of some fungus species (Neu and Gootz, 2001).
Previous studies of P. aduncum extraction showed various yields of essential oil. For example, leaves and branches samples of P. aduncum from eight different regions of the Amazon produce essential oil ranging from 1.2% to 3.3% (Maia et al., 1998). Piper aduncum leaves, native to Papua New Guinea, yields essential oil of 0.35%, while P. aduncum leaves from Cuba yields essential oil of 0.96% (Pino et al., 2004;Rali et al., 2007). The difference in yields of essential oil is probably due to different soil macronutrients in each region. Kahkashan et al. (2015) stated that soil types affected the amount of essential oil produced by Mentha arvensis L. Suryawati & Murniyanto (2011) showed that essential oil produced by Zingiber officinale L. is directly proportional to the availability of macronutrients in the soil. A lack of available groundwater can also result in changing secondary metabolites of plants than in normal conditions (Trisilawati & Pitono, 2012).

CONCLUSION
The present study concluded that essential oil of the leaves and fruits of P. aduncum contained Apiol compounds in high concentrations. The essential oil has been confirmed to inhibit the growth of Aspergillus niger and Cladosporium sp. The study promoted possibilities that essential oil of P. aduncum could be developed into industrial production of fungicides and insecticides. However, natural products should respect to cultivation, collection, and used in sustainable manner which conserves both of the plants materials and the environment.