Antimicrobial Compounds from Leaf Extracts of Jatropha curcas, Psidium guajava, and Andrographis paniculata

The present research was conducted to discover antimicrobial compounds in methanolic leaf extracts of Jatropha curcas and Andrographis paniculata and ethanolic leaf extract of Psidium guajava and the effectiveness against microbes on flower preservative solution of cut Mokara Red orchid flowers was evaluated. The leaves were analyzed using gas chromatography-mass spectrometry. A total of nine, 66, and 29 compounds were identified in J. curcas, P. guajava, and A. paniculata leaf extracts, with five (88.18%), four (34.66%), and three (50.47%) having unique antimicrobial compounds, respectively. The experimental design on vase life was conducted using a completely randomized design with 10 replications. The flower vase life was about 6 days in the solution containing the P. guajava and A. paniculata leaf extracts at 15mg/L. Moreover, solution with leaf extracts of A. paniculata had the lowest bacterial count compared to P. guajava and J. curcas. Thus, these leaf extracts revealed the presence of relevant antimicrobial compounds. The leaf extracts have the potential as a cut flower solution to minimize microbial populations and extend flower vase life. However, the activities of specific antimicrobial compounds and double or triple combination leaf extracts to enhance the effectiveness to extend the vase life need to be tested.


Introduction
A major problem in Mokara Red orchid cut flowers is reduction in water uptake which could be due to the blockage of xylem vessels by microorganisms or air bubbles, thus, causing flower senescence and shortening of vase life [1,2]. Floral senescence is an active process expressed as petal inrolling, fading of colour, and wilting, caused by programmed cell death [3]. Besides, when the stem is cut, air is immediately aspired into all opened xylem conduits. This air will at first be restricted to the opened conduits. Since vase water bacteria cannot move from one xylem vessel to the other and polysaccharides excreted by bacteria move only partially up the stem, the blockage that occurs further up the stem is mainly due to air bubbles in the xylem conduits [4]. The orchids are an extremely diverse group, in terms of species numbers and also with respect to colour, shape, and smell. Although the pollination mechanisms of several species have been studied, deceptive systems still remain to be investigated [5].
Silver nitrate (AgNO 3 ), 8-hydroxyquinoline citrate (8-HQC), sucrose, and citric acid (CA) have been used in vase solutions to extend the longevity of cut Dendrobium Pompadour flowers [6]. The sucrose acts as a food source, while CA (stabilizes pH to 3-4), AgNO 3 , and 8-HQC act as antimicrobial agents preventing the blockage of xylem vessels [7]. Currently, synthetic germicides containing AgNO 3 are no longer used in commercial vase solutions because the silver can pollute the environment and cause damage to human health [8]. Thus, it is important to develop new substances for flower preservative solutions from sources of biological origin as alternative biocides for the floriculture industry.
Extraction is the basic step in the recovery and separation of bioactive antimicrobial compounds from plant resources before component analysis. The analysis and extraction of plant constituents are beneficial for enhancing, upgrading, and controlling the quality of natural product formulations. Plant derived metabolites have been valuable sources of different antimicrobial compounds used in the production of 2 The Scientific World Journal pharmaceuticals. These natural products, with antimicrobial and antibacterial properties, are reasonably harmless to man. These natural products can negate the expensive and insufficient supply of synthetic antimicrobial compounds.
Jatropha curcas is a physic nut belonging to the Euphorbiaceae family. In many subtropical and semiarid regions, it is traditionally used for its medicinal properties. The seeds contain semidry oil, which is useful for medicinal purposes. Leaf extracts of J. curcas had demonstrated insecticidal effects against crop pests such as Helicoverpa armigera and Sitophilus zeamais [9]. Siva et al. [10] had reported antifungal activities of crude medicinal plant extracts of 20 plant species, including Ocimum sanctum, Ricinus communis, and J. curcas. Besides, Rahman et al. [11] had also reported antifungal properties of J. curcas fruit. According to Kalimuthu et al. [12], the fungal pathogen growth of Penicillium sp. was inhibited by J. curcas extracts. These findings indicate that the extracts can control fungal diseases. Yin et al. [13] found that 9hexadecenoic acid, a component present in J. curcas leaf extracts, has antifungal properties.
Andrographis paniculata belongs to the Acanthaceae family and is popularly known as king of bitters in English. It is an annual herbaceous plant which is commonly cultivated in Southern Asia, in China, and in some parts of South East Asia. A. paniculata has antimicrobial [20][21][22] and anti-snakebite activities [23]. Diterpenoids and flavonoids are the main antimicrobial constituents of A. paniculata [24].
The leaves contain alkanes, flavonoids, and terpenoids. The bitter principle in the leaves is due to the presence of andrographolide lactone and kalmeghin. Four lactones, chuanxinlian A (deoxyandrographolide), B (andrographolide), C (neoandrographolide), and D (14-deoxy-11, 12-didehydroandrographolide), were isolated from the leaves [25]. Hence, the leaf extract of A. paniculata is currently being used as a natural biocide due to the antimicrobial compounds.
Currently, there is a growing public concern on the possible risks of using synthetic fungicides on human health and environment [26]. Therefore, there is a need for alternative antimicrobial agents of biological origin that would not have adverse effects on the environment and human health. Information on the effect of extracts of J. curcas, P. guajava, and A. paniculata leaves as a biopreservative agent against microbes in vase solutions of Mokara Red orchid cut flowers is still scarce. Hence, this study was conducted to identify presence of natural antimicrobial compounds in J. curcas, P. guajava, and A. paniculata leaves extracts due to the J. curcas, P. guajava, and A. paniculata leaves extracts prospective as a cut flower preservative solution to reduce microbial populations and extend the flower vase life.

Leaf Extracts and Detection of Antimicrobial Compounds.
Leaves of J. curcas, P. guajava, and A. paniculata were collected from the University Agriculture Park, Universiti Putra Malaysia (UPM), Serdang, Selangor. Insect-and disease-free leaves of J. curcas and P. guajava from nodes seven to eight below the youngest leaves were collected at random from 25 plants, while 17-week-old A. paniculata plants were harvested at 10 cm above the soil surface and the leaves from 50 plants were used for the extraction.
The collected leaves were processed in the Postharvest Laboratory, Department of Crop Science, Faculty of Agriculture, UPM. The leaves were extracted according to the method of Rahman et al. [11], with some modifications. The samples were washed with distilled water and air-dried at ambient temperature (25 ± 2 ∘ C) to remove excess water and then oven-dried (Memmert, ULM 500, Germany) at 45 ∘ C until a constant moisture of 14% was obtained. Then the leaves were ground separately in a grinding mill (POLYMIX System MFC 13 CZ, the Netherlands). Twenty-gram crushed sample was placed into a 500 mL Erlenmeyer flask. J. curcas and A. paniculata crushed samples were soaked in 120 mL methanol solvent, while the P. guajava sample was soaked in 120 mL ethanol. Each flask was covered with a piece of an aluminium foil and then sealed with parafilm tape to prevent contamination and evaporation of the solutions. The suspended solutions were left to stand for 7 days, after which the solutions were filtered (filter paper 90 mm, Toyo Roshi Kaisha, Ltd., Japan) and evaporated under vacuum using a rotary evaporator (Model CA-1310 Eyela, Tokyo Rikakikal Co., Ltd., Japan) to remove the solvent.
Crude leaf extracts of J. curcas, P. guajava, and A. paniculata were analyzed quantitatively using a gas chromatography-mass spectrometry (GC-MS) (GC-17A/GCMS-QP5050A, Shimadzu, Japan) equipped with DB-wax fused silica capillary column (30 m × 0.25 mm i.d., 0.5 m film thickness). The method was based on Hashimoto et al. [27] with some modifications. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The injector and interface temperatures were 250 and 260 ∘ C, respectively, and solvent delay was 4 min. The column temperature was raised from 40 to 240 ∘ C (at 5 ∘ C/min) and finally to 270 ∘ C (at 10 ∘ C/min). Initial oven temperature was 50 ∘ C for 3 min, then increased to 250 ∘ C at a rate of 5 ∘ C/min, and then held at 250 ∘ C for 15 min. The J. curcas and A. paniculata leaf extracts were diluted in methanol (1/100, v/v), while P. guajava leaf extracts were diluted in ethanol (1/100, v/v). Then 1 L of the diluted extract was manually injected into the GC-MS in splitless mode. Electron ionization system with ionization energy of 70 eV and 50-500 m/z scanning range was used for detecting the antimicrobial compounds. Identification of the antimicrobial chemical compounds was based on comparison of retention The Scientific World Journal 3 times and computer matching of the mass spectra with those of the National Institute of Standards and Technology (NIST 08 and NIST 08s) library and by direct comparison with published data. Leaf extract constituents were expressed as percentage peak area normalization. Microbial populations of the preservative solutions were determined according to the method of Rahman et al. [11], with some modifications. For the microbial population determination (bacteria count and fungal growth) only the 15 mg/L preservative solutions containing leaf extracts of J. curcas, P. guajava, and A. paniculata, from the vase life study, were used. At the end of the flower vase life, 1 mL of preservative solution from the centrifuge tube was sampled into a universal bottle (100 mL) containing 9 mL of distilled water. This was used as the vase stock solution for further dilution. Then, 1 mL of the stock solution was added to 9 mL of distilled water. Each step resulted in a further 8-fold and 6-fold change, from the previous diluted preservative solution, for bacterial count and fungal growth determinations, respectively. The samples were mixed and homogenized using a vortex mixer (Model SA-8; STUART; Switzerland), at a speed of 2500 rpm for 2 min.

Determination of
Nutrient agar (NA) and potato dextrose agar (PDA) were prepared according to the manufacturer's instruction, as labelled on the bottle. Fourteen grams of NA and 20 g of PDA were diluted separately in 1 L distilled water and the solutions were boiled for 1 min, autoclaved at 121 ∘ C and 15 psi for 15 min, and cooled. Twenty five mL pre-autoclaved NA and PDA was poured into each 90 mm diameter sterile petri dish. The agars were allowed to solidify at room temperature. Then, 100 L of 8-fold and 6-fold of the final diluted vase solutions was plated onto the NA and PDA petri dishes, respectively. After the plating process, 1 L of the diluted J. curcas, P. guajava, and A. paniculata pure leaf extracts (using the same dilution as the GC-MS injection) was added into the NA and PDA petri dishes. The inoculated petri dishes containing NA and PDA were incubated upside down in the incubator for 2 and 7 days at 28 ± 2 ∘ C, respectively. Bacterial counts were determined by the standard plate counting method (by counting the number of colonies formed after incubation) to get the number of colony forming units/mL (cfu/mL) [28]. The diameter (mm) of fungus growth on PDA medium was measured with a millimeter ruler.

Experimental Design and Statistical Analysis.
The experiments on vase life, pH, and microbial populations were conducted using the completely randomized design with five treatments comprising three leaf extracts (J. curcas, P. guajava, and A. paniculata) and two controls (tap water and 8-HQC), replicated 10 times. Additionally, each stock dilution (in the petri dishes) was repeated five times (five petri dishes/treatment). All the collected data were subjected to an analysis of variance (ANOVA) to define the differences between treatments. The treatment means were compared using Duncan's multiple range test (DMRT) at ≤ 0.05 (SAS Software Version 9.1).

Antimicrobial Compounds in Leaves Extracts.
Nine peaks were detected in the GC-MS chromatograms of J. curcas methanol leaf extracts (Figure 1). There were five antimicrobial compounds that were present in considerable amounts (88.18%), and another four antimicrobial compounds comprised 11.82% (Table 1). A component 9-hexadecenoic acid [13] present in J. curcas leaf extracts has antimicrobial properties. Compounds 10-octadecenoic acid methyl ester [29], 9,12-octadecadienoic acid (Z,Z)-, 9,12-octadecadienoic acid methyl ester [30], and n-hexadecenoic acid [31] have been reported to have antimicrobial activity. The presence of these compounds makes J. curcas leaves a source of bioactive compounds. Other minor compounds like hexadecenoic acid methyl ester [30] have antibacterial properties; both 9-hexadecenoic acid methyl ester (Z)-and octadecanoic acid methyl ester [32] have antioxidant activities, whereas octadecanoic acid [33] has antimicrobial activity. All these compounds are bioactive compounds. The retention times and antimicrobial compounds detected in the methanol extracts of J. curcas leaves are presented in Table 1.
The A. paniculata leaf extracts in methanol showed 29 peaks as identified from the GC-MS chromatogram (Figure 3). There were three major (50.47%) antimicrobial compounds (Table 3). These include 24.64% of hexadecenoic acid methyl ester, 13.65% of 9,12,15-octadecatrienoic acid methyl ester (Z,Z,Z)-, and 12.18% of 9,12-octadecadienoic acid methyl ester. There were nine (38.30%) moderately active antimicrobial compounds: 9.92% of phytol, 9.75% of 10octadecenoic acid methyl ester, 5.46% of n-hexadecenoic, 5.27% of 9-hexadecenoic acid, 3.48% of octadecanoic acid methyl ester, 1.22% of heptadecanoic acid methyl ester, 1.14% of eicosanoic acid methyl ester, 1.04% of 9-hexadecenoic acid methyl ester (Z)-, and 1.02% of squalene. In addition there were 17 minor (11.23%) antimicrobial compounds ( Table 1). The antimicrobial compounds in the A. paniculata leaf extracts contributed to its medicinal activity. The retention times and the antimicrobial compounds present in A. paniculata leaf extracts are presented in Table 3. The final pH of the preservative solution was determined after the end of flower vase life. The pH of tap water at the end of vase life was significantly higher (50%) compared to the final pH of preservative solution treated with 8-HQC (Table 4). The final pH of preservative solutions with tap water was similar to the other treatments except for the pH of preservative solutions containing 15 mg/L P. guajava or A. paniculata leaf extracts. The pH of these preservative solutions containing 15 mg/L P. guajava or A. paniculata leaf extracts was lower (32-34%) than the final pH of tap water. The increase of pH could be due to the increase in OH − group resulting from metabolic activities in the flowers [34]. There was absence of bacteria in the 8-HQC preservative solution and the flower vase life was 8 days ( Table 5). The A. paniculata leaf extract preservative solution had the lowest bacterial count followed by P. guajava and J. curcas, and the flower vase life was about 6 days. The 8-HQC totally inhibited fungal growth in the preservative solution (Table 5). Preservative solution treated with J. curcas had significantly lower fungal growth than preservative solution treated with P. guajava and A. paniculata. However, the effects of these The Scientific World Journal 5  fungi to flower life were found to be less damaging than the effects of bacteria. Commonly, tap water alone is used to preserve cut flowers in a vase, resulting in a shorter vase life compared to using preservative solutions into which sugar has been added as a food source.  The Scientific World Journal

Conclusion
In the present study, the leaf extracts from J. curcas, P. guajava, and A. paniculata were found to possess antimicrobial activities. The antimicrobial activity of P. guajava and A. paniculata leaf extracts appeared to be responsible for The Scientific World Journal 7 extending the cut flower vase life by 2 days. Both the 15 mg leaf extracts/L of P. guajava and A. paniculata were able to control the microbes in the preservative solution (petri dishes), thus extending the flower vase life by reducing the pH of the preservative solution. However, further studies on the specific antimicrobial compound activities need to be tested and, possibly, a double or triple combination of the leaf extracts could enhance each of their potencies. Thus, these leaf extracts could be used as natural biocides to control microbes and extend the cut flower vase life.