Selection of Potential Plants for Saponin Extract Using Supercritical-CO2 Extraction against Golden Apple Snails (Pomacea canaliculata) for Paddy Cultivation

Abstract

Golden apple snail, (Pomacea canaliculata) has become a major constraint to the profitability of rice (Oryza sativa L.) farming by damaging rice seedlings during paddy growth. Chemical compounds such as saponin, alkaloid and flavanoid have been found in pickerel weed (Monochoria vaginalis), tea leaves, neem and their usage caused the mortality of Pomacea canaliculata. The use of Furcraea selloa var. marginata at paddy field is effective, practical, green to environment and low cost for decreasing golden apple snails. In this research, three plants have been selected which are Furcraea selloa var. marginata, Spent Tea Leaves (STL), and Monochoria vaginalis. The result of Monochoria vaginalis exhibited highest yield of 0.82 wt% followed by STL of 0.75 wt% and Furcraea selloa var. marginata of 0.52 wt% respectively at the best parameter ranges using Supercritical-CO₂ extraction. Effectiveness tests of biopesticides were carried out on golden apple snails at lab condition by converting the plant extracts as the additives to biopesticide and were sprayed onto the snails. Application of biopesticide based plant extract on golden apple snails exhibited that Furcraea selloa var. marginata and STL extracts showed higher mortality rate up to 90% compared to Monochoria vaginalis based bio-pesticide.

Keywords

Saponin, Furcraea selloa var. marginata, Compost tea, Monochoria vaginalis, Golden apple snail

Introduction

Golden apple snails (Pomacea canaliculata) were introduced in Asian countries about 30-years-ago for human consumption. But, it has been reported that the golden apple snail is one of the 100 destructive pests in the world by Ricardo San Martin, et al. [1]. The introduction of the water snails to Asia became a major constraint to the paddy growth activity and yield in paddy cultivation [2]. These snails' mollusks devour young rice seedlings, causing damage to transplant and direct-seed rice [3]. Infestation of golden apple snail on paddy was within 10 to 30 days of paddy age and normally the farmer will make the control for the pest before seedling the seeds into paddy field [4].

Development of food production to meet growing world population and global food demands have led to an increase of chemical inputs in agriculture [2]. For example, to control these snails, synthetic molluscicides chemicals are mainly used such as metaldehyde, copper sulfate, niclosamide. It is also extremely toxic to the environment and non-targeted organisms [1,2]. Ricardo San Martin, et al. reported that molluscicide based on quinoa (Chenopodium quiona) saponins had successfully eliminated 100% of golden apple snails under laboratory conditions at 24 hours with no toxicity effect on fish, such as goldfish or tilapia [1].

Saponins are natural tensoactives present in many plants. There are two main types of saponins which are triterpenic and steroidal saponins [1,5,6]. The use of saponin plants to control aquatic snails that transmit schistosomiasis and fascioliasis is well known and has been investigated for many years [1]. Chemical compounds like saponin, alkaloid and flavanoid have been found in Monochoria vaginalis, tea leaves, neem and their usage caused the mortality of Pomacea canaliculata [7,8]. Besides, Monochoria vaginalis is one of the weeds that grows in paddy field and also distract the paddy growth as it will cover the surface of water thus transferring less oxygen to paddy [9]. According to Joanne L, et al. there are four steroidal saponins that were isolated from the leaves of Furcraea selloa var. marginata which are one furostanol saponin, fucraea furostatin and three known spirostanol saponins, furostatin, yuccaloeside C and cantalasaponin-1 [10]. Besides, the use of Furcraea selloa var. marginata powder at paddy field is effective, practical, green to the environment and is cost effective to reduce the golden apple snails compared to chemical pesticide [11]. Triterpenoid saponins are important bioactive compound in tea and over 60 kinds of triterpenoid saponins have been isolated and characterized in the leaves, seeds, roots and flowers of oolong tea (Camellia sinesis) [12]. In addition, saponin is usually produced by plants as pathogen agents and herbivores as it has immune stimulatory properties, anticancer, antimicrobial, anti-fungal, anti-inflammatory and antiviral activities [13].

Moreover, the Supercritical Fluid Extraction (SFE) is one of the most useful and effective methods for extracting active components from plants because of many advantages of carbon dioxide which are non-toxic, non-flammable and cheap [14].

Thus, this preliminary test was carried out to select the potential plants which are Furcraea selloa var. marginata, STL, and Monochoria vaginalis in order to extract saponin compound using Supercritical-CO₂ extraction as an additive in biopesticide. In addition, the mortality test of Pomacea canaliculata was also performed.

Materials and Methods

Feedstock

Furcraea selloa var. marginata and Monochoria vaginalis were collected at Biosecurity Plant Unit of Agriculture Department of Parit Buntar, Perak, Malaysia. The STL were collected from a restaurant at Sri Iskandar, Perak, Malaysia. All the feedstocks were dried a temperature of 60 ℃ until the moisture content was ± 10%. The feedstocks were then ground using a grinder into selected particle sizes range from 0.15 to 0.35 mm.

Supercritical-CO₂ extraction

Supercritical-CO₂ extractions were performed at different work conditions of pressure, temperature and CO₂ flow rate. The operating procedure was carried out by filling the extraction cell with 5 ± 0.05 g of feedstock. A thermoresistance around the extractor was maintained at the desired temperature. The CO₂ gas was compressed through a high-pressured pump and preheated before flowing through the extraction vessel. The plant extract was collected in the tube covered with aluminum foil to avoid light as the properties of plant extract is light sensitive. The yields of plant extracts were calculated using Equation (1) [14,15]:

$$Yield,Y(wt\%)\text{ = }\frac{massofplantextract}{massoffeedstock}\times 100\%\text{ (1)}$$

Gas Chromatography-Mass Spectrometry (GC-MS) analysis

The plant extracts were dissolved in ethyl acetate and were analyzed by using Agilent Technologies 7890 A GC combined with a 5975 C triple quadrupole MS. The GC system was equipped with a purged tee installed after the analytical column (30 m length × 0.25 mm ID × 0.25 µmdf DB-5MS) in order to perform a back flush of low volatile material, reducing contamination of the analytical column. The oven temperature program was adjusted to 100 ℃ (3 min) - 10 ℃/min to 320 ℃ (25 min). A time-programmed 10 µL injections was performed in solvent vent mode using a Gerstel PTV. The LVI-PTV conditions were as follows: Injection speed was 0.2 µL/s and vent flow was 120 ml/min [16]. The plant extracts were compared with the saponin standards to determine the presence of saponins in the samples.

Efficacy test of biopesticides on the Pomacea canaliculata

The golden apple snails were collected from drainage system of paddy field which is located at Parit Buntar, Perak, Malaysia and the snails were kept in containers with food. According to Alexander M. Stuart, the juvenile snail is in the range of 10 to 15 mm and the adult snail is in the range of 20 to 25 mm. For this preliminary test, ten active adult golden apple snails were selected for each test. The 5 cm depth of soil was filled in the containers (20 cm × 20 cm) by adapting paddy cultivation environment [2]. Furcraea selloa var. marginata, STL and Monochoria vaginalis based biopesticides were prepared by diluting the plant extract with purified water with ratio of 1:1000 by volume with 5% of ethyl acetate solution. The conversion of plant extract as the additive in biopesticide is referred to the procedure described by Yuningsih, et al. and Ravindra C Joshi, et al. [3,17]. Four conditions were carried out in which three containers were sprayed with three different types of biopesticides and one container was sprayed with purified water as the control until all the golden apple snails immersed in the solutions of about 2 inches water level; which adopted the standard technique following the standard pest control in paddy cultivation [4]. The purified water was used as the control as there was a possibility that the water could also cause the mortality of the snail when the environment is different than paddy field. Thus, to avoid the error in this study, the water was used as control as the biopesticides were produced by diluting the plant extracts in purified water. The test was also replicated with 3 replications at each condition to increase the accuracy. The mortality rate was recorded for 12, 24, 36 and 48 hours after bio-pesticides been applied.

Results and Discussion

Yield of essential oil

The supercritical-CO₂ extractions were carried out to determine the best range of particle size, pressure, temperature, and CO₂ flow rate to have higher yields which adopted the parameter range of Supercritical-CO₂ extraction described by Kamarulzaman PSD, et al. as the guideline [18]. This study conducted extraction process using several range of particle sizes which are 0.15 to 0.19 mm, 0.2 to 0.29 mm and 0.3 to 0.35 mm; several range of pressures which are 20 to 25 MPA and 26 MPA to 30 MPA; several range of temperatures which are 40 to 49 ℃ and 50 to 60 ℃ and several range of CO₂ flow rates which are 3 to 4 ml/min and 5 to ml/min as screening data for further Research Surface Methodology (RSM) application in order to find the optimum parameters for these extractions. Table 1 described the best range of the parameters for the Supercritical-CO₂ extraction of plant extracts using selected plant which showed the highest yield compared to other range. However, the range of particle size and CO₂ flow rate showed no difference in yield compared to other parameters.

Table 1 showed Monochoria vaginalis extract produced highest yield of plant extracts of 0.82 wt% followed by STL of 0.75 wt% and Furcraea selloa var. marginata of 0.52 wt% respectively using the best parameters range of Supercritical-CO₂ extraction. The purpose of RSM measurement is to evaluate the effect of several factors and their interaction on one or more response variables [14]. By using this method, it will reduce the experimental runs and provides statistically acceptable optimum parameters to produce higher yield for these extractions. In addition, the application of supercritical-CO₂ extraction technology to the processing of polar solutes such as saponin compound is limited by low solvent power of the extracts for these solutes [19] which can be improved by using water or ethanol as cosolvent to increase supercritical-CO₂ polarity [13]. Besides, Mamata also said that for the extraction of a certain class of products, a cosolvent or an entrainer is often injected into supercritical-CO₂ to increase its polarity and hence it's solvent power. Ethanol, ethyl acetate and possibly water are the best natural entrainers for food-grade products [20], are less harmful to the environment which are also better solvents to produce biopesticide. According to Raphaela, et al., the extracts from the Supercritical-CO₂ extraction with ethanol as cosolvent with (70:30, w/w) showed the greatest ability to reduce the surface tension of water and was the best for extraction of less polar saponins in the extract [13]. This proven that a careful choice of cosolvent could be used to separate the components, not just on the basis of polarity, but also on the basis of functional groups and the ability to have specific interactions [20]. Thus, this research will study in future by selecting the best cosolvent to increase the yield of the plant extracts.

Chemical compound in plant extracts

The study was carried out using saponin standard in order to determine the saponin compound in the plant extracts. The standard was compared to each Furcraea selloa var. marginata extract, STL extract and Monochoria vaginalis extract which were dissolved in ethyl acetate solution. The GC-MS analysis is unable to detect saponin compound directly due to the high polarity of saponin. The analysis showed that most of the peaks of the sample had similar peaks with saponin standard as described in Table 2 and Figure 1. This study will analyze the sample further in future to determine the qualitative and quantitative analysis of saponin in the plant extracts using High Pressure Liquid Chromatography (HPLC) by following the procedure described by Fang Liu, et al. [6].

Table 2 showed the chemical compounds that have same retention time with the saponin standard such as cyclooctasiloxane and hexadecamethyl at the retention time of 11.79 minutes, cyclononasiloxane and octadecamethyl at the retention time of 13.508 minutes and heptasiloxane at the retention time of 20.923 minutes. All the compounds have been used in pesticide and insecticide application [21,22]. Moreover, there are also other compounds in each plant extracts that can be toxic to the insect, pest and bacteria. For examples, Imidazole which was found in Furcraea selloa var. marginata extracted at the retention time of 16.447 minutes with 0.99% of peak area was used not only in chemistry and pharmacology [23], but Imidazole was also used for derivatives process of biological activities such as antibacterial, anticancer, antitubercular, antifungal, analgesic and anti-HIV activities [24]. Benzofuranone was also found in the Furcraea selloa var. marginata extracted at a 29.264 of retention time with 2.15% of peak area. The compound is well known as antioxidant agent [25], to prevent the oxidative degradation of variety materials and improve the stability of polymer in high temperature process [26]. The Benzofuranone also was used in medicine as antinociceptive agents which are the action of blocking the pain detection by sensory neurons [27]. Phytol at retention time of 16.801 minutes with 0.43% of peak area and Beta-amyrin at retention time of 26.265 minutes with 4.94% of peak area which were found in STL extracted was used as antimycobacterial, antifungal and anti-inflammatory [28,29]. Besides, Citronellol was found in the Monochoria vaginalis extracted at a retention time of 14.118 minutes with 0.49% of peak area which was used as active compound in repellent for mice and mosquitoes [30,31]. The compound also was reported having high rate and lost lasting repellency against parasites [32]. Based on the application of the chemical compounds in plant extracts, it is proven that they have high potential to be the effective biopesticides for pests and bacteria.

Efficacy test of biopesticides against Pomacea canaliculata population

Based on three replications of efficacy test of biopesticides against golden apple snail population, biopesticides based plant extracts were successfully increased the mortality rate up to 90% within 48 hours observation as per described in Table 3 and Figure 2. Ravindra C Joshi, et al. reported that the most resistant snails corresponded to quinoa saponin have a size of 15-20 mm [3]. This study used the snails with the size range of 20 to 25 mm and the result showed that the plant extracts were successful in causing the mortality of the snails. Thus, Furcraea selloa var. marginata and STL extracts might have higher concentration of saponin compared to quinoa saponin Although Furcraea selloa var. marginata and STL extracts produced lower yields compared to the Monochoria vaginalis extract from Supercritical-CO₂ extraction, they managed to have a higher mortality rate of Pomacea canaliculata population, which are 90% mortality rate compared to Monochoria vaginalis extract with 70% mortality rate after 48 hours application of biopesticides based on the plant extracts. There is a possibility that Furcraea selloa var. marginata and STL extracts have higher saponin compound than Monochoria vaginalis extract. However, based on GC-MS analysis of plant extracts, there are also other compounds such as essential oil which can also cause the mortality of the golden apple snail. However, the analysis failed to detect the saponin compound directly it only showed that most of the peaks in the plant extracts have similar peaks with saponin standard. Therefore, the actual concentration of saponin in the plant extracts using High Pressure Liquid Chromatography (HPLC) will be determined in future. In addition, ethyl acetate solution also influences the increase of the mortality rate of the golden apple snails. Based on the positive outcome, Furcraea selloa var. marginata and STL have been chosen for further study in terms of the optimization process of extraction and formulation of biopesticide based on combination of plant extracts using Supercritical-CO₂ extraction.

Conclusion

This preliminary test was successfully proven that Furcraea selloa var. marginata and STL extracts have higher potential to be the additives in biopesticide to increase the mortality rate of Pomacea canaliculata population in paddy cultivation. Therefore, the study will continue to develop effective biopesticide based saponin by using the combination of both plant extracts as the active compound in the biopesticide. The determination for the optimum parameters using RSM, quantification of saponin using HPLC analysis and the toxicology test of paddy will be carried out further in future.

Acknowledgements

The authors would like to thank all those who have contributed for their joint efforts in conducting this research and also to Universiti Teknologi PETRONAS for supporting this work and Elsevier foundation Green and Sustainable Chemistry Challenge for the grant to undertake the research.

References

1. Ricardo San Martin, Karine Ndjoko, Kurt Hostettmann (2008) Novel molluscicide against Pomacea Calaniculata based on quinoa (Chenopodium quinoa) saponins. Crop Protection 27: 310-319.
2. Alexander M Stuart, Alvaro Nogues Palenzuela, Carmencita C Bernal, et al. (2014) Effects of fertilizer applications on survival and recruitment of the apple snail, Pomacea canaliculata (Lamarck). Crop Protection 64: 78-87.
3. Ravindra C Joshi, Ricardo San Martín, Cesar Saez-Navarrete, et al. (2008) Efficacy of quinoa (Chenopodium quinoa) saponins against golden apple snail (Pomacea canaliculata) in the Philippines under laboratory conditions. Crop Protection 27: 553-557.
4. Ismadi Zakaria (2016) Jabatan Pertanian Malaysia, K.P.d.I.A.T. Perosak Tanaman Padi. (1^st edn), Perpustakaan Negara Malaysia, Malaysia.
5. Wei MC, Yang YC (2015) Kinetic studies for ultrasound-assisted supercritical carbon dioxide extraction of triterpenic acids from healthy tea ingredient Hedyotis diffusa and Hedyotis corymbosa. Separation and Purification Technology 142: 316-325.
6. Liu F, Ma N, He C, et al. (2017) Qualitative and quantitative analysis of the saponins in Panax notoginseng leaves using ultra-performance liquid chromatography coupled with time-of-flight tandem mass spectrometry and high performance liquid chromatography coupled with UV detector. Journal of Ginseng Research.
7. Kardinan A (2005) Pestisida Nabati: Ramuan dan Aplikasi, Cetakan VI. Penebar Swadaya, Jakarta, 87.
8. Kardinan A, Suriati S (2016) Efektivitas Pestisida Nabati Terhadap Serangan Hama Pada Teh (Camellia sinensis L.). Bulai Penelitian Tanaman Rempah dan Obat 23: 148-152.
9. (2008) Jabatan Pertanian Malaysia, K.P.d.I.A.T. Pakej Teknologi Padi. (1^st edn), Perpustakaan Negara Malaysia, Malaysia.
10. Simmons-Boyce JL, Tinto WF, McLean S, et al. (2004) Saponins from Furcraea selloa var. marginata. Fitoterapia 75: 634-638.
11. Liyunan S (2011) New way to eliminate golden apple snails.
12. Shen X, Shi L, Pan H, et al. (2017) Identification of triterpenoid saponins in flowers of four Camellia Sinensis cultivars from Zhejiang province: Differences between cultivars, developmental stages, and tissues. Industrial Crops and Products 95: 140-147.
13. Raphaela G Bitencourt, Carmen L Queiroga, Ilio Montanari Junior, et al. (2014) Fractioned extraction of saponins from Brazilian ginseng by sequential process using supercritical CO₂, ethanol and water. The Journal of Supercritical Fluids 92: 272-281.
14. Dhekra Trabelsi, Abdelkarim Aydi, André Wüst Zibetti, et al. (2016) Supercritical extraction from Citrus aurantium amara peels using CO₂ with ethanol as co-solvent. The Journal of Supercritical Fluids 117: 33-39.
15. M Solana, I Boschiero, S Dall'Acqua, et al. (2014) Extraction of bioactive enriched fractions from Eruca sativa leaves by supercritical CO₂ technology using different co-solvents. The Journal of Supercritical Fluids 94: 245-251.
16. David F, Devos C, Dumont E, et al. (2017) Determination of pesticides in fatty matrices using gel permeation clean-up followed by GC-MS/MS and LC-MS/MS analysis: A comparison of low- and high-pressure gel permeation columns. Talanta 165: 201-210.
17. Yuningsih R, Firmansyah R (2014) Efektivitas Ekstrak Biji Tanaman Kemalakian (Croton tiglium) Terhadap Keong Mas (Pomacea canaliculata) Sebagai Moluskisida Botani Dalam Upaya Pengganti Moluskisida Sintetik. JITV, 19.
18. Kamarulzaman PSD, Yusup S, Mandal PC, et al. (2017) Optimization of supercritical CO₂ extraction of essential oil from neem leaves using response surface methodology. Sci Int (Lahore) 29: 25-29.
19. Sam Savage, G-S Zlem, Mazza Giuseppe (2007) Saponins: Properties, Applications and Processing.
20. Mukhopadhyay M (2000) Natural Extracts Using Supercritical Carbon Dioxide. CRC Press LLC, United States of America.
21. Carsten Lassen, Sonja Hagen Mikkelsen, Jakob Maag, et al. (2005) Siloxanes- Consumption, Toxicity and Alternatives. The Danish Environmental Protection Agency, Denmark.
22. P Gascon-Garrido, Thévenon MF, Mainusch N, et al. (2017) Siloxane-treated and copper-plasma-coated wood: Resistance to the blue stain fungus Aureobasidium pullulans and the termite Reticulitermes flavipes. International Biodeterioration and Biodegradation 120: 84-90.
23. E Lefler, DA Stevens (1984) Inhibition and Killing of Candida albicans In Vitro by Five Imidazoles in Clinical Use. Antimicrob Agents Chemother 25: 450-454.
24. Amita Verma, Sunil Joshi, Deepika Singh (2013) Imidazole: Having Versatile Biological Activities. Journal of Chemistry 2013: 329412.
25. Xin Meng, Zhong Xin, Zhi Cai, et al. (2006) Study on the antioxidant activities of benzofuranones in melt processing of polypropylene. Polymer Degradation and Stability 91: 2888-2893.
26. Xin Meng, Yongrong Li, Zhi Cai, et al. (2007) Study on the hydrogen donating abilities of benzofuranones as chain-breaking antioxidants using laser flash photolysis technique. Polymer Degradation and Stability 92: 184-188.
27. Gonçalves CJ, Pâmela Padaratz, Rogério Corrêa, et al. (2012) Benzofuranones as potential antinociceptive agents: Structure-activity relationships. Eur J Med Chem 56: 120-126.
28. MS Rajab, SG Franzblau, NH Fischer, et al. (1998) Antimycobacterial activity of (E)-phytol and derivatives: A preliminary structure activity study. Planta Medica 64: 2-4.
29. Okoye NN, Ajaghaku DL, Okeke HN, et al. (2014) Beta-Amyrin and Alpha-Amyrin acetate isolated from the stem bark of Alstonia bourei display profound anti-inflammatory activity. Pharm Biol 52: 1478-1486.
30. Tabari MA, Youssefi MR, Esfandiari A, et al. (2017) Toxicity of β-Citronellol, geraniol and linalool from Pelargonium roseum essential oil against the West Nile and filariasis vector Culex pipiens (Diptera: Culicidae). Res Vet Sci 114: 36-40.
31. Brito RG, Dos Santos PL, Quintans JS, et al. (2015) Citronellol, a natural acyclic monoterpene, attenuates mechanical hyperalgesia response in mice: Evidence of the spinal cord lamina I inhibition. Chem Biol Interact 239: 111-117.
32. Ferreiraa LL, Oliveira Filho JG, Mascarin GM, et al. (2017) In vitro repellency of DEET and β-citronellol against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma sculptum. Vet Parasitol 239: 42-45.
33. Arosha Loku Umagiliyagea, Nathalie Becerra-Mora, Punit Kohli, et al. (2017) Antimicrobial efficacy of liposomes containing D-limonene and its effect on the storage life of blueberries. Postharvest Biology and Technology 128: 130-137.
34. Mario Cesar Jucoski Bier, Adriane Bianchi PM, Carlos Ricardo Soccol (2017) Biotransformation of limonene by an endophytic fungus using synthetic and orange residue-based media. Fungal Biology 121: 137-144.
35. Zahi MR, EI Hattab M, Liang H, et al. (2017) Enhancing the antimicrobial activity of D-limonene nanoemulsion with the inclusion of e-polylysine. Food Chem 221: 18-23.