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Rajamangala University of Technology Thanyaburi (RMUTT) Sanitary Treatments Improving Safety of Wild- Caught and Farm-Raised Catfish Chananthida Thaplee Prueksa Sawardsuk Faculty of Science and Technology Faculty of Engineering Rajamangala University of Technology Thanyaburi Rajamangala University of Technology Thanyaburi Pathum Thani, Thailand. Pathum Thani, Thailand. [email protected] [email protected] Malene E. Janes Prapaporn Pongthai School of Nutrition and Food Science Faculty of Science and Technology Louisiana State University and Agricultural Center Rajamangala University of Technology Thanyaburi Louisiana, USA Pathum Thani, Thailand. [email protected] [email protected] Kittikoon Torpol* Faculty of Science and Technology Rajamangala University of Technology Thanyaburi Pathum Thani, Thailand [email protected]* Abstract— Reduction in the microbial content of raw I. INTRODUCTION catfish fillets could extend its shelf life, increase the food safety, and improve quality of the fillets. In this study, Catfish are one of the most widely cultured fish in the Wild-caught (WC) and farm-raised (FR) catfish fillets were world, ranked as top seafood species consumed not only submerged separately into antimicrobial solutions, in Asia but also Europe and United State [1]. Catfish are including calcium lactate (CL), acidified sodium chloride generally sold as live catfish, frozen steaks, and frozen (ASC), chlorinated water (CW), lactic acid (LA), and fillets. Nowadays, catfish have been gained more sodium benzoate (SB) in one-L ice water baths for 5 customer attention, as they are a protein source, having minutes. The untreated fillets and fillets submerged in moderately fats and containing high quality of proteins. sterilized water were used as fillet control and control Consequently, catfish product manufactures have been solution (H2O), respectively. The catfish fillets were continuously growing around the world. However, these analyzed for total aerobic plate counts (APC), coliform, manufactures are usually faced with a potential risk of Staphylococcus aureus, and Enterobactericeae. This study microbial hazards. As well-known, fish products are very found that the number of APC, coliform, perishable. It was reported that unpacked catfish kept at 4 Enterobactericeae, and S. aureus counts were significantly ºC for 6 days had the increase of total aerobic plate count lower than the fillet control when soaked in an ice water (APC) from 4.08 log CFU/g to 6.59 log CFU/g, and bath that contained antimicrobial solutions. The higher APC were found at 8.76 log CFU/g when the antimicrobial activity of each treatment was different catfish was packed in low-density polyethylene [2]. depended on types of bacteria and catfish sources. This Similarly, [3] reported that high APC were detected in study indicates that the selected antimicrobial substances both whole catfish (6.92 log CFU/g) and catfish fillets could help reduce the number of bacteria that have a (7.41 log CFU/g), indicating the possibility of a high rate tendency to contaminate or spoil catfish fillets, contributing of spoilage, and thus a shorter shelf life of chilled catfish to the industrial catfish production and improving the products. According to International Commission on safety and quality of catfish fillet products. Microbiological Specifications for Foods (ICMSF), good quality fresh fish should have APC less than 5 log CFU/g, Keywords— Catfish, Antimicrobial Substances, Shelf while the maximum limit of APC for acceptable fresh fish Life, Handling, Lndusty is 7 log CFU/g. These suggest that catfish have a tendency to easily get microbial spoilage. The typical spoilage microorganisms found in the catfish are Gram negative bacteria such as Aeromonas sp., Pseudomonas sp., and S. putrefaciens, causing unacceptable off-flavors The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 30

Rajamangala University of Technology Thanyaburi (RMUTT) and off-odor compounds such methyl mercaptan, II. MATERIALS AND METHODS dimethyl disulfide, dimethyl trisulfide, 3-methyl-1- butanal, trimethylamine, and ethyl esters of acetate, A. Catfish Preparation butyrate and hexanoate [4]. In addition, foodborne pathogenic bacteria have been also detected in the catfish. Catfish fillets were obtained from a local commercial Generally pathogenic bacteria in catfish can be classified harvester and a supermarket in Louisiana, called as wild- to three groups, including indigenous bacteria, the natural caught (WC) and farm-raised (FR) catfish fillets. The micro-flora of the fish, such as Enterobaceriacea, samples were stored at 4 °C and used within 24 h. Fillets Clostridium botulinum, and pathogenic Vibrio spp., were aseptically cut into 25 g pieces on a sanitized plastic enteric bacteria, non-indigenous bacteria contaminated cutting board. They were then placed into sanitized from fecal contamination (Salmonella spp., pathogenic plastic buckets. The study was performed 3 times on Escherichia coli, Staphylococcus aureus), and different days for three days. In each batch, catfish fillets contaminated bacteria during processing, mainly were newly bought to ensure that the catfish fillets were Salmonella spp., Listeria monocytogenes, fresh before testing. Staphylococcus aureus, Escherichia coli, and Vibrio spp.[5]. Environment where fish are harvested, improper B. Treatments of Catfish Fillets by Antimicrobial storage after harvesting, cross-contamination during Substances processing and fluctuation of temperatures during transportation could lead to the fish contamination, The following antimicrobial substances were used in causing food safety risk associated with consuming this experiment, including solutions of calcium lactate catfish. (CL) (200 ppm), lactic acid (LA) (200 ppm), chlorinated water (CW) (150 ppm), acidified sodium chlorite (ASC) Nowadays, food safety is become a global significant (50 ppm), and sodium benzoate (SB) (200 ppm). health concern. Consumers demand high quality and Deionized and sterilized water (H2O) was used as a safety products. This encourages fish manufactures to control solution. All solutions were measured for pH. pay more attention in fish preservation and prevention Twenty five grams of catfish fillets were placed into from microbial contaminations in order to avoid profit buckets containing 900 g of sterilized ice. One hundred losses from fish discards due to low quality and unsafe mL.of each treatment and control solution was then products. As mentioned earlier, catfish could be a harbor poured over the fillets in the buckets. The fillets were causing microbial foodborne illness. Bacteria from soaked for 5 min. They were subsequently taken out and contaminated surfaces or spoilage bacteria could transfer placed in sterile bags for microbial analysis. The catfish to the fish as soon as the fish was death [6]. In order to fillet control was only placed into a bucket with ice. reduce the risk of contamination, proper handling methods before and after fish processing are required so C. Microbiological Analysis that the quality and safety of the fish products could be maintained. Traditionally in the industry catfish fillets are After soaking the catfish fillets into different processed in a cool room with chilled water and stored in treatments, the samples were placed into sterile ice baths to prevent the growth of spoilage and stomacher bags, and 225 ml of phosphate buffered saline pathogenic microorganism during processing. However, (PBS) were added to each bag. The samples were blended it was reported that certain psychrotrophic pathogens for 1 minute in a stomacher. All samples were serial such as Listeria monocytogenes and Aeromonas diluted in phosphate buffer saline solution then plated on hydrophila could survive and grow at refrigerated to 3M™ Petrifilm™. Three WC and FR catfish pieces per temperatures [7]. This indicated that the use of only treatment per analysis day were tested for aerobic bacteria refrigeration techniques would not be enough to prevent (3M™ Petrifilm™ Aerobic Count Plate media), E. coli the catfish from bacterial hazards. Antimicrobial and Coliform (3M™ Petrifilm™ -E. coli/Coliform count substances have been reported to be able to reduce Plates media), Enterobaceriacea (3M™ Petrifilm™ pathogenic or spoilage bacteria in catfish fillets, disinfect Enterobacteriaceae plate count media) and S. aureus and kill bacteria, or mitigate the growth of (3M™ Petrifilm™ Staph Express Count media). microorganisms [8-10]. Application of antimicrobial substances into chilled water or ice bath could help D. Statistical Analysis prevent contamination or degeneration caused by bacteria after harvesting and during processing [11]. Therefore, The data of this experiment was analyzed using a the objective of this study was to determine the effects of oneway analysis of variance by the procedure of general antimicrobial substances on reducing spoilage and linear model using SPSS program. The whole pathogenic bacteria on catfish fillets, which could be used experimental procedures were triplicated. The in the catfish fillet manufacturers to improve handling differences among the mean values were compared using practices. Duncan’s multiple range test at a confidence level of p<0.05. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 31

Rajamangala University of Technology Thanyaburi (RMUTT) III. RESULTS AND DISCUSSION Calcium lactate is one of the organic acids commonly Regarding to WC catfish fillets, the result showed that used in the meat and vegetable industry to control growth only some antimicrobial solutions could reduce aerobic of aerobic and anaerobic bacterial contamination and bacteria in the fillets. CL had lower aerobic bacteria prolong their shelf life. It was found that calcium lactate counts than other treatments which were 3.12 log CFU/g. could inhibit bacteria such as Bacillus subtilis, The number of aerobic bacteria was decreased for 1.64 Lactobacillus sp. and Sarcina sp. but not yeast and molds log reduction, which was 65.55% reduction compared to [12]. This indicated that effects of calcium lactate on the fillet control (4.76 log CFU/g). CW and SB could microorganisms were different, depended on group and reduce the number of bacteria, which were 4.36 and 4.42 species of the microorganisms. [13] reported that calcium log CFU/g but less effective than H2O (4.15 log CFU/g). lactate could enhance the efficiency of low concentration However, they were not significantly different. ASC and electrolyzed water in reducing total viable cells counts in LA were not effective to reduce aerobic bacteria in the fresh pork carcass. It has been also well-known that fish samples. The aerobic plate counts were 4.79 and 4.91 chlorine compounds have wide-broad spectrum of log CFU/g, respectively (Figure 1). antimicrobial activities. They could inhibit bacteria, fungi, spores, and viruses. The mechanisms of inhibitions Figure 1. Total Aerobic plate counts of wild-caught catfish fillet could be divided into four ways, including 1) oxidization treated with antimicrobial substances including control solution of sulfhydryl (-SH) groups of enzymes and structural (H2O), calcium lactate (CL), acidified sodium chloride (ASC), proteins 2) cell membrane damages, 3) protein synthesis chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB); disruption, and 4) react to nucleic acids hence interfering Control1 means the untreated fillets. with total cell metabolism [14]. Regarding to FR catfish fillets, it was found that only Coliform could be significantly reduced by CW had the lower number of total aerobic bacteria cells submerging the WC fillets in all antimicrobial and control than the fillet control, which were 3.48 and 5.07 log solutions. The number of coliform after 5-min treating CFU/g respectively. CW could decrease the bacteria with CW, W, LA, ASC, SB, and CL were 1.18, 1.26, counts for 1.59 log reduction, which was undergone for 1.46, 1.83, 2.17, and 2.40 log CFU/g, respectively, while 68.64% (Figure 2). It was noticed that the results of FR the fillet control had 2.78 log CFU of coliform/g (Figure catfish fillets were different from the WC samples. These 3). The result demonstrated that CW significantly could be because not only types of bacteria were different provided better ability to decrease coliform when but also depended on environments and processing compared to other treatments except H2O and LA. The procedures. coliform in CW was reduced for 1.60 log CFU/g or 42.45% reduction. Similar to the WC samples, coliform could be significantly declined by treated with all antimicrobial solution as well as water or the control solution. CL had significantly lower coliform counts compared to other treatments, which was 0.78 log CFU/g. However, it was not significant with W treated FR catfish fillets, which had 0.78 log CFU of coliform/g. The percentage of coliform reduction of CL and H2O were 48.45% and 45.34% respectively. CW, LA, SB, and ASC had the number of coliform at 0.96, 1.12, 1.25, and 1.33 log CFU/g, respectively, while the coliform were found in the fillet control at 1.61 log CFU/g (Figure 4). Chlorinated water has been tentatively used as a food sanitizer for more than a decade. Although there are many researchers reported that the chlorinated water is effective in reducing the number of coliform in meat, fish, fruits, and vegetables, it was revealed that chlorine-washing systems may produce harmful by-products such as chloramines and trihalomethanes [15]. Figure 2. Total Aerobic plate counts of farm-raised catfish fillet treated with antimicrobial substances including control solution (H2O), calcium lactate (CL), acidified sodium chloride (ASC), chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB); Control1 means the untreated fillets. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 32

Rajamangala University of Technology Thanyaburi (RMUTT) Figure 3. Total coliform of wild-caught catfish fillet treated with Figure 5. Total Staphylococcus aureus of wild-caught catfish fillet antimicrobial substances including control solution (H2O), calcium treated with antimicrobial substances including control solution lactate (CL), acidified sodium chloride (ASC), chlorinated water (H2O), calcium lactate (CL), acidified sodium chloride (ASC), (CW), lactic acid (LA), and sodium benzoate (SB); Control1 means chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB); the untreated fillets. Control1 means the untreated fillets. Figure 4. Total coliform of farm-raised catfish fillet treated with Figure 6. Total Staphylococcus aureus of farm-raised catfish fillet antimicrobial substances including control solution (H2O), calcium treated with antimicrobial substances including control solution lactate (CL), acidified sodium chloride (ASC), chlorinated water (H2O), calcium lactate (CL), acidified sodium chloride (ASC), (CW), lactic acid (LA), and sodium benzoate (SB); Control1 means chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB); the untreated fillets. Control fillet mean the untreated fillets. S. aureus were also found in the WC catfish sample Enterobacteriaceae seemed to be more tolerant than (Figure 5). The huge loss of S. aureus could be observed aerobic bacteria, coliform, and S. aureus. Only CL, SB, from the catfish treated with antimicrobial and control and LA could significantly decrease the number of solutions. ASC was significantly more effective in Enterobacteriaceae in WC fillets. CL had higher reduction of S. aureus than the others. The bacteria counts reduction than SB and LA. The cell number was 2.85, were 1.82 log CFU/g after treating the fillet for 5 min, 3.31, and 3.71 log CFU/g, respectively, while the fillet while the fillet control had 4.04 log CFU/g. The reduction control had 3.89 log CFU of Enterobacteriaceae /g. The of S. aureus by ASC was 2.22 log CFU/g, which was reduction of Enterobacteriaceae by CL was undergone for 45.05% compared to the fillet control. However, the other 73.26% (Figure 7). antimicrobial substances, including CL, LA, and SB were significantly less effective in decreasing S. aureus than Figure 7. Total Enterobacteriaceae of wild-caught catfish fillet H2O and CW. The number of S. aureus in the treated FR treated with antimicrobial substances including control solution fillets were 2.27, 2.54, 3.31 log CFU/g for LA, CL, and (H2O), calcium lactate (CL), acidified sodium chloride (ASC), SB, respectively, while H2O and CW had 2.30 and 2.08 chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB); log CFU/g, respectively (Figure 6). ASC, CW, and LA Control1 means the untreated fillets. slightly reduced S. aureus but less than H2O. CL and SB failed to inhibit S. aureus in FR catfish fillets. Acidified sodium chlorite is a solution of sodium chlorite which is reduced the pH to 2.5-3.2 by using GRAS acid. It is highly effective in inactivating pathogenic bacteria such as E. coli O157:H7 and Salmonella spp. [16]. ASC was found to reduce S. aureus in beef briskets [17]. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 33

Rajamangala University of Technology Thanyaburi (RMUTT) Similar to WC catfish fillets, Enterobacteriaceae in REFERENCES FR fillets was less sensitive to the antimicrobial substances. All treatments could reduce the number of [1] Kumar, G., Engle, C.R., Hanson, T.R., Tucker, C.S., Brown, Enterobacteriaceae but was not significantly different T.W., Bott, L.B., Roy, L.A., Boyd, C.E., Recsetar, M.S., Park, J. compared with the fillet control (Figure 8). and Torrans, E.L. Economics of alternative catfish production technologies. Journal of the World Aquaculture Society, 49(6), Figure 8. Total Enterobacteriaceae of farm-raised catfish fillet 2018, pp.1039-1057. treated with antimicrobial substances including control solution (H2O), calcium lactate (CL), acidified sodium chloride (ASC), [2] Nguyen, T.T., Adhikari, A., Bhattacharya, D., Chhetri, V.S. and chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB); Kharel, K. Microbial Food Safety Risks Associated with Fresh Control1 means the untreated fillets. and Thawed Catfish Fillets during Refrigerated Storage. Food and Nutrition, 9, 2018, pp.1261-1272. IV. CONCLUSION Treatments for improving the microbiological safety [3] Ramos, M., and W. J. Lyon. \"Reduction of endogenous bacteria of Wild-caught (WC) and farm-raised (FR) catfish fillets associated with catfish fillets using the Grovac process.\" Journal were studied. Calcium lactate (CL), acidified sodium of food protection 63 (9). 2000, pp. 1231-1239. chloride (ASC), chlorinated water (CW), lactic acid (LA), and sodium benzoate (SB) were used. The study indicated [4] Gram, L. and Huss, H.H., Microbiological spoilage of fish and that different antimicrobial substances yielded different fish products. International journal of food microbiology, 33(1), results in reducing the number of bacteria that could 1996, pp.121-137. contaminate in the catfish fillets. Moreover, the activity of the antimicrobial substances also depends on the types [5] Lyhs, U. “Microbiological Methods”, In Rehbein, H. and of the catfish as well. The study indicated that the Oehlenschläger, J. (ed.), Fishery Products Quality, safety and potential contaminated bacteria in the catfish could be authenticity. 2009, pp. 318-48Wiley Blackwell. reduced by the selected antimicrobial substances. However, due to the different ability of the antimicrobial [6] Wekell, M. M., Manger, R., Kolburn, K., Adams, A and Hill, W substances, it could be suggested that the effects of “Microbiological quality of Seafoods: viruses, bacteria and mixture of the antimicrobial substances on the bacteria parasites”. In Shahidi F., and Botta R. J (Eds). Seafoods should be examined. Chemistry, Processing Technology and Quality, 1994, pp 220- 232. Glasgow: Chapman & Hall. ACKNOWLEDGMENT The authors wish to thank Rajamangala University of [7] Fernandes, C.F., Flick, G.J. and Thomas, T.B., Growth of Technology Thanyaburi for granting some expenses inoculated psychrotrophic pathogens on refrigerated fillets of during conducting the experiment at Louisiana State aquacultured rainbow trout and channel catfish. Journal of food University and Ms. Bilan C. Jessie from School of Protection, 61(3), 1998, pp.313-317. Nutrition and Food Science, Louisiana State University for all supports and suggestion. [8] Kim, T.J., Silva, J.L., Chamul, R.S. and Chen, T.C., Influence of ozone, hydrogen peroxide, or salt on microbial profile, TBARS and color of channel catfish fillets. Journal of Food Science, 65(7), 2000, pp.1210-1213. [9] Bal'a, M.F.A. and Marshall, D.L., Organic acid dipping of catfish fillets: effect on color, microbial load, and Listeria monocytogenes. Journal of food protection, 61(11), 1998, pp.1470-1474. [10] Zhuang, R.Y., Huang, Y.W. and Beuchat, L.R., Quality changes during refrigerated storage of packaged shrimp and catfish fillets treated with sodium acetate, sodium lactate or propyl gallate. Journal of Food Science, 61(1), 1996, pp.241-244. [11] Ingham, S. Lactic acid dipping for inhibiting microbial spoilage of refrigerated catfish fillet pieces. Journal of Food Quality 12 (6). 1989, pp. 433-443. [12] Baston,O. and Barna, O. Calcium lactate influence on some non- pathogenic microorganisms. Food and Environment Safety Journal, 12(3). 2013, pp.278-283 [13] Rahman, S.M.E., Wang, J. and Oh, D.H., Synergistic effect of low concentration electrolyzed water and calcium lactate to ensure microbial safety, shelf life and sensory quality of fresh pork. Food Control, 30(1), 2013, pp.176-183. [14] Ray, B. “Control of Microorganisms in Foods”.In Stern R.A, &Kaplan, L (eds.) Fundamental Food Microbiology (3rded.). 2003, pp 439-534. London : CRC. [15] Dychdala, G.R. “Chlorine and chlorine compounds. In Disinfection, Sterilization, and Preservation”, ed. Block, S.S. Philadelphia, PA: Lea and Febiger Publishers. 1991. [16] Gonzalez, R.J., Luo, Y., Ruiz-Cruz, S. and McEVOY, J.L., Efficacy of sanitizers to inactivate Escherichia coli O157: H7 on fresh-cut carrot shreds under simulated process water conditions. Journal of food protection, 67(11), 2004, pp.2375-2380. [17] Hajmeer, M.N., Marsden, J.L., Fung, D.Y.C. and Kemp, G.K., Water, sodium chloride and acidified sodium chlorite effects on Escherichia coli O157: H7 and Staphylococcus aureus on beef briskets. Meat science, 68(2), 2004, pp.277-283. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 34

Rajamangala University of Technology Thanyaburi (RMUTT) The Total Phenolic Compound in Nelumbo nucifera and Product Development of Lotus Seed Chiffon Cake Lerluck Steinrut Ariya Pakoed Faculty of Home Economic Technology, Faculty of Home Economic Technology, Rajamangala University of Technology Thanyaburi, Rajamangala University of Technology Thanyaburi, Pathum Thani, 12110, Thailand Pathum Thani, 12110, Thailand [email protected], [email protected] [email protected] Abstract—Nelumbo nucifera (Lotus) has long been used Traditional medicinal uses: Almost all part of the in Thai traditional medicine for treatment of diabetic and lotus plant are eaten as vegetable and also used in chronic disease patients. The objective of this study was to indigenous system of medicine. Rhizomes and leaves are investigate the total phenolic compound and sensory used with other herbs to treat sunstroke, fever, diarrhea, evaluation of developed chiffon cake from difference level dysentery, dizziness, vomiting of blood, hemorrhoids. of lotus seed. The total phenolic compound in 95% Moreover, the whole plant is used as an antidote to ethanolic was extracted from ten parts used of lotus. (petal, mushroom poisoning. The lotus embryonic seeds were stamen, seed, embryo, ovary, leaf, young leaf, petal stalk, used to treat high fever, cholera (in China), nervous bud and root). The petal extract exhibited the highest total disorders and insomnia; the seeds were used to stop phenolic compound with 143.06 mg/g. followed by extracts vomiting, relieve indigestion and diarrhea or just as a of ovary and petal stalk (116.63 and 91.39 mg/g). The tonic. Petals of lotus flowers were used for syphilis, chiffon cake was varied in 3 levels of lotus seed 45, 90 and cosmetic unguents (in Java), while its flower stalk was 135 grams (10 % 20% and 30%). Sensory evaluation was used with other herbs to treat bleeding from the uterus. conducted by 9-points hedonic scale test using 100 The pods contain alkaloids that stop bleeding. [1] [2] panelists. The sensory evaluation criteria composed of appearance, color, odor, taste, sour, salty and overall liking. Nelumbo nucifera extracts from leaf, root, seed, Analysis of variance was used ANOVA at 95 percent confidence level. Differences analysis was tested by and flower are reported to have significant therapeutic Duncan’s Multiple Rang Test. The results indicated 45 benefits and it possess antidiarrheal activity, grams (10 %) lotus seed chiffon cake have highest sensory psychopharmacological, diuretic activity, antipyretic, score of color (7.54±0.94), overall color (7.72±0.90), odor antimicrobial activities, hypocholesterolemic effect, (7.68±0.81), egg odor (7.67±1.16), taste (7.81±1.07), softness hypoglycemic, alleviation of hepatic steatosis.[3] [4] (7.81±1.11), sweetness (7.70±1.04), and overall liking (7.84±0.97). The study showed chiffon cake with 45 grams Antioxidant activity of various parts of Nelumbo (10 %) lotus seed was accepted in all sensory attributes. The nucifera is well established, e.g. leaf, stamen and future study should be study on other part of lotus which have high total phenolic compound and developed proper rhizome.[5] 6] Nelumbo nucifera Gaertn. (Thai name: health products to promote consumption in daily food. Bua Luang) was long used by Thai traditional doctors for the treatment of diabetic patients. There are many Keywords—Nelumbo nucifera, Lotus, Total Phenolic researches referring that the antioxidant activity herb is Compound, Sensory Evaluation always a good benefit for chronic disease patients because of inhibiting the oxidation reaction by reducing I. INTRODUCTION advanced glycation end products (AGEs). [7] Lotus (Nelumbo Nucifera) is perennial water The Objectives of this study were to investigate the total phenolic compound of ethanolic extract from 10 plant, growing from a tuberous rootstock lying in mud at parts use of Nelumbo nucifera Gaertn.(petal, stamen, the bottom of lakes and ponds. The plant produces large seed, embryo, ovary, leaf, young leaf, petal stalk, bud and leaves which float on the surface of the water. The Sacred root), to evaluate sensory evaluation of chiffon cake Lotus of India has a long history of use as a food crop and fortified with lotus seed and consumer acceptance of medicine in tropical and subtropical Asia, where it is also lotus seed chiffon cake product. seen as a symbol of purity and beauty. The plant is often cultivated for food and medicine. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 35

Rajamangala University of Technology Thanyaburi (RMUTT) II. MATERIAL AND METHOD III. RESULT A. Sample Preparation A. Ethanolic Extraction The plant materials from 10 parts use of Nelumbo The percentage of yield from extractions showed in Nucifera (petal, stamen, seed, embryo, ovary, leaf, Figure 1. The highest percentage yield of ethanolic extract was leaf by 13.82 % followed by petal and embryo young leaf, petal stalk, bud and root) were collected from of by 12.54 and 10.53 % respectively. lotus field in Nakhon pathom province. The plants were dried in well ventilation open air and ground to give the % 6.77 4.8 3.96 powder. By maceration, each 100 gm. of the powder leaf Bud 2.37 in 95 % ethanol was collected every 3 days for 3 times. 16 13.82 The extract was filtered through vacuum pump and 14 12.54 Petal Rhizome Seed evaporated by rotary evaporator then keep in -20 °C stalk before reaction testing. The percentage of 10 sample 12 10.53 9.88 extract(%yield) were calculated. 10 8.08 6.87 8 B. The Total Phenolic Compound 6 The total phenolic content was determined by using the Folin-Ciocalteu assay. An aliquot (1 ml) of extracts or 4 standard solution of Gallic acid (100, 200, 300, 400, and 500µg/ml) were added to 25 ml of volumetric flask, 2 containing 9 ml of distilled water. A reagent blank using distilled water was prepared. 1 ml of Folin-Ciocalteu 0 phenol reagent was added to the mixture and shaken. Leaf Petal Embryo Stamen Young Ovary After 5 minutes 10 ml of 7% Na2CO3 solution was added leaf to the mixture. The volume was then made up to the mark. After incubation for 90 minutes at room temperature, the Figure 1. The percentage yield of the lotus part ethanolic extract absorbance against the reagent blank was determined at 550 nm with an UV-Visible spectrophotometer. Total B. Total Phenolic Content phenolics content was expressed as mg Gallic acid Equivalents (GAE). [8] The ethanolic extract of petal showed the highest total phenolic content with 143.06 mg/g. following by extracts C. Sensory evaluation of ovary and petal stalk (116.63 and 91.39 mg/g. respectively). Acceptance testing was used to determine how much each sample was liked based on a 9-point hedonic scale mg for a set of attributes: color, odor, overall odor, taste, egg odor, softness, sweetness and overall liking where 9=like 160 143.06 extremely and 1=dislike extremely. In addition, panelists were asked what they like and dislike about each sample. 140 Sensory acceptance of chiffon cake was evaluated in 3 116.63 recipe of basic chiffon cake and 3 level of chiffon cake fortified with lotus seed 45, 90 and 135 grams (10%, 20% 120 and 30 %). Analysis of variance was used ANOVA at 95 100 91.39 85.24 79.42 percent confidence level. Differences analysis was tested by Duncan’s Multiple Rang Test. The sensory evaluation 80 was tested by 70 panelists in basic chiffon cake and 100 58.59 panelists in lotus seed chiffon cake. Lotus seed was dried and ground to give the powder before used in chiffon 60 51.08 46.72 cake. 40 D. Consumer Acceptance 19.39 Consumer acceptance of lotus seed chiffon cake 20 11.64 product was studied in 30 consumers. Analysis was used percentage and mean ± SD. 0 Leaf Stamen Young Embryo Rhizome Bud Seed Petal Ovary Petal leaf E. Nutritional Value stalk The nutritional value of lotus seed chiffon cake was Figure 2. Total phenolic content of the ethanolic extract from lotus calculated by using Thai food composition table (100 part used grams). C. Sensory Evaluation Sensory evaluation of selected 3 recipe basic chiffon cake: The results of preference sensory evaluation in 70 panelists indicated there are significant difference between 3 recipe of basic chiffon cake (P value < 0.05). The highest preference was recipe 2 with mean score of color 7.71±0.98, odor 7.84±1.01, overall odor 7.86±1.02, taste 7.63±1.06, egg odor 7.36±1.21, softness 7.71±0.93, sweetness 7.71±0.88 and overall liking 7.84±0.79 respectively. The sensory evaluation showed panelists prefer the recipe 2 most in all sensory parameters. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 36

Rajamangala University of Technology Thanyaburi (RMUTT) TABLE I. SENSORY ACCEPTANCE OF 3 RECIPE BASIC CHIFFON CAKE D. Nutritional Value of Lotus Seed Chiffon Cake Sensory Recipe 1 Chiffon cake Recipe 3 The nutritional value of lotus seed chiffon cake was parameter 5.34± 1.29c Recipe 2 6.84±0.86b showed in Table 4 by total weight of 1 recipe (931.5 5.51± 1.20 c 6.94±0.88b grams) and a small piece of each package (55 gram). The Color 5.64± 1.28 c 7.71± 0.98 a 6.77± 0.92b study showed nutritional value of lotus seed chiffon cake Odor 5.86± 1.32 c 7.84± 1.01 a 6.83± 0.88b that consist of 210 grams cake flour, 45 grams of lotus Overall odor 5.09± 1.55 c 7.86± 1.02 a 6.69± 1.19b seed flour, 112 grams of sugar, 112 grams of icing, 75 Taste 6 .14± 1.41 c 7.63± 1.06 a 6.84± 0.84b grams of vegetable oil, 70 grams of yolk egg, 198 grams Egg odor 5.97± 1.23 c 7.36± 1.21 a 6.94± 0.97b of white egg, 5 grams of baking soda, 5 grams of vanilla, Softness 5.89± 1.17 c 7.71± 0.93 a 7.03± 0.72b 2 grams ofcream of tartar. Sweetness 7.71± 0.88 a Overall liking 7.84± 0.79 a Sample with difference letter in the same row are difference (p< 0.05) TABLE IV. NUTRITIONAL VALUE OF LOTUS SEED CHIFFON CAKE Sensory evaluation of chiffon cake with 3 levels of lotus Nutritional value Package(weight) seed: The experiment exhibited significant difference (P value > 0.05) in 3 levels of lotus seed 45, 90 and 135 Energy (Kcal) Recipe (931.5 g) Piece (55 g) grams (10%, 20% and 30 %) which added in chiffon cake. CHO. (gm) Sensory evaluation in 100 panelists indicated 45 grams Prot. (gm) 3487.15 05.13 (10%) of lotus seed has the highest average score of color Fat (gm) 7.54± 0.94, odor 7.68± 0.81, overall odor 7.77±0.90, taste Fiber(gm) 365.44 21.47 7.79±1.14, egg odor 7.67±1.16, softness 7.81±1.11, Ca(mg) sweetness 7.70±1.04 and overall liking 7.84±0.97 P(mg) 95.66 5.63 respectively. The consumer prefers 45 grams (10%) of Fe(mg) lotus seed chiffon cake recipe most in all sensory Vit A(IU.) 185.15 10.89 parameters. 9.27 0.55 2438.63 143.45 2759.7 162.34 42.87 2.52 5936.39 349.20 TABLE II. SENSORY ACCEPTANCE OF LOTUS SEED CHIFFON CAKE IV.CONCLUSION Sensory 45 grams Lotus seed 135 grams The ethanolic extract of petal showed the highest total parameter (10%) ������0 grams (30%) phenolic content with 143.06 mg/g. following by extracts of ovary and petal stalk (116.63and 91.39 mg/g. Color 7.54± 0.94 a (20%) 6.58± 1.09 b respectively). Lotus seed, the most popular part of eating Odor 7.68± 0.81 a 6.80± 1.11 b 6.37± 1.16 c exhibited the lowest of total phenolic compound. The Overall odor 7.77± 0.90 a 6.74± 1.19 b 6.37± 1.16 b experiment indicated 45 grams (10%) of lotus seed has Taste 7.79± 1.14 a 6.63± 1.22 b 6.25± 1.20 b the highest average score of color 7.54± 0.94, odor 7.68± Egg odor 7.67± 1.16 a 6.40± 1.27 b 6.04± 1.26 b 0.81, Overall odor 7.77± 0.90, taste 7.79± 1.14, egg odor Softness 7.81± 1.11 a 6.37± 1.16 b 5.98± 1.27 c 7.67± 1.16, softness 7.81± 1.11, sweetness 7.70± 1.04 Sweetness 7.70± 1.04 a 6.35± 1.32 b 6.18± 1.23 b and overall liking 7.84± 0.97 respectively. The panelists Overall liking 7.84± 0.97 a 6.34± 1.24 b 6.13± 1.13 c prefer 45 grams (10%) of lotus seed chiffon cake recipe 6.55± 1.36 b most in all sensory parameter. The future study should be focused on other part of lotus which have high total Sample with difference letter in the same row are difference (p< 0.05) phenolic compound and developed proper health products to promote consumption in daily food. Consumer acceptance of lotus seed chiffon cake product: The Consumer acceptance study of 30 consumer ACKNOWLEDGMENT showed 45 grams (10%) of lotus seed ingredient was accepted by score of color (7.37± 0.45), odor (6.60± Department of Food and Nutrition, Faculty of Home 0.56), overall odor 6.60± 0.62, test 6.50± 0.57, egg odor Economics Technology. Rajamangala University of 6.47± 0.62,softness 6.67± 0.47, sweetness 6.60± 0.62 and Technology Thanyaburi, Thanyaburi, Pathumthani, overall liking 6.90±0.30 respectively. (7-point hedonic Thailand. scale) Department of Applied Thai Traditional Medicine, TABLE III. CONSUMER ACCEPTANCE OF LOTUS SEED CHIFFON CAKE Faculty of Medicine, Thammasat University, Klonglung, Pathumthani. Sensory parameter Score (mean ± SD) (7-points hedonic scale) Color Odor 7.37± 0.45 Overall odor 6.60± 0.56 Taste 6.60± 0.62 Egg odor 6.50± 0.57 Softness 6.47± 0.62 Sweetness 6.67± 0.47 Overall liking 6.60± 0.62 6.90± 0.30 The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 37

Rajamangala University of Technology Thanyaburi (RMUTT) REFERENCES of Nelumbonucifera stimulate lipolysis in the white adipose tissue of mice,” Planta Medica, vol. 73, no. 12, pp. 1255–1259, [1] H. Kitagaki, N. Ono, K. Hayakawa, T. Kitazawa, K. Watanabe, and T. Shiohara.(1997). “Repeated elicitation of contact [5] B. Halliwell. (1999).“Antioxidant defense mechanism: From the beginning to the end,” Soc. Free Radic. Biol. Med, Vol.31, pp. hypersensitivity induces a shift in cutaneous cytokine milieu 261-272. from a T helper cell type 1 to a T helper cell type 2 profile,” The Journal of Immunology, vol. 159, no. 5, pp. 2484–2491. [6] B.N Ames, M.K Shigenaga, and T.M. Hagen.(1993). “Oxidants antioxidants and the degenerative disease of aging,” Proc. Natl. [2] J. H. Oh, B. J. Choi, M. S. Chang, and S. K. Park (2009). Acad. Sci. USA, Vol.9, pp. 7915-7922. “Nelumbonucifera semen extract improves memory in rats with [7] Hyun A.H., Yu J.J. , Na Y.Y. ,Da M.J., Hyun J.B. , Dong-Wook scopolamine-induced amnesia through the induction of choline K., Dong H.N., Jae S.C.(2008). Inhibitory effects of Nelumbo acetyltransferase expression,”Neuroscience Letters, vol. 461, no. nucifera. leaves on rat lens aldose reductase, advanced glycation 1, pp. 41–44. endproducts formation, and oxidative stress.Food and Chemical Toxicology.6: 3818-26. [3] Y. Ono, E. Hattori, Y. Fukaya, S. Imai, and Y. Ohizumi. (2006). “Anti-obesity effect of Nelumbonucifera leaves extract [8] Singleton, V. L.; Rossi, Jr J.A. (1965). Colorimetry of total in mice and rats,” Journal of Ethnopharmacology, vol. 106, no. 2, phenolics with phosphomolybdic phosphotungstic acid reagents. pp. 238–244. American Journal of Enology and Viticulture, 16, 144-158. [4] E. Ohkoshi, H. Miyazaki, K. Shindo, H. Watanabe, A.. Yoshida, and H. Yajima. (2007). “Constituents from the leaves The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 38

Rajamangala University of Technology Thanyaburi (RMUTT) In Vitro and In Silico Studies on HIV-1 Protease and Reverse Transcriptase Inhibitions of Edible MushRoom (Astraeus hygrometricus) Extracts Chanin Sillapachaiyaporn Siriporn Chuchawankul* Program in Clinical Biochemistry and Molecular Department of Transfusion Medicine and Clinical Microbiology, Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, 10330, Thailand, Chulalongkorn University, Bangkok, 10330, Thailand Immunomodulation of Natural Products Research Group, Chulalongkorn University, Bangkok, 10330, Thailand Abstract— Astraeus hygrometricus (AH), an edible mushroom, has been reported for various medicinal [email protected] properties such as anti-tumor, anti-leishmanial and immunomodulatory activities. However, anti-human PR and HIV-1 RT are necessary enzymes that viruses immunodeficiency virus type-1 (HIV-1) activity of AH has need for their replication. Although several antiretroviral never been reported. HIV-1 causes acquired drugs are available, limitations of usage are observed immunodeficiency syndrome, a serious infectious disease such as drug-resistant strains (Apisarnthanarak et al., worldwide. HIV-1 protease (HIV-1 PR) and reverse 2008) and adverse effects of medication (Montessori et transcriptase (HIV-1 RT) are important enzymes needed al., 2004; Wongtrakul et al., 2016). A search for novel for viral replication. In addition, these enzymes are used as compounds from natural products is one of the important targets for anti-retroviral drug development. Herein, AH studies to develop a new class of antiretroviral drug. was extracted with hexane (AHH), ethanol (AHE) and water (AHW). The extracts were in vitro evaluated for their Astraeus hygrometricus (AH) or false earthstar HIV-1 PR and HIV-1 RT inhibitory activities. AHH mushroom is a wild edible mushroom. It has been exhibited strong inhibitions on both HIV-1 PR reported as a rich source of medicinal active compounds. (75.53±0.41%) and HIV-1 RT (48.74±1.37%). Chemical According to previous scientific studies, heteroglucan constituents of AHH were identified by gas which is a polysaccharide isolated from AH revealed chromatography-mass spectrometry (GC-MS). The antitumor and immunostimulatory activities (Mallick et identified compounds were in silico determined for their al., 2010a; Mallick et al., 2010b). Besides, drug-likeness and toxicity parameters as well as binding astrakurkurone, a triterpene isolated from AH exhibited affinity to HIV-1 PR and RT. We found that 5,7,9(11)- anti-leishmanial activity (Mallick et al., 2015; Mallick et androstatriene, 3-hydroxy-17-oxo- (Cpd21) provided a al., 2016). However, this mushroom has never been good prediction of binding affinities on both HIV-1 PR and reported for anti-HIV-1 activity. From medicinal HIV-1 RT, comparable to known inhibitors. These findings properties of AH, we hypothesized that the mushroom indicated that AH could be a potential source of compounds might be composed of certain active compounds with for anti-HIV-1 replication. Moreover, the identified HIV-1 PR and/or HIV-1 RT inhibitory activities. compounds could be beneficial data for novel anti-HIV-1 PR and RT drug development. Herein, AH was extracted by sequential maceration with hexane (AHH), ethanol (AHE) and water (AHW), Keywords— HIV-1 Protease Inhibitor, HIV-1 Reverse respectively. In vitro screening for inhibitory effects of Transcriptase Inhibitor, Astraeus hygrometricus the AH extracts against HIV-1 PR and HIV-1 RT were then evaluated. The crude extract that provides the I. INTRODUCTION strongest inhibition on both enzymes was selected. Gas chromatography-mass spectrometry (GC-MS) was then Human immunodeficiency virus (HIV) is classified utilized to display the chemical profiles. Moreover, drug- into two types: HIV-1 and HIV-2. Both can cause acquire likeness property and binding affinity to the target immunodeficiency syndrome (AIDS), by which HIV-1 is enzymes of the identified compounds were in silico the major type causing infection globally. Moreover, evaluated using ADMET online server and AutoDock 4.0 HIV-1 infection has known to result in greater severity molecular docking program, respectively. This research and progression in comparison with HIV-2 infection discovery would have an impact on antiretroviral drug (Barré-Sinoussi et al., 2013; Smith et al., 1988). HIV-1 development and additive value of the mushroom. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 39

Rajamangala University of Technology Thanyaburi (RMUTT) II. MATERIALS AND TOOLS incubation, HIV-1 PR fluorescent substrate was added. A. Mushroom Extraction Then the fluorescence intensity at excitation/emission of 330/450 nm were continuously measured at 37°C for 90 A. hygrometricus (AH) fruiting bodies were collected min using microplate reader (PerkinElmer EnSpire). The from the local market in Kanchanaburi, Thailand. The results were reported in the percentage of relative AH fruiting bodies were cleaned with water and dried inhibition of HIV-1 PR activity compared to enzymatic under sunlight then grounded into powder by a blender. control, calculated by the next equation. The AH powder was extracted by sequential maceration method as previously described (Sillapachaiyaporn and % relative inhibition HIV-1 PR = [(Slope of enzymatic Chuchawankul, 2019). Briefly, AH (100 g) was extracted control – Slope of sample)/ Slope of enzymatic control] x with 1 L of hexane at room temperature for 72 hr in an 100 incubator shaker at 225 rpm. The hexane soluble part was filtrated through a filter paper (Whatman® No.2). Then, C. HIV-1 RT Inhibitory Activity Assay the hexane was evaporated by rotary evaporator (Heidolph Instruments) to get a hexane crude extract HIV-1 RT inhibitory activities of crude extracts were (AHH, 0.91 g). The dried residue from hexane extraction determined by the colorimetric HIV-1 Reverse was macerated with 1 L of ethanol under the same Transcriptase Assay kit (Roche, Germany). Nevirapine conditions as hexane extraction to produce an ethanol (200 µM) and DMSO (1%, v/v) were used as drug control crude extract (AHE, 2.99 g). Then, the dried residue from and vehicle control, respectively. The assay was carried ethanol extraction was soaked with 1 L of water in 4ºC out according to the procedure recommended in an refrigerator for 72 hr with a magnetic stirrer. The water instruction as described in the previous report extract was filtrated and removed water by freeze-dry (Sillapachaiyaporn and Chuchawankul, 2019). Briefly, lyophilizer to get a water crude extract (AHW, 5.35 g). the 1 mg/ml of samples were incubated at 37°C with the The diagram of sequential extraction was shown in the presence of HIV-1 RT, poly-A template, biotin- figure 1. Percent yield extraction was calculated by the conjugated dUTP and DIG-conjugated dUTP. After an following equation. hour, the reaction mixtures were transferred to the pre- coated streptavidin microplate and incubated at 37 °C for % Yield = (Weigh of crude extract/ Weigh of starting an hour. Next, to remove unbound products, the dried material) x 100 microplate was washed with washing buffer. Then, POD- conjugated anti-DIG antibody was added into the wells Figure 1. Schematic representation of A. hygrometricus sequential and incubated at 37°C. After an hour, the reaction wells extraction were washed and ABTS substrate was added, then the plate was incubated at room temperature on a microplate B. HIV-1 PR Inhibitory Activity Assay shaker at 250 rpm for 15 min. Finally, the converted color HIV-1 Protease Inhibitor Screening Kit, Fluorometric was developed, the absorbance was measured at 405 nm with a reference wavelength at 490 nm. Percentage of (Biovision Incorporated, Milpitas, CA, USA) was used in relative inhibition of HIV-1 RT activity was calculated by order to evaluate HIV-1 PR inhibitory activities of crude comparing with a buffer control condition following the extracts. DMSO (1%, v/v) and pepstatin (1 mM) were equation below. used as vehicle and positive controls, respectively. The assay was performed following the previous study % relative inhibition HIV-1 RT = 100 – (Absorbance of (Ahmad et al., 2018; Sillapachaiyaporn and sample/ Absorbance of buffer control) x 100 Chuchawankul, 2019). Briefly, the samples (1 mg/ml) were incubated with HIV-1 PR at 25 °C for 15 min. After D. Statistical Analysis All experiments were performed in triplicated. The data were shown as the mean of three independent experiments ± SEM. All statistical analyses were studied by one-way ANOVA. Dunnett’s test by IBM SPSS Statistics 22 software were performed. P < 0.05 were considered as statistically significant. E. GC-MS Analysis The GC-MS analysis was performed using a Clarus 600 GC-MS (Perkin Elmer), run on fused silica capillary columns coated with a 5% diphenyl/ 95% Dimethyl Polysiloxane stationary phase (30 m x 250 mm x id 0.25). For sample preparation, the sample was dissolved in 50 µl of methoxyamine hydrochloride (MeOX) in pyridine and incubated for 90 min at 40 °C. Then, 80 µl of N- methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was added to the sample and incubated for 90 min at 40 The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 40

Rajamangala University of Technology Thanyaburi (RMUTT) °C. The mixture was transferred to a GC vial for GC-MS Besides, pepstatin, an HIV-1 PR inhibitor control analysis. The sample was analysed using parameters exhibited HIV-1 PR inhibitory activity at 81.48±0.76 %, following the previous study (Sillapachaiyaporn and data were shown in the Figure 2A. These results suggest Chuchawankul, 2019). The tentative compounds were that AH crude extracts could potentially inhibit HIV-1 PR identified by comparing their mass spectrum to the in the in vitro experiment. database using the National Institute of Standards and Technology (NIST) library. C. Anti-HIV-1 RT Activity Assay F. Evaluation of Lipinski's Rule of Five and ADMET All crude extracts significantly inhibit HIV-1 RT Parameters compared to DMSO control (P<0.001). AHH, AHE and AHW showed the percentage of relative inhibition SwissADME (http://www.swissadme.ch) and compared with enzymatic control at 48.74±1.37, preADMET (https://preadmet.bmdrc.kr/toxicity) online 43.67±1.76 and 38.99±3.71 %, respectively. Nevirapine servers were performed in order to evaluate the Lipinski's (200 µM), known HIV-1 RT drug, exhibited strong rule of five, adsorption, distribution and toxicity inhibition with 99.02±0.98 % while DMSO (1 %, v/v) parameters of identified compounds (Razzaghi-Asl et al., slightly inhibited HIV-1 RT activity (Figure 2B). These 2018). data indicate that AH crude extracts could inhibit HIV-1 RT activity in the in vitro assay. G. Molecular Docking AutoDock 4.0 program was used for molecular docking study. The X-ray crystal structure of HIV-1 PR (PDB ID: 5KR0) and HIV-1 RT (PDB ID: 3QIP) were retrieved from RCSB Protein Data Bank. For protein preparation, we removed the water molecules and all ligands and added in the missing hydrogens. BIOVIA Draw 2018 was exploited in order to draw and minimize ligand. The grid-based approach was used, the grid boxes positions were set following the original inhibitors of HIV-1 PR and RT. All parameters used in this study were set following the previous report (Sillapachaiyaporn and Chuchawankul, 2019). The Discovery studio visualizer was used to studied the protein-ligand interactions of compounds. Amprenavir (APV) and nevirapine (NVP) were used as controls. III. RESULTS AND DISCUSSION A. Mushroom Extraction A. hygrometricus (AH) dried material was extracted to obtain the hexane (AHH), ethanol (AHE) and water (AHW) crude extracts. The percent yields compared to dry weight and appearances of the extracts were shown in Table I. TABLE I. APPEARANCE AND PERCENT YIELD OF AH CRUDE EXTRACTS Crude extract Appearance % Yield AHH Yellow wax 0.91 AHE Brown powder 2.99 AHW Black paste 5.35 B. Anti-HIV-1 PR Activity Assay Figure 2. Percentage of relative inhibition of (A) HIV-1 PR and (B) All crude extracts significantly inhibited HIV-1 PR HIV-1 RT activity of DMSO (1%, v/v), crude extracts (1 activity. AHH and AHE significantly inhibited HIV-1 PR activity with percent of inhibition at 75.53±0.41 and mg/ml) and pepstatin (1 mM) or nevirapine (200 µM). All 31.44±0.56, respectively, compared with DMSO control data represent in the mean ± SEM. Statistical significance (P<0.001). While AHW significantly inhibited enzyme was studied by one-way ANOVA, Dunnett’s test. ***P < activity with 22.82±3.48 % of inhibition (P<0.01). 0.001, **P < 0.01 versus the solvent control. The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 41

Rajamangala University of Technology Thanyaburi (RMUTT) D. Chemical Profiling of AHH provided a match factor or a reverse match factor greater than 600 are listed in Table II. Three major groups could TABLE II. TENTATIVE IDENTIFIED COMPOUNDS OF AHH be classified as terpenes, fatty acids and alkanes. Moreover, these compounds have been previously found RT Area Tentative identification Match R. in natural sources, especially mushrooms. For example, (min) (%) Match fatty acids: n-hexadecanoic acid, octadecanoic acid and 26.156 0.924 3,7,11,15-Tetramethyl-2- 17-octadecanoic acid were commonly found in wild hexadecen-1-ol 825 830 edible mushroom species (Ribeiro et al., 2009). In 26.574 0.132 3,7,11,15-Tetramethyl-2- addition, astrakurkurone and astrakurkurol which are hexadecen-1-ol 780 788 classified as triterpenes with sterol backbone were 26.886 0.284 3,7,11,15-Tetramethyl-2- isolated from AH (Lai et al., 2012). Moreover, we also hexadecen-1-ol 802 810 found 5,7,9(11)-androstatriene, 3-hydroxy-17-oxo- 29.025 0.274 n-Hexadecanoic acid (Cpd21) which has a similar structure as astrakurkurone 31.446 0.645 17-Octadecynoic acid 681 687 and astrakurkurol. 31.688 1.281 Octadecanoic acid 790 792 33.057 0.158 Nonadecane, 2-methyl- 618 621 The identification of components using high- 34.381 0.172 Nonadecane, 2-methyl- 692 751 performance techniques, such as GC-tandem MS (GC- 35.665 0.247 Heptadecane, 2,6,10,15- 746 781 MS/MS) should be confirmed furthermore in order to tetramethyl- 773 788 increase the sensitivity of detection. However, GC-MS 36.905 0.312 Octadecane, 2-methyl- 38.101 0.324 Octadecane, 2-methyl- 756 820 can identify only known compounds that are available in 39.257 0.541 Heptadecane, 2,6,10,15- 779 824 the database. To discover novel compounds, purification tetramethyl- 725 773 and structural elucidation should be performed. 40.369 0.272 Heptadecane, 2,6,10,15- tetramethyl- 722 766 E. Lipinski's Rule of Five and ADMET Parameters 41.124 1.476 5,7,9(11)-Androstatriene, 3- hydroxy-17-oxo- (Cpd21) 651 680 Drug-likeness property of AHH identified 41.436 0.43 Eicosane compounds was first investigated by Lipinski's rule of 41.586 0.478 5-[2-(5-Oxopenta-1,3- 651 680 five. Briefly, molecular weight (MW), number of dienyl)phenyl]penta-2,4- 613 763 rotatable bonds (n-ROTB), number of hydrogen bond 42.482 0.577 dienal acceptor (HBA), number of hydrogen bond acceptor 5-(7a-Isopropenyl-4,5- 602 628 (HBD) and MlogP value are less than or equal 500, 10, 5, 44.47 3.826 dimethyl-octahydroinden- 5 and 4.15, respectively (Lipinski et al., 1997). All 4-yl)-3-methyl-penta-2,4- 610 655 identified compounds meet the Lipinski filter criteria dien-1-ol except tert-hexadecanethiol. Surprisingly, only Cpd21 tert-Hexadecanethiol had high human intestine absorption (HIA), capable of passing through the blood-brain barrier (BBB) and Results from the in vitro screening assays showed non-mutagenicity (Table III). For further study of demonstrated that AHH exhibited the strongest inhibitory molecular docking, we therefore selected the Cpd21 as activities on both HIV-1 PR and HIV-1RT. Thus, AHH the best choice. was selected for the chemical constituent analysis. GC- MS data revealed that approximately 50 isolated peaks were distinguished. The identified compounds that TABLE III. LIPINSKI’S RULE OF FIVE AND ADMET PARAMETERS OF CANDIDATE COMPOUNDS FROM LRH EXTRACTS Compound MW MLogP HBA HBD n-ROTB Lipinski HIA BBB AMES filter Test 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Non- 296.53 5.25 1 1 13 Yes Low No n-Hexadecanoic acid mutagen 17-Octadecynoic acid 256.42 4.19 2 1 14 Yes High Yes Mutagen Octadecanoic acid 280.45 4.57 2 1 15 Yes High Yes Mutagen Nonadecane, 2-methyl- 284.48 4.67 2 1 16 Yes High No Mutagen Heptadecane, 2,6,10,15-tetramethyl- 282.55 7.38 0 0 16 Yes Low No Mutagen Octadecane, 2-methyl- 296.57 7.60 0 0 14 Yes Low No Mutagen 5,7,9(11)-Androstatriene, 3-hydroxy-17-oxo- 268.52 7.15 0 0 15 Yes Low No Mutagen (cpd21) 284.39 3.41 2 1 0 Yes High Yes Eicosane Non- 282.55 7.38 0 0 17 Yes Low No mutagen 5-[2-(5-Oxopenta-1,3-dienyl)phenyl]penta- 2,4-dienal 238.28 2.69 2 0 6 Yes High Yes Non- 5-(7a-Isopropenyl-4,5-dimethyl-octahydroinden- mutagen 4-yl)-3-methyl-penta-2,4-dien-1-ol 288.47 4.65 1 1 4 Yes High Yes Mutagen tert-Hexadecanethiol 969.46 11.65 0 0 30 No Low No Mutagen Non- mutagen MW: Molecular weight; HBA: Number of hydrogen bond acceptor; HBD: Number of hydrogen bond acceptor; n-TOB: Number of rotatable bonds; HIA: Human intestine absorption; BBB: Blood-brain barrier The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 42

Rajamangala University of Technology Thanyaburi (RMUTT) F. Molecular Docking TABLE IV. MOLECULAR DOCKING RESULTS OF 5,7,9(11)- ANDROSTATRIENE, 3-HYDROXY-17-OXO- (CPD21) AT The molecular docking study was performed to HIV-1 PR AND HIV-1 RT ACTIVE SITES evaluate the binding affinity of Cpd21 to target enzymes. Results displayed that the Cpd21 docked into the HIV-1 Ligand ΔG Kpi Number Amino acid PR active site with a binding energy of -9.01 kcal/mol, (nM) of H- interaction which was in line with APV, a reference inhibitor (-9.73 (kcal/mol) bond (Bond length) kcal/mol) (Table IV). In addition, three-dimensional (3D) 249.88 2 schematic interactions revealed that Cpd21 and APV HIV-1 PR 73.84 4 ASP30 (1.91898), were docked in a similar binding domain (Figure 3). GLY49 (3.11815) Cpd21 could be docked into the HIV-1 RT DNA Cpd21 -9.01 46.83 1 ASN25 (3.39168), polymerase active site, comparable with the NPV as a 137.68 1 ILE50 (2.77165), referent control (Table IV and Figure 3). Interestingly, APV -9.73 ASP30 (3.24437) Cpd21 provided better binding energy and inhibition constant values compared to the reference control, data HIV-1 RT GLU138 were tabulated in Table IV. Our findings suggest that Cpd21 -10.00 (2.02117) Cpd21 could potentially inhibit both HIV-1 PR and HIV- LYS101 (3.29016) 1 RT activities. Base on chemical structure, Cpd21 is a NVP -9.36 triterpene containing sterol backbone. Interestingly, previous studies found that triterpenes could inhibit both Cpd21: 5,7,9(11)-androstatriene, 3-hydroxy-17-oxo-; APV: Amprenavia; NPV: Nevirapine HIV-1 PR and HIV-1 RT activities (Aiken and Chen, 2005). ACKNOWLEDGMENT CS was supported by Chulalongkorn University to commemorate the 72nd anniversary of his Majesty King Bhumibol Aduladeja from the Graduate School. This work was partly financially supported by Chulalongkorn University Centenary Academic Development Project and Chulalongkorn University Grant for Immunomodulation by Natural Product research group. Additionally, this work was also partially supported by Ratchadaphiseksomphot Endowment Fund: GCUGR1125602097M to CS. Figure 3. Three-dimensional schematic interactions represent REFERENCES binding site of Cpd21, APV and NVP at (A) HIV-1 PR and (B) HIV-1 RT. Red, blue and yellow molecules [1] Ahmad, R., Sahidin, I., Taher, M., Low, C., Noor, N.M., represent Cpd21, APV and NVP, respectively. Sillapachaiyaporn, C., Chuchawankul, S., Sarachana, T., Tencomnao, T., Iskandar, F. and Rajab, N.F., 2018. In conclusion, results from in vitro screening assays Polygonumins A, a newly isolated compound from the stem of showed that AH extracts exhibited HIV-1 PR and HIV-1 Polygonum minus Huds with potential medicinal activities. RT inhibitory activities. AHH displayed the highest Scientific reports, 8(1): 4202. percent inhibition on both HIV-1 PR and HIV-1 RT activities. GC-MS analysis was performed to display the [2] Aiken, C. and Chen, C.H., 2005. 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Rajamangala University of Technology Thanyaburi (RMUTT) [8] Mallick, S., Dutta, A., Chaudhuri, A., Mukherjee, D., Dey, S., virtual screening and molecular dynamics simulation. Journal of Halder, S., Ghosh, J., Mukherjee, D., Sultana, S.S., Biswas, G., Molecular Graphics and Modelling, 83: 138-152. 2016. Successful therapy of murine visceral Leishmaniasis with astrakurkurone, a triterpene isolated from the mushroom [13] Ribeiro, B., de Pinho, P.G., Andrade, P.B., Baptista, P. and Astraeus hygrometricus, involves the induction of protective cell- Valentão, P., 2009. Fatty acid composition of wild edible mediated immunity and TLR9. Antimicrobial agents and mushrooms species: A comparative study. Microchemical chemotherapy, 60(5): 2696-2708. Journal, 93(1): 29-35. [9] Mallick, S., Maiti, S., Bhutia, S., Maiti, T., 2010a. Antitumor [14] Sillapachaiyaporn, C. and Chuchawankul, S., 2019. HIV-1 properties of a heteroglucan isolated from Astraeus protease and reverse transcriptase inhibition by tiger milk hygrometricus on Dalton’s lymphoma bearing mouse. Food and mushroom (Lignosus rhinocerus) sclerotium extracts: In vitro Chemical Toxicology, 48(8-9):2115-2121. and in silico studies. Journal of Traditional and Complementary Medicine, https://doi.org/10.1016/j.jtcme.2019.08.002 [10] Mallick, S.K., Maiti, S., Bhutia, S.K., Maiti, T.K., 2010b. Immunostimulatory properties of a polysaccharide isolated from [15] Smith, T.F., Srinivasan, A., Schochetman, G., Marcus, M. and Astraeus hygrometricus. Journal of medicinal food, 13(3): 665- Myers, G., 1988. The phylogenetic history of immunodeficiency 672. viruses. Nature, 333(6173): 573. [11] Montessori, V., Press, N., Harris, M., Akagi, L., Montaner, J.S., [16] Wongtrakul, J., Paemanee, A., Wintachai, P., Thepparit, C., 2004. Adverse effects of antiretroviral therapy for HIV infection. Roytrakul, S., Thongtan, T., Janphen, K., Supparatpinyo, K., Canadian Medical Association Journal, 170(2): 229-238. Smith, D.R., 2016. Nevirapine induces apoptosis in liver (HepG2) cells. Asian Pacific journal of tropical medicine, 9(6): [12] Razzaghi-Asl, N., Mirzayi, S., Mahnam, K. and Sepehri, S., 547-553. 2018. Identification of COX-2 inhibitors via structure-based The 1st RMUTT Food Innovation and Smart Farm International Conference Page | 44

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